Triple Negative Breast Cancer Residual Tumorand Resistance ... · Triple Negative Breast Cancer Residual Tumorand Resistance to Treatment Dr Fabien REYAL (RT)2 Lab ... Micro‐ Biopsy

Triple Negative Breast Cancer Residual Tumor and Resistance to Treatment

Dr Fabien REYAL

(RT)2 LabDepartment of Translational Research

Department of SurgeryInstitut Curie

Neaodjuvant Treatment in Triple Negative Breast Cancer

Micro‐Biopsy

NeoadjuvantChemotherapy Surgery Radiotherapy

ResidualDisease

Elston Ellis GradeMitotic IndexKi67ER/PR/HER2

Diagnostic

AnthracyclinTaxan

BreastLymph Nodes

PathologicalComplete Response

Relapse?

TNBC

Clin

icalAspe

cts

TNBC

GE Classification

TNBC

WES

TNBC

CellLines

HTS

Predictive and Prognostic Factorsin Triple Negative Breast Cancer

NEOREP Cohort

H Bonsang‐Kitzis, L Belin

Retrospective cohort368 TNBC patients treated between 2002‐2012 Institut Curie, Paris‐Centre René Huguenin, St Cloud

Inclusion criteriaUnique tumorNeoadjuvant chemotherapy

Exclusion criteriaInflammatory Breast CancerM+ Loco‐Regional Recurrence

AnalysisMedian follow up: 52.4 months [8.41‐124.58] Cutoff date for analysis: 2013 July 13th




Tree Partitioning

1

2

3

4

Lymph Node

Obesity and Treatment Dose

Hormonal Environment

TILStromal Density

Prognostic Marker

Second Line TrialsPre‐Operative TrialsResponse‐Guided Trials

Biological network driven gene selection identify a stromalimmune module as a central determinant of triple negative

breast carcinoma prognosis

H Bonsang‐Kitzis, C Laurent

262"Samples"

830"Genes"

B$

Immunity"Prolifera: on"Metabolism"Matrix"

A

Immunity"Mean"correla: on"="0.37"

Prolifera: on"Mean"correla: on"="0.08"

Metabolism"Mean"correla: on"="0.19"

Matrix"Mean"correla: on"="0.22"

262"Samples"

830"Genes"

B$

Immunity"Prolifera: on"Metabolism"Matrix"

A

Immunity"Mean"correla: on"="0.37"

Prolifera: on"Mean"correla: on"="0.08"

Metabolism"Mean"correla: on"="0.19"

Matrix"Mean"correla: on"="0.22"

Validation Set: 295 samplesTraining Set: 262 samples

> 2.7

0.3

< -2.1

> 2.4

0.1

< -2.2

Metabric Data Set Application: 394 samplesIgnatidis Data Set Application: 314 samples

> 2.5

0.1

< -2.2

> 2.9

0.5

< -1.9

167 Gen

es

167 Gen

es

167 Gen

es

135 Gen

es

Pearson Correlation to Training Test CentroidsCentroid1 (0.77)Centroid2 (0.92)Centroid3 (0.84)Centroid4 (0.62)Centroid5 (0.69)Centroid6 (0.35)

Centroid1Centroid2Centroid3Centroid4Centroid5Centroid6



Genes$(number"by"module/total"number)"

Drugs$(number"by"module/total"number)"

Number"of"drugs/number"of"genes"by"

module"

1QMethylQDLQTryptophan"

Drug"examples"

Dasa=nib,"Alefacept,"Muromonab"

Bicalutamide,"Nandrolone"decanoate""

Docetaxel,"Epirubicin"

Captopril,"Urokinase"

Nadroparin,"Tinzaparin""

Genes$(number"by"module/total"number)"

Drugs$(number"by"module/total"number)"

Number"of"drugs/number"of"genes"by"

module"

1QMethylQDLQTryptophan"

Drug"examples"

Dasa=nib,"Alefacept,"Muromonab"

Bicalutamide,"Nandrolone"decanoate""

Docetaxel,"Epirubicin"

Captopril,"Urokinase"

Nadroparin,"Tinzaparin""

String©"confidence$connec1on$

Immune$prognosis$signatures$

String©"confidence$connec1on$

Immune$prognosis$signatures$


6 gene‐expression modules TNBC classification

New therapeutic interventions

Prognostic determination (Immunity2)

Stromal environment and Breast Cancer

Matrix (P Chavrier, UMR144)

Micro‐Environment (F Mechta‐Grigoriou U830)

Stromal density (T Walter U900, Les Mines)

Immunity (V Soumelis U932)

No correlation with pCR (micro‐biopsy vs FNA)

Whole Exome Sequencing of paired triple‐negative primarytumour and corresponding residual disease after

neoadjuvant chemotherapy

Laurent C, Bonsang‐Kitzis H, Sadacca B, Pinheiro A


Micro‐Biopsy

NeoadjuvantChemotherapy Surgery Radiotherapy

ResidualDisease

Diagnostic

AnthracyclinTaxan

BreastLymph Nodes

Relapse?

15 patients with paired samples

WESRNASeqIlluminaHiSeq

WESRNASeqIlluminaHiSeq



ReadsReads Quality Control(FastQC)

Mapping on the Human Genome (hg19)(BWA aligner)

Mapping Quality Control(In-House)

GATK Preprocess: Local Indel Realignment

& Base Score Recalibration

BAM

Somatic Variant CallingVarScan 2

Removing PCR duplicates (Picard) & Intersecting on Exome target (Bedtools)

read1 read2

DNA fragment

Reference Genome

Variant Annotation & Filtering(Annovar & in-House)

Variant Quality Control(In-House)

WES – EWOK Pipeline AnalysisINSERM U900


exonic

splicingexonic;splicing

UTR3UTR5UTR5;UTR3

downstreamupstreamupstream;downstream

intronic

intergenic

ncRNA_exonicncRNA_intronicncRNA_splicingncRNA_UTR3ncRNA_UTR5

Func.refGene

frameshift deletionframeshift insertion nonframeshift deletionnonframeshift insertion

nonsynonymous SNV synonymous SNV

stopgain SNV stoploss SNV

unknown

VARSCA

N ‐AN

NOVA

R| ExonicFunc.refGene

SIFTPolyPhen2 (HDIV)


EXONIC|

INDEL SNP

Filtering#AD + Tumor.Freq

frameshift

stopgain SNV stoploss SNV nonsynonymous

Filtering#AD >2 + Tumor.Freq >10%

FilteringSIFT + PolyPhen

SIFT (D) & PolyPhen (P) | SIFT (D) & PolyPhen (D) | SIFT (D) & PolyPhen (.) | SIFT (.) & PolyPhen (P) | SIFT (.) & PolyPhen (D)

FilteringAA change

Func.refGene == exonic




1 2 3 4 5 6 7 12 14

0.00

0.01

0.02

0.03

0.04

0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100

value

dens

ity

variableTP.FREQ

RES.FREQ

0.050.05




High Throughput Screening in TNBC Cell Lines

Pinheiro A, Del Nery E, Sadacca B

Hs578TMDAMB157

BT549MDAMB231MDAMB436

BT20HCC70HCC1143HCC1937MDAMB468

HCC38HCC1187

TP53KRAS

PIK3CABRAFNRASPTEN

APCRB1

TissueCluster

CCLE−1 0 0.5 1

Expression

Color Key

1234567891011

prostatestomachurinary_tracthaematopoietic_and_lymphoid_tissuekidneycentral_nervous_systemthyroidsoft_tissuesalivary_glandskinboneovarylungendometriumpancreasupper_aerodigestive_tractbreastlarge_intestineliverautonomic_gangliaoesophagusbiliary_tractsmall_intestinepleura

wtmutated

TP53KRAS

PIK3CABRAFNRASPTEN

APCRB1

TissueCluster

CCLE−1 0 0.5 1

Expression

Color Key

1234567891011

prostatestomachurinary_tracthaematopoietic_and_lymphoid_tissuekidneycentral_nervous_systemthyroidsoft_tissuesalivary_glandskinboneovarylungendometriumpancreasupper_aerodigestive_tractbreastlarge_intestineliverautonomic_gangliaoesophagusbiliary_tractsmall_intestinepleura

wtmutated

Drug Libraries

ENZO FDA Approved_V2: 800

ENZO Tyrosines Kinase inhibitors: 80ENZO Phosphatase inhibitors: 33ENZO Protease inhibitors: 53


Hs578T, MDA MB 157

BT549, MDA MB 231, MDA MB 436

BT20, HCC70, HCC1143, HCC1937, MDA MB 468

HCC38, HCC1187

Compound libraryFDA‐approved

384‐plates with cells

72 hours

Multiplexed assaysfrom the same compound

C

Luminescent Cell Viability

Fluorescent Cytotoxicity Assay

CellTox Green

Cell‐based assays

Breast‐cancer cell line models

Read

Raw CTG signal (MDA‐MB468)

Replicate 1 Replicate 2

Plate 1

Plate 2

Plate 3

Plate 4

Raw ToxGreen signal (MDA‐MB468)

Replicate 1 Replicate 2

Lysis buffer


MDA‐MB‐231 MDA‐MB‐468

MDA‐MB‐231 MDA‐MB‐468



ValidationXcelligence (Roche, Ozyme)Functional validation (in vitro, in vivo)

Cell lines defined by a combination of hits (intersection, specific)

Chemical structureIdentification of potential pathways and protein targeted(V Stoven U900)

Phenogenomics (N Saettel, UMR176)

Drugs combination


Triple Negative Breast Cancer Residual Tumorand Resistance ... · Triple Negative Breast Cancer Residual Tumorand Resistance to Treatment Dr Fabien REYAL (RT)2 Lab ... Micro‐ Biopsy

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