New breast cancer classification: traditional pathology and

molecular subtypes

Dr. PN Mainwaring

Centre for Personalised

NanoMedicine

AIBN@UQ

Disclosures

Lectures, Honoraria, Advisory Boards

- Astellas–, BMS, Gelgene, Ipsen, Janssen, Medivation, Merck, Novartis, Pfizer, Roche/Genentech

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

ABC3 ESMO Guidelines

Baselga and Norton, 2002 updated

Evolution in Breast Cancer Classification

Classical Diagnosis

Ductal infiltrating carcinoma of breast with

high grade of nuclear atypia

Protein Expression

ErbB2 over expressing breast tumour

Gene Expression Profiling

Partial two dimensional cluster analysis of17 breast tumours

MorphologicalDiagnosis

Immunohistochemicalassessment

DNA microarrayanalysis

CIRCOS Plot

Integrated Analysis

Chromosome, SNV,LSV, indel, amplification

miRNA, RNA

AJCC 8th Edition: 1st 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

Histology - T

H&E

Neoadjuvant Biopsy Surgical Specimen

Nucleoli reflect chromatincondensation

Differentiation

Well-differentiated Poorly-differentiated

Lymphovascular invasion

Anti-vascular antibodyCD34

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

Immune Context

Angiogenesis; CD34 Tumour-infiltrating lymphocytes; PD-1

Salgado Adv Anat Pathol. 2017

Intra-tumourStromal

ImmuneInfiltrateSub-typing

Special subtypes

Tubular Metaplastic

Biology – Standard Immunohistochemistry & RPPA

ER; cutoff <1% vs <10%ER-negative ER-negative; positive GATA3ER-positive

Lobular

E-cadherin

PR

• Cytoplasmic

Carroll NRC 2016

HER2

HER2

0 1 2 3

FISH

Normal Amplified

Basal

Cytokeratin 5/6 (CK 5/6) EGFR

R

Histology - N

Nodal Metastasis

Micrometastasis

Extracapsular spread

Isolated Tumour Cells

One-step CK19 mRNA Amplification

Osako BJC 2017

Molecular Classification

TCGAIGCG

Weinstein Nat Gen 2013

Now 5 years onFrom the initialpublications

Technologies

IlluminaSeq by Synth

LifeSeq by Synth

NanostringDigital PCR

BGIRolling Circle

PacBioSMART

DNA ClassificationTri NA Breast

Age 60%APOBEC 14%BRCA1/2 10%

?

APOBEC 2%

Point mutation SNV/SNA vs SNP; indel (30bp); amplif’n; LSV

Nik-Zainal Nature 2016

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

Post-translationalHistones & 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, CREBBPand SMARCA4/BRG1 it is estimated that approximately 20% of all solid malignancies harbor mutations in at least one SWI/SNF componentincl ~20% breast cancer

Morel Annals 2017

CpG island

Slinky

ER-pos enhancer regions methylation

Stone Nat Comm 2015

TNBC; Differential methylation

Stirzaker Nat Comm 2015

Epigenetic Classification

• miRNA

• lncRNA

• Others, sno/circ

• miRNA signatures

• lncRNA signatures• Prognostic

• Predictive

• Need large scale

RNA Editing

• Transcription

• Enhancers/Super-enhancers

• Splice Variants

• Fusion Genes

Perou Nature 200

Sørlie et al PNAS 2003

ER+

ER+

Original Microarray analyses

A vs BHeterogeneity

in Outcome

Increasing Complexity

Ali Genome Biol 2014

METABRIC

RNA Classification

Prat Mol Oncol 2015

Stratification of TNBC

TNBC

Luminal/AR Basal

Luminal A+B HER2-enriched Claudin-low/mesenchymal Basal-like

AR expressionLow – Immune – High

Gene expression or TILs

Chemo sensitivity

Lapatinib sensitivity

Chemo sensitivity

Proliferation

20–30% 70–80%

Perou SABCS 2016.

PI3K/Akt/mTOR inhibitors

Targeted RTK inhibitors

DNA-repair targeting agents

Cell cycle/mitotic spindle inhibitors

RAF/MEK inhibitors

Clinically targetable pathways in TNBC

~90% of all patients had an aberration in at least one of

these pathways

IMMUNOTHERAPEUTICSN

um

be

r o

f sa

mp

les

wit

h a

be

rrat

ion

s

PI3K/Akt/mTOR DNA repair Ras/MAPK Cell cycle GFRs0

10

20

30

40TSC1

PIK3CA

PTEN

PIK3R1

RICTORRAPTORAKT1AKT2

AKT3

BRCA1

BRCA2ATM

RB1

AURKA

CDNK2A

CCNE1

CCND3

CCND2

CCND1

CDK6CDK4

NF1CRAFBRAF

KRAS EGFR

MET

IGF1RKITFGFR1

FGFR2

FGFR4

Balko Cancer Discov 2014.

Protein

• Ultimate effector• capture the functional

state and dynamic properties of a cell

• Kinome• Phospho, other PTMs

• Membrane

• Cytoplasm

• Golgi etc

• Nuclear

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

KlimovBJC 2017

Metabolome

• Metabolic reprogramming inER-positive breast cancer

• Super-SILAC mix to quantifyover 10,000 proteins with high accuracy

Pozniak Cell 2016

Integration of information

• Sum is greater than the parts

HR-pos HER2-pos TNBC

Nik-Zainal ESMO 2017

Triple Negative

Basal

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

~75% of TNBC have basal gene expression

• ~5% of breast cancers• ~75-80% of BRCA1 mutation-associated BC are TN• 50% BRCA1 carriers are basal-like

• Basal but not TN (15-40%)

• Definition by gene expression

• Express basal cytokeratins

• Includes most BRCA1 mutated tumours

• 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

BRCA1/2mutated

TNBC; integrating clinical & molecular

Immune-Context

Immune gene signature

Nagalla Genome Biol 2013

Mol.Subtype

PD-L1/PD-1 IHC expression; meta-analysis

• Grade, Tumour, Lymph Nodes, ER, PgR, HER2

Kim BMC 2017

Crosstalk between DDR and Immune System: TMB & Neoepitopes

Zehir A et al. Nat Med 2017.

Future is now; ? ESMO ?

• Multi-omic analysis

• Integration into biological pathways

• Application of systems biology to decide1. Major pathway drivers

2. Major nodal points

3. Druggable nodal points

4. Integrated systemic therapy

• Monitor ‘response’• cfDNA, CTC, exosomes, proteins

“N-of-One” vs Many;