Molecular Biomarkers in the Characterization & Treatment of Colorectal Carcinoma

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1 Molecular Biomarkers in the Characterization & Treatment of Colorectal Carcinoma Andrew C. Nelson, M.D., Ph.D. Divisions of Anatomic & Molecular Pathology Department of Laboratory Medicine & Pathology

2 Objectives Review current standard of care recommendations for molecular testing in colorectal carcinoma Discuss the role of microsatellite instability as an immunotherapy biomarker Explore the potential clinical relevance of colon cancer molecular subtypes No disclosures

3 Outline Review the biologic players in CRC Guidelines for molecular testing Characterization of inherited predisposition Predictive and prognostic care planning Intersection of therapeutic decisions and molecular subtypes Past, Present, and a possible Future

Growth, differentiation, immune function ~90% of CRC

4 Biologic Pathways Tissue Homeostasis WNT, EGF, TGFB govern normal epithelial regeneration and function Growth, differentiation, immune function ~90% of CRC demonstrate activated WNT pathway signaling ~60% demonstrate 18q LOH (SMAD4)

5 Somatic Alterations MAP-Kinase Pathway Activating Mutations KRAS: ~40% NRAS: ~5% BRAF: ~10% (predominantly in MSI cases) Tend toward mutual exclusivity PI3-Kinase Pathway Activating Mutations PIK3CA: ~20% PTEN loss: ~10% Partially overlapping with RAS mutations

6 Somatic Alterations TP53 alterations Deletion of 17p: ~75% LOH by sequence mutation: ~50% Minimal current clinical value Rare alterations ERBB2 and MET amplification ALK and NTRK rearrangements EGFR mutations Primary and acquired anti-egfr therapy resistance Clinical trials

Inherited Risk for CRC* Syndrome Gene(s) Types of mutation Inheritance Age of Onset Other Cancers* Lynch MLH1, MSH2, PMS2, MSH6, EPCAM Sequence, CNA, rearrangement Autosomal Dominant 40-60

7 Inherited Risk for CRC* Syndrome Gene(s) Types of mutation Inheritance Age of Onset Other Cancers* Lynch MLH1, MSH2, PMS2, MSH6, EPCAM Sequence, CNA, rearrangement Autosomal Dominant Endometrium, ovary, stomach Familial Adenomatous Polyposis APC Sequence (90%) CNA (10%) Autosomal Dominant # Duodenum, thyroid MUTYH-Assoc. Polyposis MUTYH Sequence (99%) Autosomal Recessive Other GI, Gyn, # bladder; Het carriers: breast cancer Peutz-Jeghers Syndrome STK11 (LKB1) Sequence (80%) CNA (15%) Autosomal Dominant Variable, but pediatric Breast, lung, Gyn, other GI Juvenile Polyposis SMAD4 (JPS/HHT), BMPR1A Sequence^ (~85%) CNA^ (~15) Autosomal Dominant Variable; median CRC dx 42 Other GI Cowden (PTEN Hamartoma) PTEN Sequence (90%) Autosomal Dominant Variable; >35 Breast, thyroid, endometrial, renal * Not intended to be exhaustive # Age of onset for GI symptoms; dependent on specific mutation ^ Cause of many cases is unknown (~45%)

8 Lynch Syndrome Prevalence 1-3% CRC; 1-1.5% endometrial carcinoma Lifetime risk for CRC: 50-70% MLH1 & MSH2: 80-90% of cases Rare cases of MSH2 inversion PMS2 & MSH6: 10-15% of cases PMS2 pseudogene EPCAM: 1-3% Genomic deletion (silences MSH2)

9 Diagnostic screening, Prognosis, and Prediction GUIDELINES FOR MOLECULAR TESTING

10 Mainstays of CRC Systemic Therapy Fluoropyrmidines, oxaliplatin, irinotecan FOLFOX, FOLFIRI, CAPEOX, 5-FU/ leucovorin Anti-angiogenesis (VEGF-A) monoclonal Bevacizumab Anti-EGFR monoclonals Cetuximab, Panitumumab Anti-PD1 monoclonals Pembrolizumab, Nivolumab

11 Microsatellite Instability Testing Two purposes Lynch screening Therapy planning NCCN recommends universal screening IHC is favored for ease of implementation Newest ASCO et al. recs stress importance of analytic validation Any concern over IHC performance should be resolved with MSI-PCR High index of suspicion for LS Hallmark histologic features Samowtiz 2015; doi: /modpathol Sepulveda et al. 2017; DOI: /JCO Hegde et al. 2014; doi: /gim

12 Lynch Genetic Risk Assessment Amsterdam Criteria (all) Clinical dx (more specific, less sensitive) 3 or > relatives with LS spectrum cancers 2 successive affected generations 1 case dx d before 50 FAP ruled out Bethesda Criteria (any) Screening criteria (more sensitive, less specific CRC in patient < 50 Personal hx of LS spectrum cancer CRC with MSI-H < 60 FH: [LS-type, 1 < 50] x 1 FH: [LS-type, any] x 2

13 MSI-PCR Tumor Normal NR21 BAT26 BAT25 NR24 NR22 NCI panel: 2 mono and 3 di-nucleotide Quasi-monomorphic: 5 mononucleotide markers More readily interpreted as tumor only If 2 or more markers are shifted: MSI-High

14 Comparison of IHC and PCR Literature review within 2012 AMP report Sensitivity to detect sporadic MSI IHC ~90% MSI-PCR ~95% Sensitivity to detect Lynch IHC ~93% MSI-PCR ~91% Combination 100% Funkhouser et al. 2012; DOI: /j.jmoldx

Ruling Out Lynch MLH1 methylation in Lynch 5% of MMR germline mutation carriers 30-65% of MMR germline NEG with MSI-H Generally reported ~75% BRAF V600E mutation in Lynch 1.

15 Ruling Out Lynch MLH1 methylation in Lynch 5% of MMR germline mutation carriers 30-65% of MMR germline NEG with MSI-H Generally reported ~75% BRAF V600E mutation in Lynch 1.5% of MMR germline mutation carriers 35-63% of MMR germline NEG with MSI-H Generally reported ~50% BRAF mutated in ~5% of MSS CRC Parsons et al. 2012; doi: /jmedgenet

16 Ruling out Lynch: BRAF and MLH1 Testing NCCN and ACMG recommend both MLH1 methylation and BRAF V600E analysis Only MLH1 relevant for endometrial cancer Take Home Point Pursuit of germline testing after a positive result (either MLH1 or BRAF) should be driven by the family history and clinical phenotype

17 MSI: Prognostic & Predictive for Adjuvant Therapy Surgery Alone Stage II; MSI Stage III; MSI MSI = better prognosis, no 5-FU benefit Effectively: pt3n0 tumors managed differently by MSI/MSS status Sargent et al. 2010; DOI: /JCO

18 Predictive Molecular Testing: Metastatic CRC NCCN recommends KRAS, NRAS, and BRAF genotyping K-/N-RAS any known KRAS or NRAS mutation should not be treated with either cetuximab or panitumumab What is known?

19 Predictive Molecular Testing RAS 2017 ASCP/CAP/AMP/ASCO 12,13, 59, 61, 117, 146 in K- or N-RAS 2014 CAP Checklist (active as of October 2017) KRAS 12, 13, 61, 146 NRAS 12, 13, 61 KRAS codons 12 & 13: most extensive clinical evidence Other mutations: subset re-analysis

20 Predictive Molecular Testing RAS Biochemistry Most pathogenic mutations disrupt GTP hydrolysis, extend signaling Variability in biochemical properties Subset analyses or case reports have shown variable anti-egfr mab response KRAS G13D KRAS A59T Hunter et al. 2015; doi: / MCR

21 Predictive Molecular Testing BRAF 2017 NCCN BRAF V600E mutation makes response highly unlikely Retrospective pooled/meta analyses 2017 ASCP/CAP/AMP/ASCO Guideline 4: Insufficient evidence to recommend BRAF V600 as a predictive biomarker for anti-egfr mab therapy

22 MSI Prediction of Immune Checkpoint Inhibitor Response PD-1 inhibitors pembrolizumab (Keytruda) and nivolumab (Opdivo) Either dmmr (IHC) or MSI-H (PCR) = higher probability of response And insurance coverage! No role for PD-L1 IHC at this time MSI-H and BRAF mutant tumors are more likely to be PD-L1 positive Yet, only 20-30% of MSI-H cases are PD-L1+ PD-L1 was not associated with response More to come on this El Jabbour et al. 2017; doi: / jclinpath Rosenbaum et al. 2016; doi: /modpathol

23 Guideline Summary MMR/MSI testing recommended for all CRC IHC and PCR are both acceptable Both should be performed in cases of high suspicion or technically uncertain results BRAF mutation and MLH1 methylation recommended for dmmr (MLH1-/PMS2-) dmmr or MSI-H recommended for checkpoint inhibitor therapy selection Extended K- and N-RAS testing recommended for anti-egfr therapy response prediction

24 Emerging Paradigms with Clinical Relevance? COLON CANCER MOLECULAR SUBTYPES

25 CRC Rx: Established Paradigm One gene alteration one drug Not particularly effective in CRC Many RAS wild-type tumors don t respond Some RAS mutant tumors do respond Resistance develops in majority of cases BRAF-targeted therapy not effective

26 EGFR Therapy Optimization Retrospective analysis of 583 mcrc patients Anti-EGFR n = 255 Anti-VEGFA n = highly sensitive molecular methods for RAS, BRAF, and PIK3CA mutation detection 1% Variant Allele Fraction Association with response and survival Santos et al. 2017; doi: / MCT

27 EGFR Therapy Optimization KRAS exon 2 43% v. 24% RR Pan RAS 48% v. 23% RR Pan RAS + BRAF 50% v. 23% Santos et al. 2017; doi: / MCT

28 CRC Rx: Recent Paradigm MSI-H tumors produce excess neoantigens MSI-H tumors rely on immune checkpoint mechanisms Thus, MSI-H tumors should respond to checkpoint blockade Mehnert et al. 2017; doi: / CCR

29 MSI: Biomarker for Checkpoint Blockade NEJM MSI CRC, 21 MSS CRC, 9 MSI non-crc 70% due to Lynch 45% KRAS mut BRAF WT or unknown MSI-PCR or dmmr IHC Science patients, 12 tumor types; no MSS patients 48% due to Lynch No mention of KRAS or BRAF status MSI-PCR or dmmr IHC DOI: /NEJMoa DOI: /science.aan6733

30 40% Objective Response All Stage IV disease NOTE: PD-L1 and CD8 IHC was evaluated in this study and were NOT significantly associated with response.

31 Best radiographic response: mean 28 weeks 54% CR/PR 23% Stable 14% Progression Median PFS and OS not reached at time of publication Why a Science Paper?

32 Mechanistic Proof of Principle Investigated tumor mutation burden and patient specific T-cell responses No difference in average number of nonsynonymous mutations between responders and non-responders 1644 vs Responders had clonal T-cell responses to mutation-associated neoantigens (MANAs) All were reactive to frameshift mutation epitopes Only a minority of MANAs evoked responses

33 CRC Rx: Future Paradigm? Is there a better combination of molecular and immune-morphology biomarkers to: Better predict response to current therapies Advise different combinations of currently approved therapies Identify new pathway targets

34 Gene Expression Classifiers of Colorectal Carcinoma Clustering analysis of global gene expression patterns Multiple systems described over past ~7 years Ranging from 3 to 6 intrinsic subtypes Commonalities All identify a MSI-H/TIL subtype with an overall good prognosis All identify a mesenchymal subtype with an overall poor prognosis Poor response to therapy, irrespective of RAS status

35 Colorectal cancer subtyping consortium (CRCSC) Analysis of ~4000 (primarily) Stage II/III CRCs that had been used in other classification schemes Developed 4 subtypes CSM1 CSM 4 As well as a 5 th wastebucket unclassified Sadanandam et al. 2014; doi: /cc Dienstmann et al. 2017: doi: /nrc

36 CRCSC Subtypes 14% 13% 37% 23% Dienstmann et al. 2017: doi: /nrc

37 Clinical Translation? Distinguish CMS4 and CMS2 Mesenchymal vs. Canonical CMS4: intrinsic resistance to EGFR mabs and significantly worse overall survival CMS4 tends to have a prominent desmoplastic response, but this is not sensitive enough Develop clinical diagnostics Clinical molecular dx assays or IHC panels Drive prospective trials (combination therapy) Budinska et al. 2014; DOI: /path.4212

38 CRC Rx: Future Paradigm? Dienstmann et al. 2017: doi: /nrc

39 Summary Multi-gene testing of mcrc (K-, N-RAS, BRAF, PIK3CA) may best optimize anti- EGFR mab therapy Assessment of MSI is critical for all stages of CRC Manage disease & future risk Identification of intrinsic CRC subtypes holds promise but isn t primetime

40 Selected References NCCN Clinical Practice Guidelines, Colon Cancer, v NCCN Clinical Practice Guidelines, Genetic/Familial High Risk Assessment: Colorectal, v Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol. 2017;35(13): Hegde M, Ferber M, Mao R, Samowitz W, Ganguly A. ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis). Genet Med. 2014;16(1):

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