New breast cancer classification: traditional pathology and molecular subtypes. Dr. PN Mainwaring Centre for Personalised NanoMedicine

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1 New breast cancer classification: traditional pathology and molecular subtypes Dr. PN Mainwaring Centre for Personalised NanoMedicine

2 Outline Traditional Pathology Haematoxylin & Eosin Descriptors; Scarff Bloom Richardson Proliferation & Apoptosis Protein IHC classification Immune context Molecular Subtypes DNA classification Epigenetic classification RNA classification Integration Aim Classification Prognosis Prediction Monitoring?Screening/ Early detection

3 ABC3 ESMO Guidelines

4 Evolution in Breast Cancer Classification Morphological Diagnosis Immunohistochemical assessment DNA microarray analysis CIRCOS Plot Classical Diagnosis Protein Expression Gene Expression Profiling Integrated Analysis Ductal infiltrating carcinoma of breast with high grade of nuclear atypia ErbB2 over expressing breast tumour Partial two dimensional cluster analysis of 17 breast tumours Chromosome, SNV, LSV, indel, amplification mirna, RNA Baselga and Norton, 2002 updated

5 AJCC 8th Edition: 1 st January 2018 Combining T/N/M with biology grade,proliferation, ER/PgR/HER2/GEP NB GEP only apply to LN negative disease; T1a-bN0M0 OncotypeDx, Mammaprint, EndoPredict, PAM50, Breast Cancer Index Giuliano Breast Feb 2018

6 Histology - T

7 H&E Neoadjuvant Biopsy Surgical Specimen

8 Nucleoli reflect chromatin condensation

9 Differentiation Well-differentiated Poorly-differentiated

10 Lymphovascular invasion Anti-vascular antibody CD34

11 Proliferation & Apoptosis MIB-1 Antibody to Ki67 Protein expressed variably through cell cycle not G0 Apoptosis One form of cell death International Ki67 in Breast Cancer Working Group

12 Immune Context Angiogenesis; CD34 Tumour-infiltrating lymphocytes; PD-1 Intra-tumour Stromal Immune Infiltrate Sub-typing Salgado Adv Anat Pathol. 2017

13 Special subtypes Tubular Metaplastic

14 Biology Standard Immunohistochemistry & RPPA

15 ER; cutoff <1% vs <10% ER-positive ER-negative ER-negative; positive GATA3 E-cadherin Lobular

16 PR Cytoplasmic Carroll NRC 2016

17 HER2 HER2 FISH Normal Amplified

18 Basal Cytokeratin 5/6 (CK 5/6) EGFR R

19 Histology - N

20 Nodal Metastasis Isolated Tumour Cells Micrometastasis Extracapsular spread

21 One-step CK19 mrna Amplification Osako BJC 2017

22 Molecular Classification

23 TCGA IGCG Now 5 years on From the initial publications Weinstein Nat Gen 2013

24 Technologies PacBio SMART Illumina Seq by Synth Life Seq by Synth Nanostring Digital PCR BGI Rolling Circle

25 DNA Classification Point mutation SNV/SNA vs SNP; indel (30bp); amplif n; LSV Tri NA Breast Age 60% APOBEC 14% BRCA1/2 10%? APOBEC 2% Nik-Zainal Nature 2016

26 TCGA: Molecular characteristics of TNBC provides fuel for future therapeutics Basal-like Breast Cancer p53 mut 84% RB1 mut/loss 20% PIK3CA mut 7% MYC focal gain 40% PTEN loss 35% Global Hypomethylation INPP4B loss 30% Aneuploidy and genomic instability TCGA Nature 2012

27 CpG island Post-translational Histones & tails within the 25 genes most significantly altered among all tumour types, seven (28%) code for chromatin-modifying enzymes KMT2C/MLL3, KMT2D/MLL2, ARID1A, PBRM1, SETD2, CREBBP and SMARCA4/BRG1 it is estimated that approximately 20% of all solid malignancies harbor mutations in at least one SWI/SNF component incl ~20% breast cancer Morel Annals 2017

28 Slinky

29 ER-pos enhancer regions methylation Stone Nat Comm 2015

30 TNBC; Differential methylation Stirzaker Nat Comm 2015

31 Epigenetic Classification mirna lncrna Others, sno/circ mirna signatures lncrna signatures Prognostic Predictive Need large scale

32 RNA Editing Transcription Enhancers/Super-enhancers Splice Variants Fusion Genes

33 Perou Nature 200 Sørlie et al PNAS 2003 Original Microarray analyses ER+ A vs B Heterogeneity in Outcome ER+

34 Increasing Complexity METABRIC Ali Genome Biol 2014

35 RNA Classification Prat Mol Oncol 2015

36 Perou SABCS Stratification of TNBC TNBC 20 30% 70 80% Luminal/AR Basal Luminal A+B HER2-enriched Claudin-low/mesenchymal Basal-like AR expression Low Immune High Gene expression or TILs Lapatinib sensitivity Chemo sensitivity Proliferation Chemo sensitivity

37 Number of samples with aberrations Balko Cancer Discov Clinically targetable pathways in TNBC IMMUNOTHERAPEUTICS AKT3 PTEN PIK3CA TSC1 AKT2 AKT1 RAPTOR RICTOR PIK3R1 ~90% of all patients had an aberration in at least one of these pathways BRCA2 BRCA1 ATM PI3K/Akt/mTOR DNA repair Ras/MAPK Cell cycle GFRs NF1 KRAS CRAF BRAF RB1 CDNK2A CCNE1 CCND3 CCND2 CCND1 CDK6 AURKA CDK4 MET IGF1R EGFR FGFR4 FGFR1 KIT FGFR2 PI3K/Akt/mTOR inhibitors DNA-repair targeting agents RAF/MEK inhibitors Cell cycle/mitotic spindle inhibitors Targeted RTK inhibitors

38 Protein Ultimate effector capture the functional state and dynamic properties of a cell Kinome Phospho, other PTMs Membrane Cytoplasm Golgi etc Nuclear

39 TNBC IHC; 133 biomarkers Stratify TNBC patients into high risk groups that showed over 5, 6, 7 and 8 times higher risk of developing metastasis to the bone, liver, lung and brain, respectively, than low-risk subgroups Klimov BJC 2017

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41 Pozniak Cell 2016 Metabolome Metabolic reprogramming in ER-positive breast cancer Super-SILAC mix to quantify over 10,000 proteins with high accuracy

42 Integration of information Sum is greater than the parts HR-pos HER2-pos TNBC Nik-Zainal ESMO 2017

43 TNBC; integrating clinical & molecular ~75% of TNBC have basal gene expression Triple negative but not basal (20-30%) Definition by IHC Includes non-idc histologies 10-30% can also include claudin-low, a subtype notable for high expression of stem cell markers 90% of TNBC do not have BRCA mutations Triple Negative BRCA1/2 mutated Basal Basal but not TN (15-40%) Definition by gene expression Express basal cytokeratins Includes most BRCA1 mutated tumours ~5% of breast cancers ~75-80% of BRCA1 mutation-associated BC are TN 50% BRCA1 carriers are basal-like Gluz et al, Ann Oncol 2009; Carey et al, Nat Rev Clin Oncol 2010; Young et al, BMC Cancer 2009; Schneider et al, CCR 2008; Shah et al, Nature 2012

44 Immune-Context

45 Immune gene signature Nagalla Genome Biol 2013

46 PD-L1/PD-1 IHC expression; meta-analysis Grade, Tumour, Lymph Nodes, ER, PgR, HER2 Mol. Subtype Kim BMC 2017

47 Crosstalk between DDR and Immune System: TMB & Neoepitopes Zehir A et al. Nat Med 2017.

48 Future is now;? ESMO? N-of-One vs Many; Multi-omic analysis Integration into biological pathways Application of systems biology to decide 1. Major pathway drivers 2. Major nodal points 3. Druggable nodal points 4. Integrated systemic therapy Monitor response cfdna, CTC, exosomes, proteins