Microsatellite instability and other molecular markers: how usefulare they? Pr Frédéric Bibeau, MD, PhD Pathology Department CHU de Caen France

Content - Colorectal cancer context - CRC molecular classification - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

ptnm CRC classification MUCOSA Muscularis Muscosae --> SUB-MUCOSA MUSCULARIS SUB-SEROSA --> SEROSA --> pt Tis T1 T2 T3 T4 pn pm N0 : no positive lymph node (LN) N1 : 3 positive LN N2 : 4 positive LN M0 : No distant metastasis M1 : Distant metastasis TNM UICC 2009 7 th Classification Organe infiltration and / or visceral peritoneal perforation

Early CRC treatment N+ Stage III Chemotherapy (FOLFOX, 5-FU) pt3-4 N0 Stage II No chemotherapy But rate of relapses: 20% Need for additionnal prognostic factors

Metastatic CRC treatment 6 patients/10 Chemotherapy: 5FU/oxaliplatin/irinotecan Targeted therapies: -Cetuximab(Erbitux )(IgG1) -Panitumumab(Vectibix )(IgG2) -Bevacizumab (Avastin )(IgG1) Aflibercept(Zaltrap ), Regorafenib(Stivarga ) Need for predictive factors Anti-EGFR Epidermal Growth Factor Receptor Anti-VEGF Vascular Endothelial Growth Fractor

CRC context Sporadic (majority of cases) Hereditary (6 % of cases) Screening tools Optimal management

Content - Colorectal cancer context - CRC molecular classification - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

CRC tumour progression Aberrant crypts small polype advanced polyp avancé cancer metastases

Voies CRC d oncogenèse carcinogenesis CCR CIN pathway CIMP pathway MSI pathway Chromosomic Instabilty CpG Island Methylator Phenotype Epigenetic instability MicroSatellite Instabilty 80-85 % 20 % 15-20 %

Chromosomic instability Genomic comparative hybridization(gch array) Aneuploidy Chromosomiclosses/gains Deletions Loss of heterozygocity Mutations: RAS, TP53,.. Amplifications

Epigenetic instability CIMP: CpG Island Methylator Phenotype(tumour suppressor genes) Genes methylation: hmhl1, P16, MGMT BRAFmutation

Microsatellite instability Normal DNA MSI tumour Loss or gain of nucleotides (ou soustraction) Diploidy No /or few lossof heterozygocity

4 proteins for DNA reparation MisMatch Repair system (MMR)

DeficientMMR system 4 proteins for DNA reparation Altered apoptose, cell cycle related gened MSI CRC carcinogenesis

Terminology MSI (microsatellite instable) MSS (microsatellite stable) dmmr (deficient mismatch repair) pmmr (proficient mismatch repair) PhenotypeRER+ (Replication Error+) Phenotype RER- (Replication Error-)

Immunohistochemistry Stable tumour(mss): 4 MMR proteins expressed

Immunohistochemistry Instable tumour(msi): extinction of MMR proteins Loss of hml1 hmsh2 + Negative tumour Positive tumour hmsh6 + Parallel loss of PMS2 personna l casel F. Bibeau *MisMatch Repair

Molecular profile Microsatellite Instability Normal DNA MSI tumour Nucleotides (ou soustraction) Lossor gain

CRC molecular classification Chromosomic instability Epigenetic instability Microsatellite instability CIN pathway CIMP pathway MSI pathway 80-85 % 20 % 15-20 % Conventionnal carcinoma Serrated tumours Cancer of the elderly Lynch syndrome Lieberkühnian Serrated Médullary/ lymphocytes

Content - Colorectal cancer context - CRC molecular classifications - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

Lynch syndrome screening Autosomicdominant transmission MLH1 MHS2 MSH6 PMS2 Germlinemutation Constant MSI EarlyonsetCRC Multiple locations Familial context Othercancers

Lynch syndrome screening Othercancers Colorectal Urinarytract Small bowel Endometrium Lynch syndrome spectrum HNPCC: Hereditary Non Polyposis Colorectal Cancer Cancer risk: 75% CCR, 50% endometrium, 15% others

Lynch syndrome screening MLH1 MSH2 MSH6 PMS2 Germilemutation Time consuming Highly specialized laboratories Constant MSI

Lynch syndrome screening MLH1 MSH2 MSH6 PMS2 Germilemutation Time consuming Highly specialized laboratories Constant MSI DNA Mutation of the corresponding gene RNA PROTEINS

Lynch syndrome screening CRC < 60 ans Personal CRC history CRC familial context MSI + Clinical, endoscopic, and US (si woman) follow-up + Familial investigation Lynch diagnosis Oncogenetics team consultation Germile mutation determination Prophylactic surgery

Microsatellite instability context MSI and hmlh1 loss Sporadic cancer (15%) Hypermethylation MLH1 promotor BRAF mutation Lynch syndrome (2%) Absent Absent Elderly patient Young patient

Content - Colorectal cancer context - CRC molecular classifications - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

Identification of favorable stage II CRC Normal DNA MSI MSI tumour Loss or gain of nucleotides No adjuvant chemotherapy(5-fu)

Caracterization of high risk CRC stage II Perforation Occlusion pt4 Lymphnode< 12 Poorly differenciated tumour Venous/lymphaticInvasion Perineural invasion MSS Adjuvant chemotherapy: to be discussed (5-FU)

Immune infiltration assessement: Immunoscore?

Caracterization of agressive stage III CRC MSS KRAS mut. BRAFmut. Intensifiedchemotherapy: clinicaltrials Stratification accordingmutations? *Taieb et al JAMA Oncol 2016

Identification of agressive stage IV CRC MSS KRAS mut. BRAFmut. Metastatic setting Intensified chemotherapy: FOLFIRINOX+ Bevacizumab(BRAF mut.) Ongoing clinical trials

Content - Colorectal cancer context - CRC molecular classifications - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

Anti-EGFR targeted therapies KRAS mutations = marker of resistance Anti-EGFR antibodies Cetuximab Panitumumab Chemotherapy Radiotherapy Motility Metastases Cell cycle activation Growth Angiogenesis

Résistance: mutations KRAS Normal différenciation, proliferation and growth abnormal différenciation, proliferation and growth Adapté de Van Krieken et al. Virchows 2008;453:417-431

Resistance: RAS mutations Mutations! KRAS EXON 1 EXON 2 EXON 3 EXON 4 12 13 59 61 117 146 N/A 3,8% 5,8% NRAS EXON 1 EXON 2 EXON 3 EXON 4 12 13 59 61 117 146 3,5% 4,1% 0% BRAF EXON 1 EXON 15 EXON 16 600 + 17% Douillard et al. N Engl J Med. 2013;369:1023-34

Recommendations RAS testing mandatory before anti-egfr therapy on Primary CRC Metastasis Or

Molecular techniques

Quality of the pre-analytique step Selection Macrodissection Mutation?

Quality of the pre-analytique step Use the pretreatment biopsy

Complex molecular profile but targetable alterations Amplifications: 2,5% Mutations: 1,9% ALK/ROS/NTRK1,2,3Fusion (<1%) HER-2 FGFRAmplification (<1%) RAS and BRAFWT 40% MSI 5% Mutation KRASexon 2 RAS mut 50% BRCA2 mutation BRAF 10% Mut KRAS ex 3, 4 Mut NRAS Rankin Oncologist 2016

Immunotherapy MSI CRC : immunogenic tumour Metastatic MSI CRC Crohn like reaction Immune escape Lymphocytic infiltrate

MSI CRC : immunogenic tumour Immunotherapy Check-points immunity inhibitors T lymphocyte receptor Antigen Immune enhancement Tumor cell T lymphocyte receptor Antigen PD1 inhibitor Tumor cell Response rate: 90% (anti-pd1 pembrolizumab) PDL1 inhibitor Le DT et al. N Eng J Med 2015;372:2509-20

Immunotherapy Anti-PDL-1 treatment: overall survival CCR MSI CCR MSS Mois Selection of patients based on MSI status

Content - Colorectal cancer context - CRC molecular classifications - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

RAS and BRAF mutationnal status determination Circulating tumour DNA?

Molecular profile Signatures CIT* Carte Identité Tumeurs dm CMS** Consensus Molecular signature Poor pronosis Addition of immune signature? *Marisa et al. Plos One 2016, * * Guinney Nature Med 2015 Adapté de Pagès et al., New Engl J Med 2005

Profil moléculaire Signatures Prediction of response to FOLFIRI, Cetuximab? Reliable on fixed tissue? Reliable by using immunohistochimistry? *Marisa et al. Plos One 2016, Guinney Nature Med 2015

Quality control

Content - Colorectal cancer context - CRC molecular classification - Diagnostic value - Prognostic value - Therapeutic value - Perspectives - Conclusion

Molecular CRC classification- Useful biomarkers CIN pathway 80-85 % CIMP pathway MSI pathway 20 % 15-20 % Conventional carcinoma Serrated tumours Cancer of the elderly Lynch syndrome RAS mutation Anti-EGFR resistance (predictive factor) BRAF mutation Pronostic factor Lynch diagnosis MSI Pronostic No 5-FU efficacy Anti-PD1 efficacy

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Complex molecular profile but targetable alterations Amplifications: 2,5% Mutations: 1,9% HER-2 anti-egfr resistance? Raghac ASCO 2016 RAS and BRAFWT Mutation KRASexon 2 anti-her2 Targeted therapies? Trastuzumab + lapatinib (HERACLES) Trastuzumab + pertuzumab Sartore-Bianchi Lancet Oncol 2016 Hurwitz ASCO GI 2016 40% BRAF 10% MSI 5% Mut KRAS ex 3, 4 Mut NRAS RAS mut 50% SPECTAcolor: Folprecht ESMO 2016, abst 4580

Complex molecular profile but targetable alterations Fusion ALK/ROS/NTRK1,2,3 (<1%) HER-2 Entrectinib De BraudASCO 2015 Sartore-Bianchi JNCI 2016 RAS and BRAFWT 40% MSI 5% Mutation KRASexon 2 RAS mut 50% BRAF 10% Mut KRAS ex 3, 4 Mut NRAS Rankin Oncologist 2016