La genetica del carcinoma colo-rettale

La genetica del carcinoma colo-rettale Ivana Cataldo Ospedale Ca Foncello, Treviso ARC-NET Centro di Ricerca Applicata sul Cancro AOUI, Verona

OUTLINE What s new in colorectal cancer? Pathological Markers Molecular Markers Future perspectives: between hope and reality

CRC: Pathological Markers

CRC: Histology Report 1994 ANATOMIA PATOLOGICA 94-11032 ROSSI MARIO HISTOLOGY Histotype grading metastases Surgical margins 2016 REGIONE VENETO UNITÀ OPERATIVA DI ANATOMIA PATOLOGICA 16-32033 ROSSI MARIO HISTOLOGY Histotype Grading Metastases Surgical margins/mesorectum Budding Vascular/perineural invasion Tumor Regression Grade ptnm IHC/ISH MMR genes/cdx2/her2 Isolated tumor cells (ITC) MOLECULAR TESTS KRAS, BRAF, NRAS Microsatellite instability

CRC: Molecular Markers microsatellite instability (MSI) CpG island methylator phenotype (CIMP) somatic mutations in KRAS somatic mutations in BRAF

CRC: Molecular Markers Integrated histological and molecular classification microsatellite instability (MSI) somatic mutations in KRAS CpG island methylator phenotype (CIMP) somatic mutations in BRAF Adenoma-carcinoma pathway (conventional) MSS BRAF & KRAS wt Serrated pathway CIMP-L/CIMP-H BRAF mut Alternative pathway MSS CIMP-L & KRAS mut

Conventional pathways APC Normal mucosa APC Hypometilation, SMAD4, TP53 Tubular adenoma Tubulovillous adenoma KRAS TP53 CIMP- MSS Sensitive 5FU Sensitive aegfr KRAS, CIMP-L MSS Sensitive 5FU Resistant aegfr Bettington M, et al. Histopathology 2013

Serrated pathways BRAF, CIMP-H Normal mucosa KRAS MLH1, MSI, IGFR2, TGFRβ Sessile serrated adenoma CDKN2A, MGMT Traditional serrated adenoma Tubulovillous adenoma with serrated features Wnt pathway BRAF, CIMP-H MSI Resistant 5FU Resistant aegfr BRAF, CIMP-H MSS Sensitive 5FU Resistant aegfr KRAS, CIMP-L MSS Sensitive 5FU Resistant aegfr Bettington M, et al. Histopathology 2013

MSI related CRCs 15% CRC have defective DNA mismatch repair (dmmr) 70% sporadic MLH1 methylation 30% hereditary Mutations in MMR genes Diagnosis testing for loss of the protein products for genes involved in DNA mismatch repair, most commonly MLH1, MSH2, MSH6, and PMS2 presence of microsatellite instability (MSI) proximal mucinous diploid

Defective Mismatch Repair: a predictive marker for lack of efficacy of Fluorouracil-based adjuvant therapy untreated Stage III dmmr Stage III pmmr High-level microsatellite instability (MSI-H) or defective DNA mismatch repair (dmmr) have improved survival and receive no benefit from fluorouracil (FU)-based adjuvant therapy compared with patients who have microsatellite-stable or proficient mismatch repair (pmmr) tumors. Sargent DJ, et al. JCO 2010

CIMP is associated with response to adjuvant Irinotecan-based therapy for stage III CRC - CIMP (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) is frequently associated to dmmr due to inactivation of MLH1. - CIMP is observed in 15-20% CRC. proximal women BRAF G3 HR=1,36 CIMP neg 615 CRCs 23% CIMP+* 77% CIMP-neg CIMP + CIMP-positive tumors had shorter overall survival (FU/LV) (hazard ratio [HR]=1.36) * 4 met in at least 3 of 5 CpG island Shiovitz S, et al. Gastroenterology 2014

CIMP is associated with response to adjuvant Irinotecan-based therapy for stage III CRC - CIMP (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) is frequently associated to dmmr due to inactivation of MLH1. - CIMP is observed in 15-20% CRC. proximal women BRAF G3 HR=1,36 CIMP +/IFL CIMP +/FL CIMP+ tumors benefit most from the addition of irinotecan to fluorouracil/leucovorin therapy (for the interaction, P = 0.01). IFL: Irinotecan+Fluoruracil+Leucovorin FL: Fluoruracil+Leucovorin Shiovitz S, et al. Gastroenterology 2014

CIMP is associated with response to adjuvant Irinotecan-based therapy for stage III CRC - CIMP (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) is frequently associated to dmmr due to inactivation of MLH1. - CIMP is observed in 15-20% CRC. proximal women BRAF G3 60% 35% CIMP+/MMR-i Irin+FU-LV CIMP+/MMR-i FU-LV Stage III, CIMP+, MMR-i colon tumors have longer overall survival when irinotecan is added to combination therapy with fluorouracil and leucovorin. Shiovitz S, et al. Gastroenterology 2014

Classification of CRC based on correlation of clinical, morphological and molecular features Type 1 2 3 4 5 microsatellite H - or L - or L - or L H CIMP + + L - - KRAS - - + - - BRAF + + - - - Jass JR, et al. Histopathology 2007

Association between molecular subtypes of colorectal cancer and patient survival *Stage adjusted Disease Specific Survival* other n= 2,050 1 5 4 3 2 years type 4 is the most predominant (47%) (APC-conventional pat.) type 2 had the highest disease-specific mortality (HR=2.20, 95% CI: 1.47-3.31) (MSS-CIMP-BRAF sessile-serrated pat.) type 1 and type 5 have the lowest disease-specific mortality (HR=0.30, 95% CI: 0.14-0.66) (MSI-related pat.) the clinical importance of studies into the molecular heterogeneity of CRC!! Phipps AI, et al. Gastroenterology 2014

The CRC Subtyping Consortium Analysis of 6 independent gene-expression based classification systems Identification of 4 robust CMSs [ ] none of the subtypes is defined by an individual event, nor is any genetic aberration limited to a subtype. Guinney J,et al, Nature Medicine (12 October 2015)

Integrated histological and molecular classification Consensus Molecular Subtypes * * * Female, right colon, high grade, Superior survival after relapse Left side More advanced stage *SCNA: somatic copy number alterations Guinney J,et al, Nature Medicine (12 October 2015)

OUTLINE What s new in colorectal cancer? - Clinical and pathological factors - Molecular markers Future perspectives: between hope and reality

The future in molecular oncology: clinical genomics? Highly selective testing Stepwise, single-gene testing algorithms tailored to specific cancers KRAS mutation Multiplex testing Simultaneous multigene and multiplexed approach DNA RNA Unbiased testing Global and unbiased whole-genome approach DNA RNA NRAS mutation Multiplexed mutation testing Multiplexed RNA profiling and fusion transcript detection Whole-exome (or genome) sequencing Whole-transcriptome sequencing (including paired ends) BRAF mutation Genomic copy number profiling Present Future Taylor BS & Ladanyi M J Pathol 2011

FFPE tissue 10/40 ng DNA 1-500 genes >2,000 hotspots mutations >200 PCR in one tube

The liquid biopsy era: a short life for pathologists? Crowley E, et al. - Nat Rev Clin Oncol 2013

The liquid biopsy as a mini-invasive CRC diagnostic tool 100% 50% 0% Localized Metastatic ctdna was detected in 73% of patients with early CRC ctdna was often present without detectable circulating tumor cells Bettegowda C, et al Sci Transl Med 2014

The liquid biopsy as a miniinvasive CRC diagnostic tool n=206 KRAS exon 2 128 WT 78 mut 127 WT 1 mut 68 mut 10 WT Specificity 99.2% Sensitivity 87.2% The concordance of KRAS mut status among tissue samples and plasma was 95% (k=0,88 P>0,0001) Bettegow DA, et al Sci Transl Med 2014

The liquid biopsy : monitoring patients for resistance-conferring mutations n= 24 KRAS, NRAS, BRAF, PIK3CA & EGFR wt 23/24 (96%) presented emergent circulating mutations of at least one mitogen-activated protein kinase pathway gene during treatment More than one mutation per gene!!! METASTASES HETEROGENEITY Bettegowda C, et al Sci Transl Med 2014

micrornas: the new actors in the cancer scenario Biomarkers Diagnosis Prognosis Therapeutics Fassan M & Baffa R Curr Opin Genet Dev 2013

micrornas: the new actors in the cancer scenario one-third of protein-coding human mrnas are susceptible to this complex mirna regulatory network Normal Cancer mir-21 PDCD4 mirna t.s.gene mirna as oncogene let-7 RAS; C-MYC mirna oncogene mirna as tumor suppressor gene Fassan M, Croce CM & Rugge M World J Gastroenterol 2011

Circulating mir-182 is a biomarker of colorectal adenocarcinoma progression Plasma!! N vs K I-II vs III-IV pre vs post Perilli L, et al Oncotarget 2014

Richiesta esame istologico REGIONE VENETO CLINICA CHIRURGICA 78 Direttore Prof Rossi Maria Data nascita 01/08/1956 Codice fiscale RSSMRO89H872B ROSSI MARIO Codice a barre Comune di residenza Peschiera del Garda MATERIALE INVIATO Liver biopsy V segment NOTIZIE CLINICHE RICHIESTA Mutational profiling Dott. Giacomo

Richiesta esame istologico REGIONE VENETO CLINICA CHIRURGICA 78 Normal parenchyma Data nascita 01/08/1956 Codice fiscale RSSMRO89H872B Direttore Prof Rossi Maria ROSSI MARIO Codice a barre MATERIALE INVIATO Liver biopsy V segment Comune di residenza Peschiera del Garda Necrosis Fibrosis NOTIZIE CLINICHE CRC Liver metastasis % tumor cells RICHIESTA Histology, EGFR IHC, EGFR FISH, KRAS, BRAF, NRAS, PI3K, TP53, DPC4, ZEB2, CIP&CIOP Mutational Profiling is not the diagnosis! Dott. Giacomo

Richiesta esame istologico REGIONE VENETO Data nascita 01/08/1956 Codice fiscale RSSMRO89H872B CLINICA CHIRURGICA 78 Direttore Prof Rossi Maria ROSSI MARIO Codice a barre MATERIALE INVIATO Liver biopsy V segment Comune di residenza Peschiera del Garda the sharing of the procedures among the multidisciplinary team NOTIZIE CLINICHE CRC Liver metastasis RICHIESTA Histology, EGFR IHC, EGFR FISH, KRAS, BRAF, NRAS, PI3K, TP53, DPC4, ZEB2, CIP&CIOP Dott. Giacomo

Conclusions The histology report represent a crucial point for CRC management Not so far from a definitive CRC histo-molecular classification Next-generation diagnostics in old-generation laboratories Sharing of ideas among the multidisciplinary team Liquid biopsy: they still need us!

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