Molecular Pathological Classification Of Colorectal Cancer (CRC) Dr Molecular Pathological Classification Of Colorectal Cancer (CRC) Dr. Calypso Barbatis MD, FRCPath, PhD HBD Histobiodiagnosis ATHENS HSGO / 19-20 May 2018 ATHENS

Frequent malignancy 3rd cause of cancer-related mortality worldwide Sporadic, Hereditary, Familial Related to IBD Precursor Lesions Almost perfect stepwise model of carcinogenesis Conventional adenoma Serrated lesions CRC

Specific features of CRC Heterogeneous Disease in morphology site gender age response to treatment Source: Pol J Path. 2014; 65(4): 257-266. Bosman FT, Yan P.

Does CRC fit to the P4 medicine Predictive Personalised Preventive Participatory Source: Hood L, Friend SH. Nat Rev Clin Oncol 2011;8: 184-187

Clinicopathological parameters Still determine treatment  Classical evidence-based Clinicopathological parameters Still determine treatment But The road to molecular classification has opened and awaits full implementation when clinical translation and precision medicine are approved  

The classification of CRC is based on clinical morphological molecular features and available markers prognostic preventive

Current non-molecular classification of CRC Site Subsite (the most involved) Rectal: Inferior margin <16 cm from the anal verge or if any part of the tumor is located within the supply of the superior rectal artery

WHO Histological types Conventional adenocarcinoma Mucinous (colloid) adenocarcinoma ( > 50% mucin) Signet-ring cell carcinoma ( > 50% signet-ring cells) Medullary carcinoma Micropapillary Serrated Squamous cell carcinoma Adenosquamous Spindle cell Poorly differentiated neuroendocrine carcinoma Large cell neuroendocrine carcinoma Small cell neuroendocrine carcinoma Mixed adenoneuroendocrin Undifferentiated

Histological Grade TNM Stage (T0, T1,T2, T3, T4) Tumor budding Margins Grade 1,2,3,4 (Undifferentiated) TNM Stage (T0, T1,T2, T3, T4) Nx, N0, N1, N2 M: M0, M1a, b, c (metastasis to the peritoneum with or without other organ involvement Tumor budding Perineural/Lympho-vascular invasion Perforation Assesment of mesorectal envelope Margins Treatment effect (Tumor regression score) 0 Complete response 1 Near complete response 2 Partial response 3 Poor non response Source: Amin MB et al eds. AJCC Staging manual 8th edition 2017

Basic pathways in colorectal cancer East et al, British Society of Gastroenterology position statement on serrated polyps in the colon and rectum.GUT 2017

Pathogenesis of colorectal cancer GENETIC AND EPIGENETIC CHANGES A.GENETIC EVENTS 1.CHROMOSOMAL INSTABILITY (65-70% of sporadic CRC) a)Aneuploidy Chromosomal gains Chromosomal losses b)LOH=loss of the entire gene and the surrounding chromosomal region. CAUSES:Chromosomal segregation defects,DNA damage repair,telomere dysfunction, point mutations of oncogene or tumor suppressor genes. Mutational status:APC,K- RAS,SMAD2-SMAD4,p53 Mc Granahan et al,2012 EMBO Reports Fredericks et al, J Cancer Biol Res 3(1): 1057

GENETIC AND EPIGENETIC CHANGES B.EPIGENETIC EVENTS Epigenetic changes are those inheritable changes in gene expression with no alterations in DNA sequences. DNA methylation Histone modification Micro-RNAs (non coding RNAs) Chen et al,Oncology reports,2013 

Molecular pathways of CRC carcinogenesis 1. Classical carcinogenetic model Chromosomal instability (CIN) Marked aneuploidy 1st pathogenetic event APC gene mutations Allelic loss Somatic gene amplification Translocation Activation WNT signaling pathway

Apc/B-CATENIN PATHWAY CIN MECHANISMS=APC MUTATIONS Wnt/pathway activation B-catenin in cytoplasm and in nucleus Trascription factors promote proliferation Accumulation of mutations/EGFR activation Kras mutations Loss of SMAD2 SMAD4 Loss of p53 EMT mechanisms activation Slaby et al,Molecular Cancer2009

2nd Pathway (MMR gene system or MSI) Inactivation of MMR genes Inactivation of suppressor genes Lynch syndrome (Diploid tumors with MSI)

The Serrated pathway Hypermethylation of specific DNA regions near the “promoter genes” the CpG islands Diploid tumors Silence of Tumor Suppressor Genes

Msi pathway (1) MICROSATELLITE INSTABILITY (15% of colorectal cancer) Inactivating mutations in the DNA mismatch repair genes (MMR) or methylation of the gene promoter that codify for ATPases such as hMLH1,hMSH2 hPMS2, MSH6,MSH3. 10-15% of sporadic colorectal carcinoma CIMP-H frequent BRAF mutated. Lynch syndrome 3-5% of all colorectal carcinomas.No BRAF mutations. Imai et al,Carcinogenesis vol.29 no.4 pp.673–680, 2008

The cimp pathway CIMP stands for CpG island methylator phenotype. 1. Transcriptional inactivation of genes that have tumor suppressive roles or they are involved in cell cycle. 2.BRAF mutation is an early genetic event in this pathway 3.MLH1 hypermethylation is commonly seen. 4.Other promoters also hypermethylated are:CDKN2A,MGMT tumor suppressor genes,MUYTH polyposis syndrome-BER genes,TIMP3 ecc. 5.CIMP-H,CIMP-L,CIMP-normal classification. Tariq et al, Cancer Biol Med 2016 Schaafsma et al, Biol Med 2016

Classification by molecular subtypes The Cancer Genome Atlas (TCGA) Consensus Molecular Subtypes (CMS) Sources: Nature. 2012; 487: 330-7 (TCGA) Comprehensive molecular characterization of human colon and rectal cancer Nat Med. 2015; 21: 1350-6 The consensus molecular subtypes of colorectal cancer. Guinney J et al.

Purpose of classification “To correlate cancer cell phenotype with clinical behavior and provide guidance to rational treatment with specific targeted therapies” (CRC subtyping Concortium) This was attempted by the knowledge of gene expression data based on “epigenomic, transcriptomic, microenviromental, genetic and clinical characteristics of tumors’’

Consensus Molecular Subtypes of colorectal cancer (CMS) Assessment methods Mutations Chromosomal number alterations Methylation status Posttranslational Gene regulation Clinical analysis (site, gender, grade, stage) DFS (Disease Free Survival) RFS (Relapse Free Survival) SAR (Survival After Relapse) IMPORTANT!! “No subtype was solely defined by a genetic aberration” wild-type KRAS may be found in all subtypes

Clinical implications of CMS CMS1 (MSI) Better prognosis before dissemination Worst prognosis after relapse Stages 1,2 perhaps no adjuvant chemotherapy Stage 3 no response to 5-FU Importance of the immunogenic microenvironment Treatment? Check point inhibitors

CONSENSUS MOLECULAR GROUP 1 https://familyhistorybowelcancer.wordpress.com/hereditary-colorectal-cancer-syndromes

CMS2 Canonical (adenoma – CRC sequence) APC loss KRAS activation P53 loss CIN increased copies of oncogenes reduced copies of suppressor genes low mutation rate ( < 8 mutations / 106 bases) adjuvant chemotherapy

Consensus MOLECULAR GROUP 2 http://www.imagingpathways.health.wa.gov.au/index.php/imaging-pathways/gastrointestinal/gastrointestinal/staging-of-colorectal-cancer

Tubulovillous adenoma with serration CMS3 metabolic subtype 30% hypermutated KRAS mutation Tubulovillous adenoma with serration Precursor lesion (?) Enriched in RNA (9-10 metabolic pathways) Glutamine, fatty acid, phospholipid metabolism Poor prognosis of KRAS mutated cases Her-2 mutations Resistance to EGFR mAb

CONSENSUS MOLECULAR GROUP 3 http://www.thepinsta.com/adenocarcinoma_8zBRakAGoyf3SGdDDmkNXug7gO9Fo6YlJVf7hsFfvWU/

Resistant to anti-EGFR Regardless of KRAS mutation status CMS4 (mesenchymal) Advanced stage in diagnosis Poor survival (62% in 5 years) Resistant to anti-EGFR Regardless of KRAS mutation status Difficult to treat ? use of microenvironment features ? anti avβ6 integrin Anti-CAF Anti-macrophage Source: Thanki K et al. IBBJ 2017; 3(3) 105-111

CONSENSUS MOLECULAR GROUP 4 DAWSON et al Reviews in medicine 2015

Molecular features of CRC Molecular Heterogeneity Genomic, epigenomic define Molecular subtypes Implementation of Personalized therapies Improvement of management

Acknowledgement My gratitude to Dr.Bouklas for providing some of the tables and additional literature

Thank You For Your Attention