BY: - DR. ADDISU ALEMU

Molecular Diagnostics of Colorectal Cancer

Outline of PresentationIntroductionEtiology Types of CRC Molecular pathogenesis of CRC Molecular diagnosis for colorectal cancer

Introduction

CRC accounts for about 9 percent of all cancer deaths; ranks third in both incidence and cause of cancer death in both men and women.

Approximately 782,000 new cases are diagnosed worldwide each year, of which 70% originate in the colon and the rest in the rectum

Etiology The only certain way to avoid cancer is not to be born, as to live is to incur risk. Genetic mutations inherited

can be transmitted from parent to offspring occurs at or before fertilization

Acquired occurs spontaneously in the sperm, ovum, or zygote -

future progeny may inherit such mutation somatic mutation - during the growth and/or

development Common in CRC (65%)

Types of CRCSporadic = 70% of cases

there is no family history, common above age of 50Inherited = 1 – 6% of cases

Hereditary NonPolyposis CRC (HNPCC, Lynch syndrome) Multiple polyps CRC

familial adenomatous polyposis (FAP) hamartomatous polyposis syndromes (e.g. Peutz-Jeghers, juvenile polyposis) MUTYH-associated polyposis (MAP)

Familial CRC = 25% of cases have a family history of CRC, but the pattern is not consistent and the

risk is not as high as with the inherited syndromes. single affected first-degree relative = 1.7 ↑ed risk two affected first-degree relatives or if Dx before age 55 = Further ↑ risk

The adenoma-carcinoma sequenceMost CRCs arise from adenomas

(adenomatous polyps) which are formed when normal mechanisms regulating epithelial renewal are disrupted.

the accumulation of multiple germ-line or somatic mutations determines the behavior of a tumor

clonal nature of tumorscancers result from the stepwise

accumulation of multiple somatic mutations. Therefore, many CRCs remain asymptomatic for years before diagnosis.

Comprehensive exome sequencing has revealed that individual CRCs harbor an average of 76 gene mutations and the mutated genes in the 2 tumors overlap to only a small extent; a few genes such as APC are mutated at high frequency, whereas a much larger number of genes are mutated at relatively low frequency.

MOLECULAR PATHOGENESIS /Molecular tumorigenesis/

CRC can arise in more than one molecular pathways.

1. Chromosomal instability (CIN), 2. mismatch repair pathway 3. epigenetic gene silencing

1. The chromosomal instability (CIN/APC) pathway

encompasses 80% to 85% of all CRC and adenomaResult in abnormal karyotypes, gross chromosomal

abnormalities, such as aneuploidy, chromosome rearrangement, oncogene activation and loss of heterozygosity of tumor suppressor genes.

Results "gain of function" mutations which may result in loss or mutation of tumor suppressor genes such as APC, TP53 activation of oncogenes such as KRAS apoptotic pathways

CRC caused by CIN usually have poor prognosis

APCIn 90% of CRCs inactivation of the Wnt signaling

pathway /APC or β-catenin gene/; usually by mutation of one copy of the APC gene (70%).

a tumor suppressor adenomatous polyposis coli gene mutation + (allelic deletion or an additional mutation → inactivation of the other allele) → development of dysplasia in aberrant crypt foci and early adenomas → accumulation of additional genetic mutations (KRAS, DCC, p53 and others) → tumor progression

kRAS oncogene encode proteins that regulate cellular signal transduction of

extracellular growth signals(e.g., epidermal growth factors) to the nucleus

point mutations resulting in a constitutively active GTP-bound protein and a continuous growth stimulus.

The activation of RAS genes can promote cell survival and suppress apoptosis

RAS mutations are found in up to 50% of sporadic CRCs and 50% of colonic adenomas larger than 1 cm; they are rarely seen in smaller adenomas → acquired during later adenoma progression

The presence of a RAS mutation in CRC is significantly associated with the absence of response to agents targeting the epidermal growth factor receptor (EGFR) such as cetuximab and panitumumab.

Causes mutation of serine/threonine protein kinase which is involved with that acts as a downstream effector of the KRAS gene.

substitution of valine for glutamate Not detected in any LS/HNPCC tumors occurs in approximately 12% of all CRC,

mutually exclusive of KRAS mutation a prognostic and predictive marker to predict

resistance to anti-EGFR therapy

BRAF gene mutation

Role and Expression Pattern of Epidermal Growth FactorReceptor in Colon Cancer

• EGFR is expressed in normal colon epithelium and in 80- 100% of colorectal cancers (by IHC)

• Up to 40% response rate to anti-EGFR in wild type tumors

• BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate

P53DNA damage → ↑ p53 protein levels intracellular → up

regulation of cyclin dependent cell cycle inhibitor p21Waf1/Cip1→ cell cycle arrest → cells undergo DNA repair

if the cell is unable to repair the DNA injury, p53 leads to an increase in the levels of the proapoptotic protein BAX and the cell undergoes apoptosis.

p53 may also promote apoptosis by baseline suppression of the inhibitor of apoptosis (IAP)

Somatic mutations in both alleles are present in 80 percent of sporadic CRCs, and a single germ-line mutation in this gene is responsible for FAP

Loss of heterozygosity (LOH) can be defined as the manifestation that results from the loss of large genomic regions.

Lack of amplification of a particular segment of DNA can be identified by PCR.

Amplification of two to ten segments of interest is performed.

Loss of heterozygosity (LOH)

DCC (deleted in colorectal cancer)

Tumor suppressor genes, affected as a result of 18q mutations

DCC is an adhesion molecule of the immunoglobulin family, responsible for homotypic binding between cells.

DCC protein loss can be examined by immunohistochemistry.

2. mismatch repair pathway/The mutator phenotype /

accounts for 15% of CRCMismatch repair corrects errors made when DNA is

copied. For example, a C could be inserted opposite anA, or the polymerase could slip or stutter and insert twoto five extra unpaired bases.

If a mismatch or small loop is found, endonuclease (MSH2 protein recognizes, MSH6 or MSH3) cuts the strand bearing the mutation and exonuclease (MLH1 and PMS2) then digests this strand.

Finally it will be filled in by normal cellular enzymes.

microsatellites are short, sequences of 1 to 6 nucleotide base pairs which are repeated dozens to hundred times throughout the genome.

aka simple sequences or short tandem repeats

are a ubiquitous component of the genome of higher organisms.

The most common microsatellite in humans is a dinucleotide repeat of the nucleotides C and A, which occurs tens of thousands of times across the genome.

microsatellite loci with many repeats are rarely detected in the genome

Microsatellites

Microsatellites…

due to their repetitive nature they are liable for backward slippage during DNA replication so that the same short sequence is copied twice a higher number of repeats

causes a higher mutation rates

single-stranded loop or insertion/deletion loops

Microsatellite instability (MSI) due to the presence of the mismatch repair system, in vivo

microsatellite mutation rates range from 10-6 to 10-2 per generation.

Mutation of both alleles of mismatch repair (MMR) system→ DNA strand (microsatellites) might be displaced, and realigns out of register creating small loop of unpaired DNA → unable to remove the loops → microsatellite increases or decrease in size due to either insertion or deletion of repeating units when compared to the normal cell’s = Microsatellite instability (MSI)

MSI is implicated in 50–60% of inherited condition HNPCC(Lynch syndrome) → germline mutations

How does faulty mismatch repair result in colon cancer?

tri- (and hexa-) nucleotide repeats are overrepresented in coding sequences.

If the mutation do NOT encompassed a codon triplet → frameshift mutations → affecting critical areas of cell growth regulation genes → promotion of tumorigenesis.

These kinds of tumors are diploid

3. epigenetic gene silencing (Hypermethylation phenotype (CIMP+)) pathway

DNA methylation is involved in normal cellular control of gene expression (CpG island) are usually found in the regions close to promoters

characterized by methylation of a number of genes rich in CpG islands → silencing of multiple genes or promoter region → loss of gene function or transcriptional inactivation (e.g. tumor-suppressor genes, APC)

If MMR system is silenced→ MSICRCs with high frequency of methylation of some CpG

islands are referred to as CpG island methylation phenotype (CIMP) positive tumors

3. epigenetic gene silencing…

encompasses 35%-40% of sporadic CRCbegin with serrated polyps, especially sessile serrated

adenomas (SSAs) bearing an activating mutation in the BRAF gene.

Epigenetic changes are potentially reversible by drugs.CIMP+ CRCs

microsatellite stable (MSS) cancer (60% of CIMP+ ) or MSI-H (40% of CIMP+)

Methylation occur in hMLH1 methylation may occur in tumor suppressor genes

The adenoma-carcinoma…

FAP

FAP is the most common polyposis syndrome. the risk of CRC by the age of 40 years is almost 100%. It is autosomal dominant and is caused by de novo germline

mutations. The presence of FAP can be diagnosed by direct sequencing

of the germ-line mutations in APC gene on chromosome 5q21.

Somatic APC mutations are also present in most sporadic colorectal adenomas and cancers.

HNPCC (Lynch syndrome)

The presence of HNPCC is defined as the presence of germ-line mutation found in one of the four MMR genes, namelyMLH1, MSH2, MSH6 and PMS2.

HNPCC is the most common hereditary colon cancer syndrome. It is autosomal dominant. 2-hit hypothesis = germline mutation in 1 copy of 1 MMR

gene represents the “first hit,” and somatic inactivation of the wild type allele the “second hit.”

The BRAF gene is almost never mutated in Lynch syndrome–associated CRCs; however, KRAS and p53 mutations can be present.

90%

Bethesda guidelines Bethesda guidelines can be used as a screening tool for

HNPCC.(1) CRC in a patient younger than 50 years of age; (2) synchronous or metachronous colorectal or other HNPCC-

related tumors, (3) CRC with histologic features associated with MSI-H status in a

patient younger than 60 years of age; (4) CRC in 1 or more first-degree relatives with an HNPCC-

associated tumor, with one of the patients being diagnosed before age 50 years; and

(5) CRC in 2 or more first- or second-degree relatives withHNPCC-related tumors, regardless of age.

Molecular testing of CRC Screening Diagnosis Prognosis Targeted drug therapy (personalized cancer care) Follow up

Molecular Diagnosis for CRC

Screening of CRCThe risk of recurrence and subsequent death due to CRC is closely

related to the stage of the disease at the time of the first diagnosis.A good marker helps the detection of disease at earlier stage so that

diseases can be cured effectively.Screening of CRC1. Fecal occult blood testing (FOBT)

is simple, non-invasive and inexpensive false positive results might be yield by diet and medication.

2. Immunological FOBT detects human haemoglobin Specific but low sensitivity at detecting adenomas and CRC

3. Colonoscopy and sigmoidoscopy more effective and sensitive in screening invasive, high cost and inconvenience (privacy, extensive bowel preparation…)

Screening of CRC…

4. molecular markers testingFollowing the better understanding in the molecular basis

of CRC, Molecular markers that detect gene mutation in the early stages of CRC can be used as non-invasive screening tests for early detection of CRC, followed by invasive confirmatory tests such as colonoscopy for individuals with positive results.

e.g. Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)

Screening of CRC…

4.1 Carcinoembryonic antigen (CEA) glycoproteins initially found in embryonic tissue and colon

malignancies. may not be detected until the cancer is in advanced stages found in patients with IBD, malignancies of breast, pancreas,

stomach and lung. So, better used as prognostic markers and for follow up (Half

life of CEA is approximately 2 weeks) rather than screening

Screening of CRC…

4.2 Blood samplesapoptotic release of DNA from tumor cells into the

bloodstream → elevated level of circulating methylated SEPT9 DNA (oncogen) sequences was found.

Screening of CRC…

4.3 Fecal DNA Tests (Cologuard™) during apoptosis of normal colonic cell some DNA fragments of 180-

200 bps long is released into stool. However, CRC cells show a decreased rate of apoptosis and DNA

sequences with 1800-2400 bp are detected in stool samplesCRC cells gene assay for

APC (mutated in up to 70% of specimens), K ras (mutated in up to 60% of specimens) and p53 (mutated in up to 60% of specimens).

for screening specificity 95%; sensitivity 60 - 90% not currently used – expensive, low sensitivity

MOLECULAR DIAGNOSTICS METHODS

Detection of gene mutations with known positions and base changes

o Sanger Sequencingo Allele Specific PCR

Name/Method Target

Intended Use Detected Property

Source Material

Molecular Method Use /Availability

KRAS therapeutic decision EGFR targeted therapy

KRAS mutations FFPE or snap frozen tissue

Sequencing clinical routine

BRAF chemotherapeutic susceptibility

BRAF mutations >> >> sequencing, Real-time PCR clinical routine

BRAF chemotherapeutic susceptibility

BRAF mutations >> >> Digital PCR, COLD-PCR studies

MSI status PCR chemotherapeutic susceptibility

MSI status >> >> PCR clinical routine

MSI status IHC chemotherapeutic susceptibility

MSI status >> >> IHC clinical routine

TP53 mutation screening p53 mutation analysis

>> >> sequencing clinical routine

TP53 mutation screening >> >> >> >> oligonucleotide microarray studies

CIMP probable screening /staging

methylation >> >> methylation microarray studies

miRNA assay for blood/stool

screening miRNA expression level

plasma, stool micro array studies

Multitarget stool DNA test

screening, increasing sensitivityfor colonoscopy

KRAS mutation, NDRG4, BMP3methylation, hemoglobin immunoassay

stool mutation and methylationanalysis, immunoassay

under approvalfor clinical use

Epi proColon early detection assay

screening, increasing sensitivity for colonoscopy

Septin 9 DNA methylation assay

blood plasma Real-time PCR available forclinical use

CIMP = CpG island methylator phenotype, FFPE = formalin fixed paraffin embedded, IHC = immunohistochemistry, miRNA = micro ribonucleic acid,MSI = microsatellite instability, PCR = polymerase chain reaction. ARMS= amplification refractory mutation system

Sanger sequencing

uses dideoxy nucleotides to terminate DNA synthesis, yielding a series of DNA fragments whose sizes can be measured by electrophoresis. The last base in each of these fragments is known, because we know which dideoxy nucleotide was used to terminate each reaction.

directly detects nucleotide sequences of regions of interest. Base substitution, insertion and deletion

mutations, could be detected long turnover time and high running cost

Allele Specific PCR

allele-specific PCR, also known as amplification refractory mutation system (ARMS) that detects specific known mutated form of a gene i.e. primers bind to mutant DNA but not WT DNA so that you only get amplification if the mutant allele is present..

could be adopted to a real-time PCR platform in order to increase the speed and accuracy of the detection Employs probe consists of a fluorophore and a quencher attached

covalently on both ends; that is specific to the targeted DNA sequence. As this probe is broken down by the 5’ to 3’ exonuclease activity of DNA

polymerase, a fluorescent tag is separated from a quenching tag, so fluorescence increases, and this increase can be measured in real time in a fluorimeter.

the amount of target DNA molecules can be measured.

turnover time is shorter than that of Sanger sequencing

Compared to conventional PCR, real-time PCR is quantitative, fast and sensitive.

As no post-PCR manipulation, such as gel or capillary electrophoresis, is required, the risk of laboratory cross-contamination can be minimized.

Identification of MSI-H CRCmicrosatellites are marker of choice

are highly liable for mutation high variability in size and number of repeats from one individual to another, co-dominance and distribution over the euchromatic genome

MSI status determination could be done by immunohistochemical staining for MMR protein expression PCR (A fluorescent multiplex PCR-based method, RT-PCR)

TESTING MSI IN TUMORS PROTOCOL

1. Unstained sections from formalin fixed paraffin embedded tissue (5-7 micron); overlap with H&E stained section• From same patient select one or two tissue

blocks containingo Viable Tumoro Noneoplastic tissue

• Scrape selected areas for DNA extraction at least 1cm2

o Tumor Area (T); Nonneoplastic tissue (N)

PCR Identification of MSI-H …

2. A tumor tissue specimen and normal tissue specimen are amplified using PCR for 5 to 7 microsatellite markers

3. Fluorescently labeled products are sized by capillary electrophoresis

mononucleotide loci

dinucleotide loci

PCR Identification of MSI-H …4. Analyze 5 loci (2 mononucleotide repeats and 3 dinucleotide repeats) for change of any length

1. microsatellite-high(MSI-H) = instability 2 loci 5-fluorouracil is not effective in treating betterprognostic factor sporadic (~12%) or Lynch syndrome–associated (~3%)

2. microsatellite-low (MSI-L) = instability at 1 locus If that locus is dinucleotide → additional analysis of one

mononucleotide repeat with BAT-40 ) is recommended3. microsatellite-stable (MSS) = no microsatellite

mutations

DNA hypermethylation detection

DNA hypermethylation can be detected in primary colorectal carcinomas using bisulfite conversion of DNA samples followed by methylation-specific PCR.

Methylation-Specific PCR (MSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR. However, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated.

Microarray Analysis

is a technique that permits the study of up to thousands of genes concurrently in a single experiment. Multiplex lab on chip

Genes are represented by 15–20 different 25 mer oligonucleotides on GeneChip® that serve as unique, sequence–sequence detectors

Evolved from southern blotting

Is collection of microscopic DNA spots attached to solid surface (glass or silcon chip).

The identity of the the feature is known by its position

Each DNA spot contain 10-12 moles of specific DNA probs which is used to hybridize c DNA cRNA

Prob target hybridization is detected & quantified by fluorophore labled targets

microRNAs in CRC

are a class of small non-coding single-stranded RNAs with important posttranscriptional regulatory functions by binding to their target mRNAs.

E.g. involvement of miRNA-133a in regulating the EGFR pathway

Personalized Cancer Medicine /Targeted therapy/

Reference

http://www.en.wikipedia.org/wiki/Microsatellite_instabilityhttp://www.gradworks.umi.com/33/30/3330991.htmlhttp://www.wjgnet.com/1007-9327/pdf/v20/i14/3847.pdfUpToDate 21.2Harper's Illustrated Biochemistry - Robert K. MurrayENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing

Group / www.els.netwww.medscape.com/viewarticle/575850Arch Pathol Lab Med—Vol 135, May 2011

Thank you