Friday 16th NovemberOsteosarcoma

Poster discussionL 1425144M 1426725N 1427271O 1433633P 1425094

Bass Hassan

University of OxfordSir William Dunn School of Pathology

Oxford Sarcoma Service at the Oxford University Hospitals TrustOxford Cancer and Haematology Centre

Nuffield Orthopaedic Centre

Osteosarcoma Genomics- Highly Complex

The HALLMARKS

Paper Presentation Summary

Adjuvant chemotherapy plus early >90% necrosis : OS=57% at 25 years

miR-133a suppression improves outcome in mice

Caprin 1 promotes SaOS-2 cell metastasis in mice

Intercalary allografts not recommended

Need for a chemotherapy trial age >40 year

RNA Interference

miRNA Families and targets

L 1425144 Micro-RNA expression and functional profiles of osteosarcomaKobayashi et al National Cancer Research Centre, Tokyo

24 fresh frozen OS8 cell lines

miRNA microarrays (933)anti-miR library

Targets proteins identified (liquid chromatography and mass spec- 2DICAL)

• Blocking let-7 suppressed proliferation• Main targets Serpin H1 and vimentin

24 OS samples

108 commonly expressed miRNAs in OS samples

Identification of miRNA targeted protein

Identification of miRNAs regulating OS cell proliferation

microRNA microarray analysis

Anti-miR inhibitors library, MTT assayIn 8 OS cells

Proteomic approach (2DICAL) In U2 cells

Flow diagram

200 400 600 800

OS67OS65OS64OS52OS51OS50OS49OS48OS46OS45OS44OS38OS36OS35OS32OS28OS27OS26OS25OS20OS18OS17OS13OS03

Sam

ple

Index

0

25

50

75

100

Freq

uenc

y of

miR

NA

exp

ress

ion

515 miRNAs 108 miRNAs

Number of miRNAs (n=939)

Global expression of miRNAs in OS samples

L 1425144

hsa-miR-1274a

hsa-miR-1826

hsa-miR-296-5p

hsa-miR-342-3p

hsa-let-7a hsa-let-7b hsa-let-7f hsa-let-7g hsa-let-7i-40

-30

-20

-10

0

10

20

30

40

50

60

U2 NOS1 MNNGHOS SaOS2

9N2 HsOS1 3N1 MG63

Ch

ang

e o

f A

TP

pro

du

ctio

n (

%)

Inhibited miRNA

Survey of miRNAs regulating OS cell proliferation L 1425144

Serpin H1-Vimentin-

1,093.924m/z (1,093.824~1,094.024)

Vimentin

-Actin-

Serpin H1

let-7a

let-7f

let-7g

let-7i

NC

1

NC

2

let-7a

let-7f

let-7g

let-7i

NC

1

NC

2

let-7a

let-7f

let-7g

let-7i

NC1

NC2

let-7a

let-7f

let-7g

let-7i

NC1

NC2

-Actin-

Identification of proteins regulated by let-7a family miRNAs

M 1426725 Evaluation of the role of metadherin in osteosarcoma metastasisZhu et al University of Texas MD Anderson Cancer Centre

Metadherin (MTDH) Expression is Up-regulated in Osteosarcoma Cells

A

C

B

MTDH Knockdown Decreases Cell Motility and Invasiveness

Blockade of Cell Surface MTDH Impedes Migration and Invasion

Invasion Migration

Invasion Migration

M 1426725

Knockdown of MTDH Inhibits Pulmonary Metastasis of Osteosarcoma Cells

A B

C D IHC-MTDH

M 1426725

N 1427271 Targeting liposomal nanoparticles to a mouse model of osteosarcomaVaghasia et al UCLA, Los Angeles

Federman N. Targeting liposomes toward novel pediatric anticancer therapeutics. Pediatr Res. 67(5), 514-9

ALCAM (CD 166) has been previously shown to be highly expressed in osteosarcoma cell lines

Previous in vitro work demonstrated targeting and improved cytotoxicity in ALCAM targeted nanoparticles when compared to a conventional liposomal Doxorubin.

Federman N. Enhanced growth inhibition of osteosarcoma by cytotoxic polymerized liposomal nanoparticles targeting the alcam cell surface receptor. Sarcoma. 2012; 2012:126906.

Osteosarcoma cell lines KHOS and MNNG were transduced with a CMV-luciferase-puromycin lentiviral plasmid

N 1427271

N 1427271

O 1433633 Prediction of a patient survival by MMP1 and P16 immunoreactivity In osteosarcoma tumour samples before and after neoadjuvant chemotherapyRobl et al Zurich Switzerland

Methods:Tissue micro array (TMA): samples of 86 patients, taken before (biopsy, Bx) and after neoadjuvant chemotherapy (resection, Rx) to analyze 36 known tumor marker proteinsOnly non-necrotic tissue sections were graded (controlled by a pathologist)

semi-quantitative grading performed in MATLAB1

Mean values of grades (Bx and Rx) were used for further analysis: downregulation / no change / upregulation “proportion of change” (PoC) = (downregulation + upregulation) / total # of samples

1Ruifrok, AC, Johnston, DA. (2001). Anal Quant Cytol Histol, 23:291-299.

• All markers showed a change in expression before and after neoadjuvant chemotherapy

• The PoCs ranged from 3 to 87 % based on samples fulfilling all criteria:• -Bx and Rx available• -no metastasis at diagnosis• -neoadjuvant chemotherapy

Results

Fig. 1: Markers ranked according to the largest PoC in immunoreactivity (Bx vs Rx).

0

5

10

15

20

25

30DownregulationNo changeUpregulation

Nu

mb

er

of

Pa

tie

nts

Vim

en

tin

Pte

n C

ap

rin

CX

CR

4 C

D4

4 M

MP

1

CC

N1

C

XC

R7

_11

G8

Irx

4

Ne

uro

pil

in2

P

16

PoC (87%) (84%) (79%) (77%) (73%) (73%) (69%) (67%) (67%) (67%) (59%)

O 1433633

• If correlated with 5-YS (overall five-year survival) only expression changes of P16 (P=0.044) and MMP1 (P=0.012) showed a significant correlation

Fig. 2: Change in immunoreactivity (Bx vs. Rx) of P16 and MMP1 in non-metastatic OS patients related to patient status after five years.

Down-

regulation Down-

regulationNo change

Up-regulation

Up-regulation

No changeSeries10

5

10

15

20

25

P16-immunoreactivity(Ntotal = 37 patients)

deceasedalive

Nu

mb

er

of

pa

tie

nts

Series10

5

10

15

20

25

MMP1-immunoreactivity(Ntotal = 33 patients)

deceasedalive

Nu

mb

er

of

pa

tie

nts

ResultsO 1433633

P 1425094 Micro RNA expression profiling indentifies mir-15B as a regulator of multi-drug resistance in human osteosarcoma cell linesDuann et al MGH, Boston

KH

OS

U-2

OS

KH

OS R

2

U-2

OS M

R

P<0.01

KHOSKHOSR2U-2OSU-2OSMRSKOV-3SKOV-3TR

Val

ues

of

expr

essi

on

Abso

rban

ce

Doxorubicin (μM)

U-2OSMR

U-2OSMR+ miR-15b 1nM

U-2OSMR+ miR-15b 5nM

U-2OSMR+ miR-15b 10nM

U-2OSMR+ miR-15b 20nM

Doxorubicin (μM)

Abso

rban

ce

U-2OSMR

U-2OSMR+ nonspecific miR precursor 5nM

U-2OSMR+ nonspecific miR precursor 10nM

U-2OSMR+ nonspecific miR precursor 20nM

P 1425094

Wee1 98kD

0 1 5 10 20 40

miR-15b precursor (nM)

U-2

OS

MR

10 20 40

miR- precursor nonspecific control

Actin 42kD

Wee1 98kD

Actin 42kDKH

OS

R2

P 1425094

Wee1 target gene

microRNA

Thank you to all the speakers, poster presentersand organisers