Colorectal cancer Chapelle, J Clin Oncol, 2010
Early-Stage Colorectal cancer: Microsatellite instability, multigene assay & emerging molecular strategy Asit Paul, MD, PhD 11/24/15
Mr. X: A 50 yo asymptomatic male, MrX, underwent staging colonoscopy. Colonoscopy showed a 3 cm-mass in the sigmoid colon. Staging scans showed no adenopathy or distant metastases. CEA was 2.0 ng/ml (normal <3 ng/ml). Patient underwent sigmoid colonic resection. Pathology showed moderately differentiated adenocarcinoma, invading upto muscularis propria (pt2). 20 lymph nodes were retrieved and negative for metastasis. What is the next step to do? 1. Observation 2. Adjuvant chemotherapy 3. Testing for MMR protein 4. Testing for RAS and BRAF status
Mr. Y: A 45 yo M presented with 3 episodes of FBPR within a month. Colonoscopy with EUS showed a 4-cm ulcerating-mass obstructing the sigmoid colon, but no enlarged LN. Staging scans showed no adenopathy or distant metastases. CEA was 4.2 ng/ml (normal <3 ng/ml). Patient underwent R hemicolectomy with diverting colostomy. Pathology showed a mucinous adenocarcinoma with involvement of serosa (pt4). 27 lymph nodes were retrieved and negative for metastasis. What is the next step to do? 1. Observation 2. Adjuvant chemotherapy 3. Testing for MMR-protein 4. Multi-gene expression analysis
Current standard of care for non-metastatic CRC Stage I (T1/T2, Node negative): Surgery alone Stage II (T3/T4, Node-negative): Surgery ->?? Stage III (any N, Node positive) : Surgery + adjuvant chemotherapy
Adjuvant chemotherapy is stage II CRC could be beneficial, but the absolute benefit is small (5.4% highrisk, 3.6% low risk, over 5 years) Lancet, 2007, 370:2020-29
Adjuvant Chemotherapy Guidelines for stage II CRC High-risk stage II Colon cancer pt4 Tumor perforation Obstructing tumor Inadequate node sampling (<12 nodes)
Beyond the traditional measures of risk stratification.
Risk of recurrence in Stage II CRC patient in QUASAR study (n=1436) T4 status, MMR protein status & Recurrence score by multigene assay are the significant prognostic factors for recurrence Gray RG, J Clin Oncol, 2011
MMR & MSI Basics Testing strategy Clinical relevance
CRC carcinogenesis pathways Villar & Gruber, Nat Rev Oncol, 2010
Microsatellites Microsatellites are short (1-7 bp) repetitive nucleotide sequences scattered throughout human genome. There are estimated 500,000 microsatellites in human genome. Most common MS in human genome is a dinucloide-repeats of A & C Length of microsatellites varies from personto-person, but have a set length for an individual person ( DNA finger print ).
Microsatellite Instability (MSI) Microsatellites are prone to replication error Erroneous replication process leads to increase or decrease in number of repeats in MS known as microsatellite instability (MSI) Gruber, J Nat Cancer Inst, 2003
Consequence of MSI In most cases, MSI may not have any consequences. If MSI occurs in an important gene, it leads to disease. MSI was first described in XP. MSI in an oncogene or tumor suppressor gene increases the risk of cancer. Lynch syndrome is the classic example of MSI as a result of germline mutation of MMR gene
Mismatched Repair Proteins (MMR) MMR proteins are responsible for surveillance & correction of replicationerrors MMR proteins are products of 4 genes: MLH1, MSH2, MSH6, PMS2 Deficiency of MMR protein (MMR-d), due to mutation of MMR gene (germ line or sporadic), leads to replication error & MSI
Testing of MMR/MSI IHC of tumor tissue for MMR protein expressions MSI testing of tumor tissue A panel of microsatellite markers, is compared in tumor tissue and normal tissue. Core panel of five markers are generally used: BAT25, BAT26, D2S123, D5S346, and D17S250 DNA-based testing, PCR, gene-sequencing analysis (blood or tumor tissue) Chapelle, J Clin Oncol, 2010
MMR Proteins MMR proteins make heterodimers MLH1 & MSH2 are the dominant partner of their pair MSH6 & PMS2 proteins are unstable in absence of their dominant partners
IHC patterns of MMR protein expression Richman, Int J of Oncol, 2015
Mr. Z: A 51 yo M underwent staging colonoscopy. Colonoscopy showed a non-obstructing 3 cm-mass in the ascending colon. Staging scans showed no adenopathy or distant metastases. CEA was 3.0 ng/ml. Patient underwent colonic resection & anastomosis. Pathology showed mucinous adenocarcinoma, invading through muscularis propria to adjacent pericolonic tissue (pt3). 27 lymph nodes were retrieved and negative for metastasis. What is the next step to do? 1. Observation 2. Adjuvant chemotherapy 3. Testing for MMR proteins 4. Testing for RAS and BRAF status
Who should get testing for MMR/MSI Testing for MMR protein and/or MSI should be done for all patients with newlydiagnosed colorectal cancer ASCO, ESMO & ACG guidelines, 2014 Patients who are diagnosed with colorectal cancer <70 years, stage II tumors, or who meet the Bethesda Criteria NCCN, 2015
Phenotype of d-mmr/ MSI Infiltrating Mucinous differentiation Poorly differentiated with medullary growth pattern Crohn-like lymphocytic reaction Early age of onset, R sided tumors, synchronus or metachronus tumors, high-grade/low-stage
Mr. Z Mr. Z with pt3pn0m0 (stage II) colon cancer. His tumor resected tumor was tested for MMRprotein expression by IHC. Staining showed absence of MLH1 & PMS2-proteins, staining for MSH2 & MSH6 are positive. What to do next? 1. Observation 2. Adjuvant chemotherapy 3. MSI-testing of tumor tissue 4. Testing for BRAF mutation
IHC pattern in MMR deficiency MMR-intact: (MMR-i) All MMR proteins are present ->MSS MMR-deficient (MMR-d): 1 or more MMR protiens are deficient -> MSI
15% of CRCs are MMR-d (MSI) More commonly, MMR-d is sporadic & is due to hypermethylation of MLH1 gene promoter. Sporadic tumors often carry the BRAF V600E somatic mutation. Others have germ line mutation of MMR gene (MLH1,MSH2, MSH6, PMS2) i.e Lynch syndrome In case of MLH1-loss by IHC, BRAF testing should be done prior to testing for Lynch Syndrome
Majority of MMR deficiency are sporadic, not Lynch Syndrome Loss of MLH1: Check BRAF mutation first. If wt, genetic testing for LS Loss of MSH2, MSH6, PMS2: genetic testing for LS
Prognostic value of MMR/MSI status in CRC
N=607, 50 y or younger 17% patients have MSI MSI has independent survival advantage over other risk factors MSI tumors tend to metastasize to LNs less Gryffe, NEJM, 2000
Patients with stage II & III CRC & MSI-status have better prognosis n outcome HR CI Data type Dienstmann R, J Clin Oncol. 2015
MSI as a predictor of response to 5FU based chemotherapy Villar & Gruber, Nat Rev Oncol, 2010
Mr. Z Mr. Z with pt3pn0m0 (stage II) colon cancer. Patient s resected tumor was tested for MMR-protein expression by IHC. Staining showed absence of MLH1 & PMS2-proteins, staining for MSH2 & MSH6 are positive. BRAF mutation testing of the tumor was done, which came out to be mutated for BRAF 600E Based on the molecular markers, who has the best & worst prognosis 1. MMR-i (MSS), BRAF wt, R sided tumor 2. MMR-i (MSS), BRAF V600 E mutated, L sided tumor 3. MMR-d (MSI), BRAF wt, R sided tumor 4. MMR-d (MSI), BRAF V600E mutated, R sided tumor
Prognostic value of BRAF V600E mutation MSS- All site MSS- Left side MSS- Right side In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of poor survival only in subpopulations involving microsatellite stable and left-sided tumors (n=1423, PET-ACC3 cohort). KRAS status had no prognostic value. Popovici, BMC cancer, 2013
BRAF V600E is considered poor prognostic factor in CRC Interpretation of BRAF V600E mutation should always be done in the context of MSI status in early CRC The prognostic effect of MSI in early CRC overrides poor prognosis determined by BRAF Worst prognosis is seen in patient with MMR-i (MSS) & L sided tumor Dienstmann R, J Clin Oncol. 2015
Mr. Q A 51 yo M underwent staging colonoscopy. Colonoscopy showed a non-obstructing 3 cm-mass in the sigmoid colon. Staging scans showed no adenopathy or distant metastases. CEA was 3.0 ng/ml. Patient underwent sigmoid colonic resection. Pathology showed mucinous adenocarcinoma, invading through muscularis propria to adjacent pericolonic tissue (pt3). 27 lymph nodes were retrieved and negative for metastasis. IHC showed expression of all 4 MMR proteins (MMR-i). 27 lymph nodes were retrieved and negative for metastasis. What is the next step to do? 1. Observation 2. Adjuvant chemotherapy 3. Molecular/genetic testing for MSI-status 4. Multi-gene assay
Multigene assay in CRC Dienstmann R, J Clin Oncol. 2015
Risk of recurrence in Stage II CRC patient who were treated with edrecolomab or observation in CALGB 9851 study (n=162) Recurrence score & 5 year risk of of recurrence Venook, J Clin Oncol. 2013
Incorporating RS & MMR-status in decision making of stage II CRC T4 tumor: High risk, independent of RS & MMR status Gene expression analysis in not needed Patient should receive adjuvant treatment T3 Tumor, MMR-deficient (MSI) Good outcome, adjuvant chemotherapy is not indicated T3 Tumor, MMR-intact (MSS) Standard risk patient Multigene assay can identify high risk patient
IHC of MMR proteins is recommended for all new patients with CRC MLH1 loss by IHC can be associated with germ-line mutation or sporadic mutation. Loss of MLH1 should have BRAF testing to rule out sporadic mutation Loss of MLH2, MSH2, PMS2 are due to germ line mutation. Germ line mutation of MMR-gene is the hall-mark of Lynch Syndrome. Patients with MMR-d/ MSI have better prognosis Patients with BRAF V600E mutation has poor prognosis, when combined with MSS state
Adjuvant chemotherapy in stage II CRC Dienstmann R, J Clin Oncol. 2015
Adjuvant chemotherapy Stage III (node-positive), CRC patients are at high risk & should receive adjuvant chemotherapy. In Stage II standard risk patients, MMR status & Multigene assay should be taken into consideration in deciding chemotherapy MMR-d (MSI) has good prognosis without adjuvant treatment in stage II CRC. Stage II, MMR-i(MSS) can be risk stratified using multigene assay To date, none of the multi-gene assay are predictive of treatment benefit & should always be used in conjunction with MMR status & clinico-pathologic variables
Emerging Molecular Strategy in CRC
Herzig & Vassiliki, J Surg Onc, 2015
Hereditary Colorectal Neoplasms The Genetics of Colorectal Cancer Jasperson K & Burt RW, Surg Oncol Clin N Am, 2015: 24:683 703 ACG clinical guideline Syngal et al, Am J Gastroenterol, 2015; 110: 223-262 Genetic/familial high risk assessment, NCCN GeneReviews@ NCBI
Hereditary CRC 5-6% of all CRCs are associated with germ-line mutations, causing hereditary predisposition Lynch syndrome is the most common, accounting for 2-3% of all CRCs FAP patients account for <1% of CRCs Other hereditary CRCs are very rare
Jasperson, Sur Clin NA, 2015
Jasperson, Sur Clin NA, 2015
Life-time Risk of Cancers in Lynch Syndrome Kohlmann & Gruber, GeneReviews, 2011
6342 individuals from 147 families 31 families (21%) had at least 1 pancreas CA Kastrinos, JAMA, 2009
Indications of MSI testing to rule out Lynch Syndrome
Jasperson, Sur Clin NA, 2015
Questions?