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

2. 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

3. 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

4. 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%)

5. 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

6. 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.

7. 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

8. 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

9. 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

10. 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.

11. 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 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 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, (October 2005)
BUB1
N-APC: N-terminal APC fragments of Tumor APC gene protein

12. 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

13. 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

14. 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

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

16. 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, 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

18. 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%

19. 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.

21. 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 genes involved in DNA repair.

22. 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

23. 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

24. + 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.

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

26. Features of MSI associated CRC

27. 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

28. 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,

29. These groups are genetically, clinically & histomorphologically distinct

30. Utility of Knowing Molecular Biology

31. 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

32. Prognostic Value of Molecular classification

34. 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

35. Thank you