Molecular subtyping: how useful is it?

Molecular subtyping: how useful is it? Daniela E. Aust, Institute for Pathology, University Hospital Dresden, Germany Center for Molecular Tumor Diagnostics at the NCT-Partner Site Dresden CMTD

Disclosure slide I Member of advisory boards for AMGEN, ROCHE, BOEHRINGER I Speaker honoraria from FALK Pharma, Pfizer, Lilly and ROCHE I Third party funds from MERCK for immunohistochemistry in a clinical trial

What can (molecular) pathology offer for clinical decisions in colorectal cancer? Better understanding of the disease Prognostic markers Predictive markers

Therapeutic Targeting of the Hallmarks of Cancer Institut für Pathologie Weinberg R & Hanahan D, 2011

GOAL:

Molecular subtyping of CRC why? Institut für Pathologie To select stage II patients who are at riskof recurrence (~15%) To select stage III patients who are at low risk of recurrence (~50%) To select stage II and III patients who will benefit from adjuvant chemotherapy To select patients for targeted or intensified therapies

UICC Stage II and Stage III prognosis Institut für Pathologie I Inside each tumour stage the risk of recurrence is depending of various risk factors I For UICC stage II : obstruction/perforation, emergent admission, T4 stage, high-grade, less than 12 LN are indicative of poor prognosis I For stage III the number of positive lymph-nodes are associated with the risk of recurrence I The only validated prognostic biomarker is the MSI status in stage II patients O Connor J Clin Oncol 2011;29:3381-88 Weisser J Clin Oncol 2011; 29:4796-802 Roth J Cin Oncol 2009; 28:466-74

Molecular subtyping of CRC how? Institut für Pathologie By sequencing analyses (DNA, genetics) By copy number variation analyses (DNA, genetics) By methylation analyses (DNA, epigenetics) By expression analyses mrna mirna Protein (IHC, mass spectometry)

Molecular subtyping of CRC Institut für Pathologie CIMP-L CIMP-H SCNA high SCNA low Serrated pathway Traditional pathway Alternate pathway MSI-H MSS/MSI -L hypermutated RASmutated BRAFmutated Rightsided RAS- /RAF-WT MSI immune mesenchymal invasive Leftsided canonical metabolic

TCGA subclassification of CRC (DNA) Hypermutated vs. nonhypermutated phenotype Hypermutated phenotype is associated with MSI-H, CIMP-H, MLH1- BRAF 46% mut hypermethylation or Somatic mutations in MMR-genes or POLE Cancer Genome Atlas Network, Nature 2013

TCGA subclassification of CRC (RNA) Institut für Pathologie RNA-signatures associated with MSI/CIMP (more prevalent in hypermutated tumors) SCNA/CIN (exclusive to non-hypermutated tumors) Invasive (more prevalent in non-hypermutated tumors), assoc. with higher stage & grade Cancer Genome Atlas Network, Nature 2013

International Colorectal Cancer Subtyping Consortium, ICCSC Bionetwork Sage initiative (Guinney, S. Friend) Institut für Pathologie

TCGA MSI/CIMP CIN Invasive Group F Netherlands 1.1. 1.2 1.3 2.1. 2.2. Group D Swiss Inflammator y Goblet Transit Amplifying Stem-like Enterocyte Group A Petacc3 Surface crypt Lower crypt CIMP+ Mesenchymal Mixed Group E AMCAJCCII CCS1 CCS2 CCS3 Group B French Group C Agendia CIN immune down dmmr KRAS CSC CIN Wnt up CIN m normal A-type B-type C-type Rodrigo Dienstmann (version 3, Jan 3rd

Consensus Molecular Subtypes (RNA) Institut für Pathologie SCNA = somatic copy number aberrations Guinney et al., Nature Med 2015

Clinical consequences of subtyping Right sided tumors, mucinous histology, elderly females In stage II: reduced risk of relapse after surgery, no improvement with 5-FU based chemotherapy In stage IV uncommon, but associated with poor prognosis, no prediction of chemotherapy nonresponse Therapeutic options: BRAF-inhibitors in combination with MEK- and EGFR-inhibitors Immune checkpoint inhibition relationship with response to radiotherapy not yet established Biswas et al., Clinical Oncology 2016

MSI and 5-FU I Several studies showed the absence of benefit for adjuvant chemotherapy in MSI-H patients Ribic et al. N Engl J Med 2003 570 cancers, 95 (16,7 %) with MSI-H. Interaction chemotherapy*msi status p=0.009 Jover et al. Gut 2006 505 patients stage II-III 125 stages II (42.2%) 135 stages III (64.5%) with adjuvant chemotherapy Interaction chemotherapy*msi status p=0.007

Dan Sargent et al. Institut für Pathologie 1027 patients included in trials demonstrating the effect of FU in adjuvant settings MSI + (dmmr) 185 pts (18%) No chemotherapy 5FU chemotherapy Dan Sargent et al, JCO 2010

Stage II Stage III MSI MSS Dan Sargent et al, JCO 2010

Chemotherapy 5FU No chemotherapy Lynch syndrome Sporadic MSI 2141 patients, 344 MSI + (16%) ; positive effect limited to the group of lynch syndrome

Immune checkpoint inhibitors Sunshine et al, Current Opinion in Pharmacology 2015 20

Tumormicroenvironment in colorectal cancer Llosa Cancer Discovery 2015 21

Tumormicroenvironment in colorectal cancer Llosa Cancer Discovery 2015 22

Mismatch repair deficiency predicts response to pembrolizumab (Le et al, NEJM 2015) I Phase II-study with 41 patients: 11 mismatch repair deficient CRC 10 mismatch repair deficient non-crc 21 mismatch repair proficient CRC I Primary endpoints: Immune-related response Immune related progression free survival I Significant difference in immune-related response and immunerelated progression-free survival between mismatch repair deficient and mismatch repair proficient CRC I Whole exome sequencing revealed 1782 somatic mutations in mismatch repair deficient tumors and 73 in mismatch repair proficient tumors 23

Clinical benefit of Pembrolizumab treatment acc. to MMR-status Objective response to PD-1 blockade: all CRCs = 14% MSI-H CRCs = 40%

Immune-checkpoint-inhibitors for mismatch-repairdeficient colorectal cancer But: only 4% of mcrc are mismatch repair deficient! Kelderman, Cancer Cell 2015 25

Clinical consequences of subtyping Predominantly left sided tumors Traditional pathway with APC-mutation and high SCNA Worse prognosis than MSI-H tumors, but better than mesenchymal tumors Therapeutic consequences No specific treatment options SCNA = somatic copy number aberrations Biswas et al., Clinical Oncology 2016

Clinical consequences of subtyping 75% RAS-mutations, often in combination with PIK3CA-mutations No association with gender, location, age Intermediate prognosis Alternate pathway? Therapeutic consequences: No response to EGFR-antibodies PIK3CA-inhibition? SCNA = somatic copy number aberrations Biswas et al., Clinical Oncology 2016

RAS-/RAF-pathway Institut für Pathologie

KRAS-mutation as a negative predictor for anti-egfr-treatment BRAF + PIK3CA mutation NRAS mutation 2% 3% BRAF mutation 5% KRAS + PIK3CA mutation 8% PIK3CA mutation / PTEN loss 12% KRAS, BRAF, NRAS, PIK3CA wild type, no PTEN loss Response Non-Response 85% Molecular aberration not yet identified 23% KRAS mutation 32%

KRAS and NRAS Mutations in Colorectal Cancer Institut für Pathologie KRAS 1 2 3 4 5 6 exon 12/13 59/61 117/146 mutation position NRAS 40% 4% 6% frequency (Amgen) 43-49% TCGA 1 2 3 4 5 6 7 exon 12/13 59/61 117/146 mutation position 3.5% 4% 0% frequency (Amgen) 5-9% TCGA non-coding exon coding exon

Douillard JY & Oliner KS et al, NEJM 2013

From Tissue to biomarker Institut für Pathologie

Extended RAS-Analyses Institut für Pathologie KRAS-Mutational analyses codon 12/13 n=5000 Extended RAS: cases versus analyses Mutationen: 1.423 Analyses = 40% WT Mut KRAS Mutation 117/146: 4% KRAS Mutation 59/61: 3% NRAS Mutation 12/13: 2% Wild type: 2.166 Analyses = 60% Extended RAS -Analyses n=1000 NRAS Mutation 59/61: 1% NRAS Mutation 117/146: 0% Wildtyp: 50% 1600 1400 1200 1000 800 600 400 200 0 125 RAS cases 1.179 RAS Analyses KRAS Mutation 12/13: 40% KRAS Fälle 01.07.2013-01.09.2013 KRAS Analysen 01.07.2013-01.09.2013

Clinical consequences of subtyping EMT-signature Worst prognosis, more advanced stage tumors, younger age Resistant to anti-egfr-therapy (independent of RAS-mutational status) Traditional serrated adenomas are thought to be precursor lesions Therapeutic consequences:??? SCNA = somatic copy number aberrations Biswas et al., Clinical Oncology 2016

Proteogenomic subtyping of colorectal cancer Institut für Pathologie 5 proteomic subtypes of CRC 2 subtypes overlap with MSI/CIMP transcriptomic subtype SCNA show strong correlation with RNA abundance, but not with protein abundance Zhang et al., Nature 2014

Can these subclassifications be utilized in daily clinical practice? DNA-sequencing: yes SCNA: maybe RNA-expression analyses: not really Proteomic expression analyses: really not What do we do?

Immunohistochemical subtyping of colorectal cancer Institut für Pathologie Classification into 4 subtypes is done with MSI-analyses + 5 immunohistochemical markers (CDX2 for epithelial; FRMD6, HTR2B, ZEB1 for mesenchymal phenotype) Concordance with RNAsubclassification (Guinney et al.) is 85% No distinction between CMS2/3 possible No response to anti-egfr-therapy in CMS4, independent of RASstatus (CAIRO2-cohort) TMA-study Trinh et al., Clin Cancer Research 2016

Immunohistochemical subtyping of colorectal cancer CDX2: marker for differentiation, expected to be highly expressed in epithelial-like tumors HTR2B: FRMD6: ZEB1: high expression in mesenchymal-like tumors marker for goblet cells, expressed in mesenchymal-like tumors marker for EMT Markers derived from mrna-analyses perhaps better markers by proteomics 38

CDX2 as a Prognostic Biomarker in Stage II and III Colon Cancer Institut für Pathologie Dalerba P, et al., N Engl J Med. 2016 Jan 21; 374(3): 211 222..

CDX2 as a Prognostic Biomarker in Stage II and III Colon Cancer Institut für Pathologie Dalerba P, et al., N Engl J Med. 2016 Jan 21; 374(3): 211 222..

Water in the wine: tumor heterogeneity Morris and Kopetz, Clin Cancer Research 2016 Institut für Pathologie Mamlouk, Aust, Sers, unpublished data

Water in the wine: Discordance between primary tumor and metastases Concordance of driver mutations between PT and MET very good Analysis of PT adequate Possible discordance of SCNA between PT and MET Analysis of MET may be necessary Mamlouk, Aust, Sers, unpublished data

Molecular classification of CRC: what you need to know CRC can no longer be treated as a homogeneous disease, gene expression classification suggests four major subgroups Patients with MSI tumors (CMS1) Improved prognosis in stage II No benefit of adjuvant 5-FU therapy Benefit from immune checkpoint inhibitors in stage IV Expanded RAS-testing is required to screen for non-sensitivity to anti- EGFR-therapy BRAF-mutation predicts poor prognosis in stage IV and patients shoud be considered for combination targeted therapies Prospective molecularly driven trials are needed to prove the impact of subclassification

HE I: label tumor area HE II: check for tumor Institut für Pathologie FFPE tumor block gdna MSI block adequate on entry? (samples) microstallelite instability (MSI) adequate for analysis (FFPEQ) tumor cell content etc. (DNA) amount DNA

Thank you for your attention. 45