malignant mesothelioma - nordiqc 2015nordiqc2015.dk/.../mesothelioma-nordiqc-2015-for-web_4.pdf ·...
TRANSCRIPT
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1
bullMalignant Mesothelioma
bullEpidemiology and molecular
biology Oluf Dimitri Roslashe MD PhD
Assoc Prof
Levanger Hospital
Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU)
Trondheim Norway
Aalborg University Hospital Denmark
10062015
No disclosures
3
Short CV
bull My name Oluf Dimitri Roslashe
bull Trondheim Norway
bull Mykonos Greece
bull 10 years GP
bull 15 years Oncology
bull PhD 2008- on mesothelioma
bull Now Consultant oncologist
bull Postdoc NTNU
bull Ass Prof at Aalborg University Aalborg Denmark Hospital
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2
Department of Cancer Research and Molecular Medicine Faculty of Medicine
Norwegian University of Science and Technology TrondheimNorway
A day at the lab center
6
Overview
bull Asbestos
bull Mesothelioma Epidemiology
bull Mesothelioma Molecular Biology
bull Biomarkers Early Biomarkers
bull Cancer-Biomarker HUNT - Where are we
One of our patients with Pleural mesothelioma A woman 42 years old The reason She worked as a hair-dresser And used an Asbestos containing hair dryer For 10 years
History of Asbestos
Asbestos (Greek inextinguishable) a family of naturally occuring minerals that readily separate into thin fibers and are found in many parts of the world Amiantus (Greek untaintable currently used in modern Greek for asbestos) was used by Pliny the Elder (23-79 AD) described that this was mined in the Montains of Arcadia could be spun and woven to material and was resistant to fire [1] The Greek geographer Strabo (6364 BC-ca24 AD) described similar use of the so-called Karystian stone from the ancient quarry in Evvia island of Greece Dioscorides (ca 40-90 AD) described in his Materia Medica the use of this stone for for weaving napkins that could be cleaned and whitened by fire [1]
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In Finland asbestos was used in pottery 4500 years ago In Norway bucket-shaped pottery tempered with crushed asbestos was used for storing and making food in the Late Roman and Migration periods (c 350-475 AD) [2] Marco Polo (1254-1324 AD) also described asbestos mining in China [3] 1 Dilek Y Newcomb S Geological Society of America Meeting (2003) Geological Society of America xii 504 p p 2 Engevik A (2008) Oxford Archaeopress xiv 240 p p 3 Rapp GR (2009) Archaeomineralogy Berlin London Springer xv 348 p p
History of Asbestos
Bucket-shaped pottery contains 20-25 clay and 65-80 asbestos Norway 400-575 AD
Mesothelioma etiology
asbestos inhalation WHO and the IARC monograph (2012) all
types of asbestos class I carcinogens Crocidolite Amosite Anthrophyllite Chrysotile
12
Massive production and use started in the 20th century due to its properties as insulation against heat fire corrosion and its tensile strenght Asbestos was widely used for insulation of water- and combustion pipes materials used for house construction and shipbuilding car brakes and gaskets even toys jewellery and cigarette filters At its peak 3000 products were registered
History of Asbestos
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0
5000
10000
15000
20000
25000
30000
35000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
Norway
Denmark
Sweden
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
World
Europe
Former Soviet Union
USA
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
2013
2 mill tonn asbestos mined in 11- 12 countries
Russia 50 China 20 Brazil 12 Kazakhstan 10
Canada stopped 2013
A meso epidemic is coming in the 3rd World
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17
Asbestos consumption in Denmark 1930-84
Metric tons
Aringr
18
Mesothelioma incidens i North Jutland 5100000
bull 13-fold incrase in 4 decades (1970-2013) and increasing
Panou V Meristoudis C Roslashe OD
26th European Pathology Congress London 2014
MM tilfaeliglde
Aringr
19
Asbestos exposure in North Jutland
46
14
20
20
Types of asbestos exposure for females with MM
DOMESTIC
ENVIRONMENTAL
OCCUPATIONAL
UNCERTAIN
Panou V Omland Oslash Langhoff MD Meristoudis C Weinreich UM Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
20
Overrepresentation of Peritoneal Mesothelioma in
Women
80
20
Females with MM
MM in pleura
MM in peritoneum
91
9
Males with MM
MM in pleura
MM in peritoneum
Panou V Omland Oslash Vyberg M Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
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Type of asbestos exposure and location of
mesothelioma in women
15
85
MPM and types of asbestos exposure
primary
secondary
71
29
MAM and types of asbestos exposure
primary
secondary
Panou V Omland Oslash Meristoudis C Hoffmann L Roslashe OD
Abstract submitted to the 16th World Conference on Lung Cancer Denver 2015
23
Asbestos and Malignant Mesothelioma
bull Mainly by asbestos inhalation first documented by
Dr Wagner South Africa 1960
bull Both pleural and peritoneal mesothelioma is induced by asbestos inhalation
bull Risk of mesothelioma is dependent on dose and duration of asbestos inhalation but there is no lower limit-
one fiber can be enough
bull Occupational exposure
heavily exposed- max 10 got mesothelioma
bull Several occupations with low exposure but still risk (plumber carpenter car mechanichelliphellip)
24
Asbestos and Malignant Mesothelioma
Non-occupational exposures with known risk
Second-hand exposure-
Wife cleaning dusty clothes of husband or son children indirectly exposed
Langhoff et al Dan Med J 2014
Neighborhood exposure- living by an asbestos plant
bull In Italy in Casale Monferrato (Eternit) and Bari (Fibronit)
bull Risk- OR 10 and 525 (lt500m from factory) respectively
bull Barbieri et al Ann Occup Hyg 2012
Environmental exposure- asbestos or similar minerals in the
soil
Greece Turkey China USA Italy
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Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
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29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
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33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
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mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
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DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
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Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
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Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
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microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
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57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
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BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
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65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
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69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
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73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
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77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
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MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
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85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
2
Department of Cancer Research and Molecular Medicine Faculty of Medicine
Norwegian University of Science and Technology TrondheimNorway
A day at the lab center
6
Overview
bull Asbestos
bull Mesothelioma Epidemiology
bull Mesothelioma Molecular Biology
bull Biomarkers Early Biomarkers
bull Cancer-Biomarker HUNT - Where are we
One of our patients with Pleural mesothelioma A woman 42 years old The reason She worked as a hair-dresser And used an Asbestos containing hair dryer For 10 years
History of Asbestos
Asbestos (Greek inextinguishable) a family of naturally occuring minerals that readily separate into thin fibers and are found in many parts of the world Amiantus (Greek untaintable currently used in modern Greek for asbestos) was used by Pliny the Elder (23-79 AD) described that this was mined in the Montains of Arcadia could be spun and woven to material and was resistant to fire [1] The Greek geographer Strabo (6364 BC-ca24 AD) described similar use of the so-called Karystian stone from the ancient quarry in Evvia island of Greece Dioscorides (ca 40-90 AD) described in his Materia Medica the use of this stone for for weaving napkins that could be cleaned and whitened by fire [1]
692015
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9
In Finland asbestos was used in pottery 4500 years ago In Norway bucket-shaped pottery tempered with crushed asbestos was used for storing and making food in the Late Roman and Migration periods (c 350-475 AD) [2] Marco Polo (1254-1324 AD) also described asbestos mining in China [3] 1 Dilek Y Newcomb S Geological Society of America Meeting (2003) Geological Society of America xii 504 p p 2 Engevik A (2008) Oxford Archaeopress xiv 240 p p 3 Rapp GR (2009) Archaeomineralogy Berlin London Springer xv 348 p p
History of Asbestos
Bucket-shaped pottery contains 20-25 clay and 65-80 asbestos Norway 400-575 AD
Mesothelioma etiology
asbestos inhalation WHO and the IARC monograph (2012) all
types of asbestos class I carcinogens Crocidolite Amosite Anthrophyllite Chrysotile
12
Massive production and use started in the 20th century due to its properties as insulation against heat fire corrosion and its tensile strenght Asbestos was widely used for insulation of water- and combustion pipes materials used for house construction and shipbuilding car brakes and gaskets even toys jewellery and cigarette filters At its peak 3000 products were registered
History of Asbestos
692015
4
0
5000
10000
15000
20000
25000
30000
35000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
Norway
Denmark
Sweden
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
World
Europe
Former Soviet Union
USA
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
2013
2 mill tonn asbestos mined in 11- 12 countries
Russia 50 China 20 Brazil 12 Kazakhstan 10
Canada stopped 2013
A meso epidemic is coming in the 3rd World
692015
5
17
Asbestos consumption in Denmark 1930-84
Metric tons
Aringr
18
Mesothelioma incidens i North Jutland 5100000
bull 13-fold incrase in 4 decades (1970-2013) and increasing
Panou V Meristoudis C Roslashe OD
26th European Pathology Congress London 2014
MM tilfaeliglde
Aringr
19
Asbestos exposure in North Jutland
46
14
20
20
Types of asbestos exposure for females with MM
DOMESTIC
ENVIRONMENTAL
OCCUPATIONAL
UNCERTAIN
Panou V Omland Oslash Langhoff MD Meristoudis C Weinreich UM Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
20
Overrepresentation of Peritoneal Mesothelioma in
Women
80
20
Females with MM
MM in pleura
MM in peritoneum
91
9
Males with MM
MM in pleura
MM in peritoneum
Panou V Omland Oslash Vyberg M Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
692015
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21
Type of asbestos exposure and location of
mesothelioma in women
15
85
MPM and types of asbestos exposure
primary
secondary
71
29
MAM and types of asbestos exposure
primary
secondary
Panou V Omland Oslash Meristoudis C Hoffmann L Roslashe OD
Abstract submitted to the 16th World Conference on Lung Cancer Denver 2015
23
Asbestos and Malignant Mesothelioma
bull Mainly by asbestos inhalation first documented by
Dr Wagner South Africa 1960
bull Both pleural and peritoneal mesothelioma is induced by asbestos inhalation
bull Risk of mesothelioma is dependent on dose and duration of asbestos inhalation but there is no lower limit-
one fiber can be enough
bull Occupational exposure
heavily exposed- max 10 got mesothelioma
bull Several occupations with low exposure but still risk (plumber carpenter car mechanichelliphellip)
24
Asbestos and Malignant Mesothelioma
Non-occupational exposures with known risk
Second-hand exposure-
Wife cleaning dusty clothes of husband or son children indirectly exposed
Langhoff et al Dan Med J 2014
Neighborhood exposure- living by an asbestos plant
bull In Italy in Casale Monferrato (Eternit) and Bari (Fibronit)
bull Risk- OR 10 and 525 (lt500m from factory) respectively
bull Barbieri et al Ann Occup Hyg 2012
Environmental exposure- asbestos or similar minerals in the
soil
Greece Turkey China USA Italy
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7
Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
692015
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29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
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41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
3
9
In Finland asbestos was used in pottery 4500 years ago In Norway bucket-shaped pottery tempered with crushed asbestos was used for storing and making food in the Late Roman and Migration periods (c 350-475 AD) [2] Marco Polo (1254-1324 AD) also described asbestos mining in China [3] 1 Dilek Y Newcomb S Geological Society of America Meeting (2003) Geological Society of America xii 504 p p 2 Engevik A (2008) Oxford Archaeopress xiv 240 p p 3 Rapp GR (2009) Archaeomineralogy Berlin London Springer xv 348 p p
History of Asbestos
Bucket-shaped pottery contains 20-25 clay and 65-80 asbestos Norway 400-575 AD
Mesothelioma etiology
asbestos inhalation WHO and the IARC monograph (2012) all
types of asbestos class I carcinogens Crocidolite Amosite Anthrophyllite Chrysotile
12
Massive production and use started in the 20th century due to its properties as insulation against heat fire corrosion and its tensile strenght Asbestos was widely used for insulation of water- and combustion pipes materials used for house construction and shipbuilding car brakes and gaskets even toys jewellery and cigarette filters At its peak 3000 products were registered
History of Asbestos
692015
4
0
5000
10000
15000
20000
25000
30000
35000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
Norway
Denmark
Sweden
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
World
Europe
Former Soviet Union
USA
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
2013
2 mill tonn asbestos mined in 11- 12 countries
Russia 50 China 20 Brazil 12 Kazakhstan 10
Canada stopped 2013
A meso epidemic is coming in the 3rd World
692015
5
17
Asbestos consumption in Denmark 1930-84
Metric tons
Aringr
18
Mesothelioma incidens i North Jutland 5100000
bull 13-fold incrase in 4 decades (1970-2013) and increasing
Panou V Meristoudis C Roslashe OD
26th European Pathology Congress London 2014
MM tilfaeliglde
Aringr
19
Asbestos exposure in North Jutland
46
14
20
20
Types of asbestos exposure for females with MM
DOMESTIC
ENVIRONMENTAL
OCCUPATIONAL
UNCERTAIN
Panou V Omland Oslash Langhoff MD Meristoudis C Weinreich UM Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
20
Overrepresentation of Peritoneal Mesothelioma in
Women
80
20
Females with MM
MM in pleura
MM in peritoneum
91
9
Males with MM
MM in pleura
MM in peritoneum
Panou V Omland Oslash Vyberg M Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
692015
6
21
Type of asbestos exposure and location of
mesothelioma in women
15
85
MPM and types of asbestos exposure
primary
secondary
71
29
MAM and types of asbestos exposure
primary
secondary
Panou V Omland Oslash Meristoudis C Hoffmann L Roslashe OD
Abstract submitted to the 16th World Conference on Lung Cancer Denver 2015
23
Asbestos and Malignant Mesothelioma
bull Mainly by asbestos inhalation first documented by
Dr Wagner South Africa 1960
bull Both pleural and peritoneal mesothelioma is induced by asbestos inhalation
bull Risk of mesothelioma is dependent on dose and duration of asbestos inhalation but there is no lower limit-
one fiber can be enough
bull Occupational exposure
heavily exposed- max 10 got mesothelioma
bull Several occupations with low exposure but still risk (plumber carpenter car mechanichelliphellip)
24
Asbestos and Malignant Mesothelioma
Non-occupational exposures with known risk
Second-hand exposure-
Wife cleaning dusty clothes of husband or son children indirectly exposed
Langhoff et al Dan Med J 2014
Neighborhood exposure- living by an asbestos plant
bull In Italy in Casale Monferrato (Eternit) and Bari (Fibronit)
bull Risk- OR 10 and 525 (lt500m from factory) respectively
bull Barbieri et al Ann Occup Hyg 2012
Environmental exposure- asbestos or similar minerals in the
soil
Greece Turkey China USA Italy
692015
7
Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
692015
8
29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
4
0
5000
10000
15000
20000
25000
30000
35000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
Norway
Denmark
Sweden
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
1920 1930 1940 1950 1960 1970 1975 1980 1985 1990 1995 2000
World
Europe
Former Soviet Union
USA
AC-1956-78
Trends of asbestos consumption in metric tons 1920-2000 based on ldquoWorldwide Asbestos Supply and Consumption Trends from 1900 through 2003rdquo by Robert L Virta
2013
2 mill tonn asbestos mined in 11- 12 countries
Russia 50 China 20 Brazil 12 Kazakhstan 10
Canada stopped 2013
A meso epidemic is coming in the 3rd World
692015
5
17
Asbestos consumption in Denmark 1930-84
Metric tons
Aringr
18
Mesothelioma incidens i North Jutland 5100000
bull 13-fold incrase in 4 decades (1970-2013) and increasing
Panou V Meristoudis C Roslashe OD
26th European Pathology Congress London 2014
MM tilfaeliglde
Aringr
19
Asbestos exposure in North Jutland
46
14
20
20
Types of asbestos exposure for females with MM
DOMESTIC
ENVIRONMENTAL
OCCUPATIONAL
UNCERTAIN
Panou V Omland Oslash Langhoff MD Meristoudis C Weinreich UM Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
20
Overrepresentation of Peritoneal Mesothelioma in
Women
80
20
Females with MM
MM in pleura
MM in peritoneum
91
9
Males with MM
MM in pleura
MM in peritoneum
Panou V Omland Oslash Vyberg M Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
692015
6
21
Type of asbestos exposure and location of
mesothelioma in women
15
85
MPM and types of asbestos exposure
primary
secondary
71
29
MAM and types of asbestos exposure
primary
secondary
Panou V Omland Oslash Meristoudis C Hoffmann L Roslashe OD
Abstract submitted to the 16th World Conference on Lung Cancer Denver 2015
23
Asbestos and Malignant Mesothelioma
bull Mainly by asbestos inhalation first documented by
Dr Wagner South Africa 1960
bull Both pleural and peritoneal mesothelioma is induced by asbestos inhalation
bull Risk of mesothelioma is dependent on dose and duration of asbestos inhalation but there is no lower limit-
one fiber can be enough
bull Occupational exposure
heavily exposed- max 10 got mesothelioma
bull Several occupations with low exposure but still risk (plumber carpenter car mechanichelliphellip)
24
Asbestos and Malignant Mesothelioma
Non-occupational exposures with known risk
Second-hand exposure-
Wife cleaning dusty clothes of husband or son children indirectly exposed
Langhoff et al Dan Med J 2014
Neighborhood exposure- living by an asbestos plant
bull In Italy in Casale Monferrato (Eternit) and Bari (Fibronit)
bull Risk- OR 10 and 525 (lt500m from factory) respectively
bull Barbieri et al Ann Occup Hyg 2012
Environmental exposure- asbestos or similar minerals in the
soil
Greece Turkey China USA Italy
692015
7
Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
692015
8
29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
5
17
Asbestos consumption in Denmark 1930-84
Metric tons
Aringr
18
Mesothelioma incidens i North Jutland 5100000
bull 13-fold incrase in 4 decades (1970-2013) and increasing
Panou V Meristoudis C Roslashe OD
26th European Pathology Congress London 2014
MM tilfaeliglde
Aringr
19
Asbestos exposure in North Jutland
46
14
20
20
Types of asbestos exposure for females with MM
DOMESTIC
ENVIRONMENTAL
OCCUPATIONAL
UNCERTAIN
Panou V Omland Oslash Langhoff MD Meristoudis C Weinreich UM Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
20
Overrepresentation of Peritoneal Mesothelioma in
Women
80
20
Females with MM
MM in pleura
MM in peritoneum
91
9
Males with MM
MM in pleura
MM in peritoneum
Panou V Omland Oslash Vyberg M Roslashe OD
12th International Mesothelioma Conference Cape Town 2014
692015
6
21
Type of asbestos exposure and location of
mesothelioma in women
15
85
MPM and types of asbestos exposure
primary
secondary
71
29
MAM and types of asbestos exposure
primary
secondary
Panou V Omland Oslash Meristoudis C Hoffmann L Roslashe OD
Abstract submitted to the 16th World Conference on Lung Cancer Denver 2015
23
Asbestos and Malignant Mesothelioma
bull Mainly by asbestos inhalation first documented by
Dr Wagner South Africa 1960
bull Both pleural and peritoneal mesothelioma is induced by asbestos inhalation
bull Risk of mesothelioma is dependent on dose and duration of asbestos inhalation but there is no lower limit-
one fiber can be enough
bull Occupational exposure
heavily exposed- max 10 got mesothelioma
bull Several occupations with low exposure but still risk (plumber carpenter car mechanichelliphellip)
24
Asbestos and Malignant Mesothelioma
Non-occupational exposures with known risk
Second-hand exposure-
Wife cleaning dusty clothes of husband or son children indirectly exposed
Langhoff et al Dan Med J 2014
Neighborhood exposure- living by an asbestos plant
bull In Italy in Casale Monferrato (Eternit) and Bari (Fibronit)
bull Risk- OR 10 and 525 (lt500m from factory) respectively
bull Barbieri et al Ann Occup Hyg 2012
Environmental exposure- asbestos or similar minerals in the
soil
Greece Turkey China USA Italy
692015
7
Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
692015
8
29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
6
21
Type of asbestos exposure and location of
mesothelioma in women
15
85
MPM and types of asbestos exposure
primary
secondary
71
29
MAM and types of asbestos exposure
primary
secondary
Panou V Omland Oslash Meristoudis C Hoffmann L Roslashe OD
Abstract submitted to the 16th World Conference on Lung Cancer Denver 2015
23
Asbestos and Malignant Mesothelioma
bull Mainly by asbestos inhalation first documented by
Dr Wagner South Africa 1960
bull Both pleural and peritoneal mesothelioma is induced by asbestos inhalation
bull Risk of mesothelioma is dependent on dose and duration of asbestos inhalation but there is no lower limit-
one fiber can be enough
bull Occupational exposure
heavily exposed- max 10 got mesothelioma
bull Several occupations with low exposure but still risk (plumber carpenter car mechanichelliphellip)
24
Asbestos and Malignant Mesothelioma
Non-occupational exposures with known risk
Second-hand exposure-
Wife cleaning dusty clothes of husband or son children indirectly exposed
Langhoff et al Dan Med J 2014
Neighborhood exposure- living by an asbestos plant
bull In Italy in Casale Monferrato (Eternit) and Bari (Fibronit)
bull Risk- OR 10 and 525 (lt500m from factory) respectively
bull Barbieri et al Ann Occup Hyg 2012
Environmental exposure- asbestos or similar minerals in the
soil
Greece Turkey China USA Italy
692015
7
Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
692015
8
29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
7
Denmark Aalborg Eternit factory 1927-1986 used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus 1936-1986
In Aalborg PathDept 436 mesotheliomas from 1973-2013 In Cyprus unknown number of mesotheliomas
50 of the mortality was of mesothelioma In the Karain village Genetical tendency One village was moved
Cappadocia in Turkey Erionit and Asbestos on the earth surface
Environmental exposure
27
Other possible causes
Confirmed
bull The contrast medium Thorotrast- alpha- emitter (not in
use any more)
bull Secondary cancer many years after thorax-peritoneal
irradiation for lymphoma testicular cancer and breast
cancer
Hypothetical
bull SV40 virus- contaminated poliovaccine
Stella Biointerphases 2011
Future challenges Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future
692015
8
29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
8
29
Incidence
bull There are no reliable world-wide statistics on mesothelioma
bull 0-6 cases 100 000
15000-20000 deaths year very unreliable figures lack of registries
bull USA 4000 year UK 2200 year Denmark 120 year
bull Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world
bull (5100 000) In Aalborg 25-35 cases yearly
bull Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press
bull Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural
mesothelioma in women Analysis of 28 cases in Southeast China American
Journal of Industrial Medicine May 2015
30
Malignant Mesothelioma Facts
bull Latency from asbestos exposure ndash cancer median 40 years
bull Diagnosis is often challenging and
bull Patients are rarely diagnosed in an early stage
bull Pleural Mesothelioma has 12 months median
survival with pemetrexedplatinum chemotherapy
bull 5-years overall survival 0-5
Schematic presentation of
mesothelioma the parietal
and visceral pleura
Representative
histology showing the most
abundant cell types
Mesothelioma with gt80
tumour cells (brown)
Alveolar cells
Vascular
endothelium
Mesothelium
Stroma with
Fibrocytes
fibroblasts
and collagen
Fat cells
Parietal pleura
(Mesothelial cells
stain dark brown)
Visceral pleura
Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
9
33
Mesothelioma pathophysiology
bull Asbestos inhalation
Asbestos fibers -----gtLung----gtPleura Peritoneum -----gtCytotoxicity
-----gtDNA-damage---gtChronic inflammation-----gtMesothelioma
(Latency 10-60 yrs median 40 yrs)
multiple deletions eg p16 (CDKN2A) NF2-Merlin
Inactivation of p53 and pRB but rarely mutation
Differential gene (mRNA) expression
Differential DNA methylation
Differential microRNA expression
Asbestos effects on mesothelium
bull Clastogenic and cytotoxic in vitro
bull Abnormal segregation at mitosis
bull Deletions in 1p 3p21 6q 9p21 15q11-15
22q
bull Promoter hypermethylation at tumor
suppressor loci
Known molecular changes in
mesothelioma
bull AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression
bull HGF and c-Met are highly expressed
bull PI3K AKT and the downstream mTOR are involved in cell growth and survival and are often found to be activated
bull p16INK4a and p14ARF are frequently inactivated and approximately 50 contain NF2 mutation
Crocidolite fiber inhibits microvilli formation in the mesothelial cell
Chrysotile fibers entangle In lung cancer cell division
Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C)
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
10
mRNA expression of mesothelioma
Early Microarray studies-classifiers
Lopez-Rios Cancer Res 2006
PV
PP T
Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other
samples
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
11
41
DNA repair systems Repairs
HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrongdefect bases NER (nucleotide excision repair) Wrongdefect nucleotides
DNA repair pathways are differentially expressed
ICL DSB
BRCA2FANCD1
FANCI FANCF
FEN1
RAD51AP1
FANCA FANCD2
PCNA
XRCC4
NHEJ HR
RAD50
DNA
SHFM1
BCCIP RAD21
DAMAGE RESPONSE
CHEK1
UBE2V2
RAD54L RAD18
CELL CYCLE ARREST
RAD50
TELOMERE MAINTENANCE
EME1 TOP2A
NATURAL VANILIN CURCUMIN
SURVIVAL
VARIOUS INHIBITORS
RAD50
SG2
CHEMOTHERAPY IRRADIATION
GTF2H2p44
CIB1DNA-PK
13 PROTEASOME
GENES MSH6
BORTEZOMIB
PCNA PCNA
CCNOUng2
MMR BERSP BERLP NER
UHRF1
RAD50 RAD18
Searching for molecular resistance mechanisms in the individual patient
One of our cases of Pleural mesothelioma A woman 42 years old 3rd line Alimta Carbo 39 courses Biopsy before and after resistance gt5 years later Genome-wide mRNA expression
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
12
Genes changed m ore than 2 - fold after resistance
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
SP
AR
CL
1S
PA
RC
L1
IG
FB
P7
IT
M2
AS
EP
P1
FS
TL
1S
RP
XS
OX
18
CX
CL
12
DC
NL
OC
72
93
45
CR
TA
PO
SR
1C
LD
N1
CC
L1
5O
GN
SN
AI2
CM
BL
PC
OL
CE
2Z
FP
36
L2
AD
D3
C4
orf1
8C
AV
2IL
33
BC
HE
GP
R1
77
PM
P2
2E
CM
2
KR
T7
EE
F1
A2
PIT
X1
CO
MP
MY
OM
2S
DC
1D
AP
L1
SY
NP
O2
LC
OL
10
A1
MM
P3
GU
CY
1B
2H
FE
2
Gene sym bols
Ge
ne
ex
pre
ss
ion
- a
ve
ra
ge
sig
na
l
Expression before
Expression after
Change down Change up
CHEK1 TYMS NQO1 CK7 TYMP SPARCL1
Tumour before resistance (above) and at acquired resistance (below)
Cell cycle changes in tumour versus normal and in acquired resistance
Next-generation sequencing
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
13
Transcriptomesecond generation
sequencing of mesothelioma
bull 15 new mutations were discovered in 4 mesothelioma
patients
bull All had a different mutational profile
bull Significant aneuploidy and numerous translocations
bull Novel large-scale inter- and intra-chromosomal deletions
inversions and translocations Nearly all candidate point
mutations appeared to be previously unknown SNPs Thirty
tumor-specific fusionstranslocations were independently
validated with PCR and Sanger sequencing
bull Take home message the deeper you search the more
complex and individual changes you find
Sugarbaker et al PNAS 2007 Bueno et al PlosOne 2010
EPIGENETICS OF MESOTHELIOMA
Methylation and microRNAs
DNA methylation distinguishes
mesothelioma adenocarcinoma and
normal lung and pleura
Christensen et al Cancer Res 2009
microRNA expression of mesothelioma
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
14
microRNA origins from non-coding or ldquojunk-DNArdquo
54
microRNA facts
bull gt1800 have been discovered
bull One microRNA can regulate several hundred genes
bull One gene can be regulated by many microRNA
bull Can act as tumor suppressors and oncogenes
bull Can classify tumors
bull Can show cancer risk
bull Diagnosticprognosticpredictive markers potential
bull microRNA are stable in body fluids and tumors and easy to measure by PCR
Detection and analysis
of cancer-specific
microRNA
expression patterns
Benjamin et al J Mol Diagn 2010 November 12(6) 771ndash779
microRNA in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
15
57
microRNA in serum for diagnosing
mesothelioma
bull Increased circulating miR-625-3p was found to be a
potential biomarker for patients with malignant pleural
mesothelioma
Kirschner et al
J Thorac Oncol 2012 Jul7(7)1184-91
microRNA can be a prognostic marker of mesothelioma in serum
Elevated hsa-miR-29c was associated with a significantly longer TTP and survival time
Pass H I et al Cancer Res 2010701916-1924 copy2010 by American Association for Cancer Research
Genetic mesothelioma syndrome ndash
BAP1(BRCA1 associated protein 1)
bull BAP1 is a tumor supressor gene located in the 3p21
bull BAP1 mutations cause a new cancer syndrome characterized by mesothelioma uveal melanoma and melanocytic tumors
bull BAP1 is also related to other cancers like renal cancer
bull BAP1 provides researchers with a novel target for prevention and early detection
Carbone et al Nat Rev Cancer 2013
BAP1 mutation
families and cancer
bull In these two families
bull (n=28)
bull Mesothelioma (12)
bull Uveal Melanoma (1)
bull Skin cancer (2)
bull Pancreatic cancer (1)
bull Prostate cancer (2)
Testa et al Nat Genetics 2011
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
16
BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal
tumorsrdquo (MBAITs) or epitheloid atypical Spitz
tumors
Carbone et al J Transl Med 2012
MOLECULAR BIOMARKERS
63
Clinical Prognostic factors
bull Epithelial vs non-epithelial types (biphasic and
sarcomatous)
bull Sex female better prognosis
bull Age younger better prognosis
bull But still we talk about differences in months
64
WHAT are Molecular Biomarkers
bull ldquoa molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal) a condition or disease rdquo (httpwwwcancergov)
bull Single molecules or signatures of DNA RNA microRNA proteins metabolites that may
bull diagnose and subtype a disease
bull prognose the natural disease path
bull predict effect of certain treatments for this disease
bull Ideally sensitivity and specificity higher than 80
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
17
65
Why are diagnostic biomarkers
problematic
bull Tumor types and subtypes are heterogenous
bull Clinically
bull Histology-Morphology
bull Molecular
bull Genes
bull Epigenetic
66
Biomarkers for early diagnosis
bull Most cancers have a better prognosis by early
detection
bull Non-invasive methods based on blood
analysis is an ideal method
bull Non-invasive methods with SINGLE markers have
repeatedly failed (CEA CA125 CYFRA 19-9
mesothelin osteopontin etc etc)
bull BUT THERE IS HOPEhellip
67
44 Mesothelioma Patients
228 lsquoControlsrsquo
Robinson et al The Lancet 2005
Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC)
68
Studies of biomarkers in blood samples of
Mesothelioma (Panou et al Cancer Treatment Reviews May 2015)
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
18
69
Current Status
There are no clinically validated
non-invasive proven diagnostic markers
for mesothelioma with
high sensitivity and specificity
Translational research urgently needed
70
Studies of microRNA in pre-diagnostic
blood samples of Mesothelioma
ZERO studies
Studies only reached phase 2 of biomarker discovery and validation
71
Our project
Cancer-Biomarkers in HUNT Biomarker discovery for early diagnosis of
human mesothelioma and lung cancer
間皮瘤 amp 肺癌
microRNA regulates mRNA post-translationally Cartoon from the Illumina homepage
72
HUNT Research Centre
HUNT Biobank
Levanger Norway
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
19
73
What is HUNT
bull HUNT is the abbreviation of Helse-Undersoslashkelsen i Nord-
Troslashndelag (Health Survey of Nord-Troslashndelag County)
bull HUNT is a prospective Population-based biobank
bull 78 000 participants (70 of the population)
bull Serum DNA collected 1998 and 2007
bull gt400 clinical parameters registered pr patient
bull gt500 lung cancer and 30 mesothelioma cases with
blood samples 1-15 years PRIOR to clinical disease
74
New biomarkers urgently needed
bull Early diagnostic markers can save many lives world-wide
bull Existing markers are not good enough or not validated for early diagnosis
bull MicroRNA sequencing metabolomics and proteomics of serum has not been combined and may identify unknown new markers
bull Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank
bull We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex age smoke history no cancer
75
What is Cancer-Biomarker-HUNT
Clinical data collection
Clinical history
Physical exams Results collection
Exams results
Biological sample collection
Serumblood sample
Tissue sample
Nucleic acid extraction
Cancer-Biomarker HUNT From clinical samples to biomarkers
RNA
DNA
microRNA mRNA
Epigenetics DNA Methylation
Bioinformatics analyses
Combined Clinical
information and
Validation
Biomarkers
SerumampsamplesampcollectedampandampaccumulatedampMesotheliomaamp
cases
1 1 2 3 4 5 6 7 8 9 10 11 12 14 16
00
20
40
60
80
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
Mesothelioma
0
5000
10000
15000
20000
25000
30000
35000
1741
6487
110133
156179
202225
247275
306347
388
6
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
20
77
Techniques used in this study
bull Next Generation Sequencing (NGS)
-can detect rsaquo 2000 microRNAs
bull Magnetic Resonance Spectrometry (MRS)
-can detect rsaquo 40 metabolites
bull MS Metabolomics -can detect rsaquo 1500 metabolites
bull MS Proteomics -can detect rsaquo 1000 proteins
78
Is this study different
bull Probably the first study with up-to-date
comprehensive high-throughput analysis of
different classes of molecules in the same
pre-diagnostic samples
bull Relations between microRNA proteins AND
metabolites has been little studied especially in
SERUM
79
Next-Generation sequencing (NGS) of
serum microRNA (Illumina)
bull Small RNA sequencing is a powerful application enabling the
discovery and profiling of microRNAs and other non-coding
RNA without prior genome annotation
bull Using low RNA inputs one can profile the differential
expression of known microRNAs as well as detect novel
microRNA targets and wide-ranging sequence variation or iso-
miRs miRBase accessions
bull Illuminas small RNA sequencing methods enable accurate
detection and quantification of rare small RNA sequences
80
RNA isolation and library preparation
bull RNA was isolated from 100 uL serum
bull MicroRNA sequencing libraries were generated using the
Illumina TruSeq small RNA Sample prep kit
Small RNAs
Ribosomal RNAs Marker
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
21
MR based serum
metabolomics
Use 100microl serum
Avance III 600MHz for proton
Equipped with a QCI cryoprobe (for
increased sensitivity) and a Bruker
SampleJet (for automation)
gt 40 small molecular metabolites
can be quantified (Chenomx)
MR Core Facility NTNU httpwwwntnuedudmfmr-corefacility
82
Orbitrap - MS proteomics and
metabolomics
Proteomic approach
Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum
Tryptic digestion of proteins lyophilization and reconstitution for LC-MSMS
Mass spectrometry for ID andor (metabolomics) relative quantitation at PROMEC NTNU
Metabolomic approach
Protein precipitation using cold methanol and centrifugation Lyophilization and
reconstitution for LC-MSMS
84
We correlate serum microRNAs with proteomics and metabolomics
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
22
85
Chemosensitivity Prediction of Tumours Based on Expression miRNA and
Proteomics Data
I Tsamardinos1 2 G Borboudakis1 2 E G Christodoulou3 O D Roslashe4 5 1 Bioinformatics Laboratory ICS-FORTH Heraklion Crete Greece
2 Computer Science Department University of Crete Heraklion Crete Greece 3Cellular Networks Group BIOTEC TU Dresden Dresden Germany
4 Cancer Clinic Levanger Hospital Nord-Troslashndelag Health Trust Levanger Norw ay 5 Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology (NTNU) Trondheim Norway
How to analyze huge amounts of data
from 3-4 datasets bull Comprehensive data analysis by a group of dedicated
bioinformaticians (University of Crete Prof Tsamardinos)
bull Using novel bioinformatics methods
bull mine data for diagnostic signatures pathways and combinations
bull Search for biological meaning
86
Conclusions I
bull Asbestos (Greek inextinguishable) is the term of a family of naturally occurring minerals
bull 1960 J C Wagner C A Sleggs and P Marchand from South Africa linked mesothelioma to asbestos
bull All types of asbestos are class-I carcinogens according to the WHO
bull Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years
bull Other etiological factors are environmental exposition to erionite ionizing radiation and SV40 virus as a putative co-factor
bull Pleural mesothelioma has increasing incidence worldwide
bull Most mesotheliomas are of epithelial type while the sarcomatous and biphasic types are less common
87
Conclusions II
early and combined markers
bull There were no pre-clinical sample studies for mesothelioma
bull The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis
bull including
global microRNA and other non-coding RNAs
metabolomics
proteomics
Immune markers
bull Urgent need for large-scale multicenter studies for miRNA profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid miRNA signatures for timely cancer diagnosis
88
Conclusions III - where are we
bull With a limited sample size we showed that our study design
can identify potential early diagnosis biomarkers for lung
cancer and mesothelioma ca 3 years prior to diagnosis
bull Probably detect subgroups as well
bull Differential expression in metabolites and proteins and
validation studies are ongoing
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
23
European Respiratory Journal ERR 2015
90
PUBLICATIONS ON MESOTHELIOMA
bull 34 Zhibin Gao Kenzo Hiroshima Oluf D Roslashe Xing Zhang Kenji Morinaga
bull Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women Analysis of 28 cases in Southeast China American Journal of Industrial Medicine May 2015
bull
bull 33 V Panou M Vyberg U M Weinreich C Meristoudis U Falkmer O D Roslashe
bull The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews Online 8th May 2015
bull
bull 32 Oluf D Roslashe Giulia M Stella Malignant pleural mesothelioma- history controversy and future of a man-made epidemic review Eur Respir Rev 2015 Mar
bull
bull 13 Xiao-feng Chen Guo-qiang Ping Oluf Dimitri Roslashe Yong-qian Shu Ren-hua Guo Case report Recombinant human endostatin (endostar) decreased recurrent ascites pleural fluid and ascitic VEGF in a case of advanced mesothelioma
bull J Chemother 2012 Aug
bull
bull 12 Oluf Dimitri Roslashe Adam Szulkin Helmut Sandeck Endre Anderssen Tore Amundsen Sten Even Erlandsen Katalin Dobra Stein Harald Sundstroslashm Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma
bull PLoS One 2012
bull 5 Sandeck HP Roslashe OD Kjaeligrheim K Willeacuten H Larsson E Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry Diagn Pathol 2010 Jul
bull
bull 4 Roslashe OD Anderssen E Sandeck H Christensen T Larsson E Lundgren S
bull Malignant pleural mesothelioma Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets Lung Cancer 2010 Jan
bull
bull 3 Oluf D Roslashe Endre Anderssen Eli Helge Caroline Hild Hakvaag Pettersen Karina Standahl Olsen Helmut Sandeck Rune Haaverstad Steinar Lundgren Erik Larsson Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura The emerging gene portrait of the mesothelioma phenotype PLoS One 2009 Aug
bull
bull 2 Roslashe OD Creaney J Lundgren S Larsson E Sandeck H Boffetta P Nilsen TI Robinson B Kjaerheim K Mesothelin-related predictive and prognostic factors in malignant mesothelioma A nested case-control study Lung Cancer 2008 Aug
bull
bull 1 Kjaerheim K Roslashe OD Waterboer T Sehr P Rizk R Dai HY Sandeck H Larsson E Anderssen A Boffetta P Pawlita M Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples Int J Cancer 2007 Jun
bull
bull
bull
AcknowledgementsCollaborators
Norwegian University of Science and Technology (NTNU) Norway
The HUNT research center
Head Prof Kristian Hveem
Dept of Cancer Research and Molecular
Medicine
Prof Hans Krokan
Prof Frank Skorpen
Robin Mjelle PhD student
Lars Hagen PhD
Animesh Sharma MD PhD
Charalampis Giannadakis MD
Dept of Computers and Information
Science
Prof Paringl Saeligtrom
MRI-Cancer Group
Prof Tone F Bathen
Trygve Andeassen PhD
Riyas Vettukatil PhD
University of Crete (UOC) Greece
Dept of Computer Science
Prof Ioannis Tsamardinos
AssProf Vincenzo Lagani
Kostas Kerkentzes PhD student
Nanjing Medical University (NMU) China
Prof Jianwei Zhou
Wu ChenXiaofei Zhi PhD stud
Aalborg University Hospital (AUH) Denmark
Prof Ursula Falkmer
Prof Hans E Johnsen
Prof Mogens Vyberg
Prof Oslashyvind Omland
Overlege Christos Meristoudis
Vasiliki Panou MD PhD stud
Nikolaj A Jensen MD PhD stud
Uppsala University (UU) Sweden
Prof Erik Larsson
692015
24
Takk og god sommer
692015
24
Takk og god sommer