Molecular biology of Colorectal Carcinoma (CRC) Dr. Anurag Mehta RGCI & RC

CRC is a major health issue. Rekindling of interest because mol bio is increasingly exhibiting to us that CRC is not one disease but a group of diseases arising from differing pathways with different intrinsic and extrinsic qualities Incidence of CRC in Males Incidence of CRC in Females

Knowing the molecular basis of CRC can help To deal with this major problem Effective prevention & screening strategy. Strategy for early detection. Appropriate treatment. Knowing the molecular basis of CRC can help

Distribution CRC- inheritance Adapted from Burt RW et al. Prevention and Early Detection of CRC, 1996 Sporadic (75%–80%) Familial (15%–20%) Hereditary nonpolyposis colorectal cancer (HNPCC) (5%) Familial Adenomatous Polyposis (FAP) (1%) Rare CRC syndromes (<0.1%)

OTHERS- RARE like Mut YH, PJ, JP, SSP, POLE, POLD FAMILIAL- 20% CRC SPORADIC- 75% CIN – 80% CIMP-15% INHERITED- 5-8% LYNCH-5% FAP-1% OTHERS- RARE like Mut YH, PJ, JP, SSP, POLE, POLD FAMILIAL- 20% POORLY DEFINED POLYGENIC & MULTIFACTORIAL CIN: Chromosomal instability CIMP: CpG island methylator phenotype FAP: Familial Adenomatous Polyposis Feuer EJ: DEVCAN: National CA Inst. 1999

Arise from at least two different carcinogenic Pathways Sporadic CRC Arise from at least two different carcinogenic Pathways The traditional pathway suppressor or chromosomal instability pathway(CIN). CIN is defined as persistent mis-segregation of whole chromosomes and is caused by defects during mitosis. ~80% sporadic cases The CpG island methylator pathway(CIMP); 15% of all cases of sporadic CRC. Characterized by widespread CpG island methylation in promoter sequences of genes → epigenetic silencing of several genes including MLH1 gene from MMR gene group → Rapid accumulation of genetic alterations with or without MSI→ Carcinogenesis CRCs develop from apparently normal mucosa into a benign precursor stage, the premalignant polyp and can progress to invasive disease. The molecular mechanisms driving this process and the important pathological landmarks that characterize CRC, however, are being continually refined.

Hereditary CRC Lynch Syndrome: ~5% Caused by germline mutation in MMR gene →Microsatellite instability (MSI). Familial Adenomatous Polyposis ~1% Caused by germline mutation in APC gene. Follows the same path as sporadic CIN CRCs. Others

Sporadic Traditional (CIN) Molecular Pathogenesis of CRC Epigenetic Others Sporadic Traditional (CIN) Sporadic CIMP Lynch FAP Sporadic CIN CRC & Hereditary FAP CRC – overlapping molecular pathogenesis Sporadic CIMP and lynch CRC have partly overlapping pathogenesis In all three major pathways of CRC carcinogenesis Genetic

Pino MS, Chung DC. Gastroenterology 2010;138:2059 CIN Pathway BRAF(5-12%) SMAD 2 SMAD 4 Vogelstein’s model: at least 7 mutations are required to form cancer. Recent: genome-wide sequencing – 80 mutations/colon tumor, but <15 genes are true “driver” APC: driver in human and mouse, K-ras: cannot initiate cancer in vivo (only combined with APC) K-ras oncogene is described in 9% of small adenomas, 58% of adenomas larger than 1 cm, and 46% of colorectal Sporadic CRC Gatekeeper Pino MS, Chung DC. Gastroenterology 2010;138:2059

B Catenin Accumulation ROLE of APC loss in CRC – Proliferation & Tumor Progression APC loss B Catenin Accumulation Cyclin D1, Myc, Jun MMP7, MMP26 In the absence of Wnt ligand, beta-catenin is sequestered in a multiprotein degradation complex containing the scaffold protein Axin, the tumor suppressor gene product APC, as well as the kinases CK1 and GSK3beta, among others. Upon sequential phosphorylation, beta-catenin is ubiquititylated by the beta-TrcP-E3ligase complex and subsequently degraded by the proteasome machinery. Wnt ligand associates with Frizzled/LRP co-receptors. By an as yet unknown mechanism, Dishevelled now inactivates the destruction complex. Beta-catenin accumulates in the cytoplasm and subsequently travels to the nucleus. Wnt target gene transcription is initiated following binding of beta-catenin to TCF/Leftranscription factors.

APC mutation role in mitotic check point - genetic instability Jiayin Zhang, Roberto Neisa, and Yinghui Mao: Oncogenic Adenomatous Polyposis Coli Mutants Impair the Mitotic Checkpoint through Direct Interaction with Mad2. Mol Biol Cell. 2009 May 1; 20(9): 2381–2388. BUB1 BUB1 BUB1 Mitotic Origins of Chromosomal Instability in Colorectal Cancer W. Brian Dalton, BA and Vincent W. Yang, MD, PhD. Curr Colorectal Cancer Rep. 2007 April ; 3(2): 59–64. BUB1 Defects in the attachment of microtubules to kinetochores activate the spindle checkpoint to delay mitotic progression by transiently inhibiting the anaphase-promoting complex (also called the cyclosome) Genes involved in the spindle checkpoint were first isolated from Saccharomyces cerevisiae and include MAD1, MAD2, and MAD3 (mitotic arrest–deficient) (Li and Murray, 1991); BUB1, BUBR1 and BUB3 (budding uninhibited by benzimidazole [a microtubule depolymerizing drug]) (Hoyt et al., 1991); and MPS1 (monopolar spindle) In human cells, the defects reported in APC-compromised cells include irregular spindle architecture, abnormal dynamics of spindle microtubules, reduced stretching between sister chromatids, abnormal positioning and segregation of chromosomes, an excess of spindle microtubules which remain unattached to chromosomes, and CIN Geert J.P.L. Kops; Beth A.A. Weaver; Don W. Cleveland. On the Road to Cancer: Aneuploidy and the Mitotic Checkpoint. Nature Reviews Cancer 5, 773-785 (October 2005) BUB1 N-APC: N-terminal APC fragments of Tumor APC gene protein

Role of APC loss in tumor cell migration Inactivation of APC Accumulation of β-Catenin Retention of stem cell features No maturation/ no shedding off Amassing of undifferentiated cell Polyp

APC ↓ Transcription activation- proliferative genes and microenvironment genes Failure of mitotic check point- genomic instability β Catenin induced maturation loss. Cells fails to mature and shed Tumorigenesis

Pino MS, Chung DC. Gastroenterology 2010;138:2059 CIN Pathway BRAF(5%) SMAD 2 SMAD 4 Vogelstein’s model: at least 7 mutations are required to form cancer. Recent: genome-wide sequencing – 80 mutations/colon tumor, but <15 genes are true “driver” APC: driver in human and mouse, K-ras: cannot initiate cancer in vivo (only combined with APC) K-ras oncogene is described in 9% of small adenomas, 58% of adenomas larger than 1 cm, and 46% of colorectal Sporadic CRC Gatekeeper Pino MS, Chung DC. Gastroenterology 2010;138:2059

Several sites of genetic alterations promoting tumorigenesis in EGFR pathway. Relevant to use of Cetuximab /Panitumumab

Effect on anti EGFR immunotherapy Gene Nature of alteration (n) Effect on anti EGFR immunotherapy EGFR Over expression Amplification Mutation ~90% ~50% Rare None KRas/ NRas Gain of function mutations in Codon 12, 13, 59-61 and 146 ~40% No response BRAF V600E- Gain 4-12% PIK3CA Exon 9, 20. Gain 14-18% No response if 20 mutated PTEN Loss of protein expression 13-19% Present recommendation: KRas, NRas , BRAF Testing (NCCN- 2016) Likely that best result if all five are WT

p53 (17p13) Loss of p53, usually through allelic loss of 17p A late event in the traditional pathway Heralds the transition from pre-invasive to invasive disease. Adenomas - 4%-26%, Adenomas with invasive foci - 50% CRCs - 50%

LOH at 18q SMAD2, SMAD4 SMAD2 and SMAD4 are at 18q21.1 Allelic loss at this site is found in up to 60% of CRCs. SMAD2 and SMAD4 are involved in the TGF-β signalling pathway. Regulate growth Apoptosis. SMAD4 germline mutation – Juvenile polyposis syndrome & increased risk of CRC TGF-β pathway is being pursued actively for drug targeting.

Direct enzymatic repair Base excision repair THE LYNCH & CIMP PATHWAYS. ( REPLICATION ERROR REPAIR) DNA repair pathways Direct enzymatic repair Base excision repair Nucleotide excision repair Mismatch repair(MMR): proof reads replicated DNA. Corrects slippages Double-strand break repair Non-homologous end joining Homologous recombination DNA repair very important. 1000 genes involved in DNA repair.

Mismatch repair pathway Defects in mismatch excision repair lead to colon and other cancers. MSH2:MSH6 complex binds the mismatch and identifies newly synthesized strand. MLH1 endonuclease and other factors such as PMS2 bind, recruiting a helicase and exonuclease, which together remove several nucleotides including the lesion. The gap is filled by Pol δ and sealed by DNA ligase. There are at least seven genes in the MMR system: hMLH1, hMLH3, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2

Lynch Syndrome~5% One germline loss of function mutation in anyone of MLH1, MSH2, MSH6, PMS 2. Subsequently the second allele is also lost Failure to repair replication error. Amassing of genetic errors Surrogated as Micro-Satellite Instability. Loss of function mutations in TSG like TGFR-BII, BAX, APC, CASP5, P53 Gain of function mutations in KRAS, CCTNB1 CARCINOMA No BRAF Mutation

+ BRAF Mutation CIMP Pathway ~ 15% 50% genes -CpG islands in promoter sequence Methylation of these islands- gene silencing Microsatellites number altered (MSI-H) Mutations in several genes induced by repair errors P16ink4, p14ARF, THBS1, COX2 SSA CRC BRAF Mutation + One of the silenced gene is MLH1 No MLH1 protein Loss of ability to repair replication Errors Those which undergo BRAF mutation follow sessile serrated adenoma pathway and are high CIMP. CIMPL are ass with K RAS mutation and produce Tubulovillous adenoma. The genes which are methylated in Tumors are called MINT. CONSENSUS for MINT for CRC yet to develop. According to Issa group, the definition of cimp+ is: 1. concordant methylation of a group of genes 2. the methylation is cancer specific If a tumor is defined as cimp+ without referring to what genes are in the group, then cimp+ simply doesn't make any sense because the tumor could be cimp+ for another group of genes.

Premalignant serrated polyps more frequently arise in the proximal colon and are associated with microsatellite instability and aberrant DNA methylation at CpG islands.

Features of MSI associated CRC

CIMP Pathway ~ a smaller fraction Methylation of MGMT - silenced Microsatellites number LESS altered (MSI- L) KRAS Mutation P53 Mutation Tubulovillous / TSA CRC Some can even be MSS despite being CIMP + and BRAF + Those which undergo BRAF mutation follow sessile serrated adenoma pathway and are high CIMP With K RAS mutation are low on CIMP and produce Tubulovillous adenoma

The proportions of each molecular subtype CIMP+ MSI+ 75-80% CIMP- MSI- 5% K RAS Gp 5 (Lynch) 70-75% Aneuploid K-RAS Gp 4 Traditional Adenoma – Carcinoma) 10% BRAF Gp 1 Common CIMP + 5-10% Gp 3 <5% Gp.2 . Molecular classification of CRC; JMD January 2008, vol10, No 1 Jass JR, Histopathology2007,50, 113-130

These groups are genetically, clinically & histomorphologically distinct

Utility of Knowing Molecular Biology

Predictive Value of Molecular classification Biomarker (n) Drug Selection Evidence status K Ras (12,13) 40% Resistance to ant EGFR strong validated K Ras ( 61, 17,146) 1% Probably Moderate In use, not fully BRAF V600E 5% PIK3CA 20% Possibly Limited Not in use PTEN loss 30% possibly MSI 15% Adverse outcome to 5FU -18q/LOH 50% May predict resistance to 5FU

Prognostic Value of Molecular classification

Algorithm to Identify Lynch Syndrome Tumor Section IHC for MLH1, MSH2, MSH6, PMS2 or Look for MSI using NCI or Promega Panel of 5 Microsatellite sites MSI – H(≥2 of 5) Negative staining for MMR protein except MLH1 MLH1 is not expressed Could be CIMP or Lynch BRAF Methylation study on MLH1 Germline testing (Blood / Saliva) for the gene whose protein has been found deficient Negative Positive either or both Counseling Lynch. Carry out germline testing for mutation in MLH1 Sporadic CIMP Not Lynch

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