MOLECULAR BIOLOGY OF

COLO-RECTAL CANCERS

Colorectal Cancer (CRC) results from the progressive accumulation of genetic and epigenetic alterations that lead to the transformation of normal colonic epithelium to colon adenocarcinoma.

“Polyp – cancer” progression sequence

Most CRCs are located in sigmoid colon & rectum but the proportion of cancers that are more proximally located increases with age.

Molecular pathology has site the differences :

Tumors with High levels of microsatellite instability (MSI-H) & CpG island methylation microsatellite stable tumors ; Caecum , ascending colon & transverse colon.

CpG island methylation microsatellite stable tumors ; both right & left colon.

Microsatellite stable tumors without CpG island methylation ; left colon.

WHY STUDY THE MOLECULAR PATHOLOGY OF CRC ? Paradigm definer. Heterogeneity of CRC. Differences in precursor lesions. Different morphology. Prognosticate / Predict. Identifying targets for treatment. Screening policy.

INHERITANCE OF CRC

Sporadic (65Sporadic (65%–%–85%)85%)

Rare CRC Rare CRC syndromes (<0.1%)syndromes (<0.1%)

Familial adenomatous Familial adenomatous polyposis (FAP) (1%)polyposis (FAP) (1%)

Hereditary Hereditary nonpolyposis colorectal nonpolyposis colorectal

cancer (HNPCC) (5%)cancer (HNPCC) (5%)

Familial Familial (10(10%–%–30%)30%)

Cancer associated genes & biomarkers can be grouped into categories based upon the type of information they provide.Predisposition : Germline variants that result in

increased risk.Profile : Somatically mutated in expression within the

tumors.Prognostic : Associated with natural history & clinical

outcome.Predictive : Associated with responsiveness or

resistance of tumors to specific therapies.Pharmacogenomic : Associated with metabolism &

may affect levels & effectiveness of specific therapies.

GERMLINE VARIANTS ASSOCIATED WITH POSSIBLE RISK OF COLORECTAL CARCINOMAS

SyndromeFeatures Commonly Seen

in Affected Individuals Gene DefectSyndromes with adenomatous polypsFamilial adenomatous polyposis (FAP)

Multiple adenomas (>100) and colorectal carcinomas; duodenal polyps and carcinomas; gastric fundus polyps; congenital hypertrophy of retinal epithelium

APC (>90%)

Gardner syndrome Same as FAP, with desmoid tumors and mandibular osteomas

APC

Turcot syndrome Polyposis and CRC with brain tumors (medulloblastoma, glioblastoma)

APC, MLH1

Attenuated adenomatous polyposis (AAPC)

Less than 100 polyps, although marked variation in polyp number (from ~5 to >1,000 polyps) seen in mutation carriers within a single family

APC (5′ mutations)

Hereditary nonpolyposis colorectal cancer (HNPCC)

Colorectal cancer with modest polyposis; high risk of endometrial cancer; some risk of ovarian, gastric, urothelial, hepatobiliary, and brain cancers

MSH2, MLH1, MSH6(together >90%), may bePMS2

MYH-associated polyposis (MAP)

Multiple gastrointestinal polyps, autosomal recessive

MYH

Syndromes with hamartomatous polypsPeutz-Jeghers syndrome Hamartomatous polyps

throughout the gastrointestinal (GI) tract; mucocutaneous pigmentation; estimated 9- to 13-fold increased risk of GI and non-GI cancers

STK11 (30%–70%)

Cowden disease Multiple hamartomas involving breast, thyroid, skin, brain, and GI tract; increased risk of breast, uterus, thyroid, and some GI cancers.

PTEN (85%)

Juvenile polyposis syndrome

Multiple hamartomas in youth, predominantly in colon and stomach; variable increase in colorectal and stomach cancer risk; facial changes

BMPR1A (25%), SMAD4(15%), ENG

PROFILE GENES & BIOMARKERS

CRC is a paradigm for multi-step carcinogenesis with morphologic genomic association in this adenoma-carcinoma sequence

4-STEP SEQUENTIAL PATHWAY-CIN PATHWAY Step 1 : APC inactivation causes adenoma development. Step 2 : KRAS mutations promote adenomatous growth. Step 3 : Genetic alterations of chromosome 18q allowed

progression with biallelic loss. Step 4 : p53 inactivation triggers the final transition to

carcinoma.

CHROMOSOMAL INSTABILITY (CIN) PATHWAY Most syndromic CRCs other than lynch syndrome develop

along CIN pathway.

These cancers are characterized by gross chromosomal abnormalities : Aneuploid karyotype Large chromosome segment deletion & duplication Increased nuclear DNA content.

APC mutation (>90%) , 18q allelic loss (~80%) , TP53 mutation (~70%) , KRAS mutation (~50%).

Some tumors harbour BRAF mutation as an alternative to KRAS mutation.

ROLE OF APC LOSS IN CRC PROLIFERATION & TUMOR PROGRESSION

APC LOSS ß CATENIN

ACCUMULATION CYCLIN D1 , MYC , JUN ,

MMP7 , MMP26

ROLE OF APC IN TUMOR CELL MIGRATION

Inactivation of APC has also been related to the promotion of tumorigenesis , through loss of cell adhesion.

It has been shown that a mutation in APC in mice can decrease the level of E-cadherin at the cell membrane.

LOH at 18q DCC , SMAD2 & SMAD4 are all located at 18q21.1.

Allelic loss at this site is found in upto 60% of CRCs.

SMAD2 & SMAD4 are involved in the TGFβ signalling pathway. Regulate growth. Apoptosis.

SMAD4 germline mutation – Juvenile polyposis syndrome & increased risk of CRC.

MICROSATELLITE INSTABILITY (MSI) PATHWAY

MMR PATHWAY There are 4 important MMR genes : hMLH1, hMSH2,

hMSH6, hPMS2. Loss of function mutation in any one of the four genes. 90% in hMLH1, hMSH2 (MSI Pathway) Epigenetic sliencing – almost always of hMLH1 (CIMP

Pathway) Results in genetic instability giving rise to mutations ; CRC.

MICROSATELLITE INSTABILITY (MSI) PATHWAY Around 15% of sporadic CRCs & almost all CRCs in HNPCC

patients develop along MSI pathway.

SPORADIC TUMORS High levels of microsatellite instability (MSI-H) & loss of

mismatch repair genes (MMR) Due to methylation of MLH1 gene promoter Resultant epigenetic loss of protein expression of MLH1 &

its binding protein PMS2

Sporadic MSI-H cancers have more extensive alteration of microsatellites than Lynch Syndrome CRCs.

LYNCH SYNDROME Loss of MMR genes Occurs due to germline mutation in one of four MMR genes (

MLH1 , MSH2 , MSH6 , PMS2) or TACSTD1 regulatory gene. Increased risk of extra-colonic malignancies : endometrial

cancers , followed by gastric , small intestinal, hepatic & pancreaticobiliary , ovarian & brain tumors.

In these tumors , 2nd allele of affected DNA MMR gene is somatically mutated ; biallelic gene inactivation.

Usually there is loss of expression of corresponding gene product such as MSH6 with MSH2 & PMS2 with MLH1.

MSH6 can also be lost as a result of defective MMR.

TUMORS WITH HIGH LEVELS OF MICROSATELLITE INSTABILITY (MSI-H)

Right sided Mucinous or occasionally Medullary adenocarcinoma Crohns like peritumoral lymphocyte infiltrate Devoid of necrosis Lower stage Expansile growth Better stage specific prognosis Less responsive to certain chemotherapeutic drugs such

as 5-FU but more responsive to irinotecan

CpG ISLAND METHYLATOR PHENOTYPE (CIMP) Methylation of cytosine residues in these CpG islands Change in chromosomal structure Inhibits gene expression (including tumor suppressor

genes) Resultant epigenetic loss of function

Methylation of gene promoters can occur as An age related phenomenon : Type A methylation Specific widespread phenomenon : Type C methylation

Tumors with CIMP often have MSI-H due to methylation of MLH1 MMR gene ; however >50% of CIMP cancers are microsatellite stable.

CIMP status clusters with MSI status & mutations in KRAS , BRAF & TP53 Tumors with frequent MSI-H & BRAF : CIMP 1 Tumors with KRAS mutation but microsatellite stable :

CIMP2 Tumors with TP53 mutation but microsatellite stable :

CIMP3

PROGNOSTIC & PREDICTIVE FACTORS Anatomic extent of disease - TUMOR STAGE ; strongest

prognostic factor for CRCs.ADVERSE FEATURES OF PRIMARY TUMOR

ADVERSE VESSEL INVASION

ADVERSE SURGICAL TECHNIQUES

FAVOURABLE HOST RESPONSE

Greater extent of circumferential involvement

Muscular vein Incomplete mesorectal excision

Intratumoral infiltrate

Bowel obstruction Lymphatic vessel Incomplete excision with residual tumor

Peritumoral infiltrate

Bowel perforation Perineural space Desmoplasia

Poor differentiation

Reactive lymph nodes

Infiltrative pattern of invasion / budding

PREDICTIVE GENES & BIOMARKERS KRAS MUTATION & EGFR MUTATION EGFR is expressed in most CRCs

Located on cell surface Attractive target

Anti-EGFR monoclonal antibodies(cetuximab , panitumumab) Block ligand binding

EGFR signals through RAS/RAF Pathway Mutations in downstream KRAS leads to constitutive

activation Renders tumor unresponsive to upstream inhibition by

anti EGFR antibodies Testing for KRAS mutation is therefore required before

antibodies are used.

Resistance to Anti-EGFR therapy BRAF V600E activating mutation KRAS mutation at codon 61 & 146 PIK3CA exon 20 mutation PTEN protein inactivation

MSI-H & 5-FU , OXALIPLATIN , IRINOTECAN MSI-H predicts poor response to 5-FU & Oxaliplatin.

MMR detects rearrangements produced by 5-FU into DNA & by formation of Oxaliplatin adducts.

Induce apoptosis if these alterations cannot be repaired.

In the absence of MMR Apoptosis may not occur Resistance to these agents seen.

Cell lines with MSI-H are better responsive to Irinotecan.

METHODS OF TESTING FOR MSI-CRC MOLECULAR TESTING

Evaluation of certain loci within human genome that are known to harbour microsatellites

DNA extracted from both normal tissue & tumor tissue After PCR amplification of selected microsatellites , size

of PCR products obtained from normal & tumor tissue are compared.

MSI is defined as change of any length due to either insertion or deletion of repeating units in a microsatellite within a tumor.

As per Bethesda panel , Consists of 2 mono-nucleotide repeats (BAT25 & BAT26)

and 3 di-nucleotide repeats (D5S346 , D2S123 , D17S250)

MSI – H : ≥ 2 out of 5 loci. MSI – L : Instability at one locus. MSS : No loci with instability.

IMMUNOHISTOCHEMISTRY Loss of nuclear immunoreactivity for one or more of MMR

proteins.

Among protein interactions , MLH1 recruits its binding partner PMS2 to the site of DNA repair.

If normal expression of MLH1 is lost , PMS2 shall also be lost.

( MSH2 ~ MSH6)

CONCLUSION MSI + cancers have a better prognosis and CIN + cancers do

worse.

Recent studies reported the combined use of MLH1 & MSH2 by IHC achieved 92% sensitivity & 100% specificity for identification of MSI – H tumors.

More recently , due to heterodimeric pairing properties of MMR , 2–Antibody panel of PMS2 & MSH6 has been as effective as 4-Antibody panel.

IHC testing is readily available while molecular testing is not.

Demonstration of germline mutation is gold standard for diagnosis of Lynch Syndrome.

On IHC , Exclusively described for patients with Lynch Syndrome Loss of MSH2 Individual loss of MSH6 Individual loss of PMS2

MLH1

MSH6

PMS2

MSH2

Loss of MLH1 expression is seen with MSI-H tumors

BRAF Mutation analysis BRAF Mutation present in 40-50% of Sporadic MSI-H

tumors Virtually excludes the possibility of Lynch Syndrome

EGFR blockade is useful provided KRAS mutations are absent and possibly also BRAFV600E mutation.

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