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?
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Pearson Correlation to Training Test CentroidsCentroid1 (0.76)Centroid2 (0.93)Centroid3 (0.74)Centroid4 (0.64)Centroid5 (0.85)Centroid6 (0.76)
Pearson Correlation to Training Test CentroidsCentroid1 (0.90)Centroid2 (0.83)Centroid3 (0.72)Centroid4 (0.91)Centroid5 (0.63)Centroid6 (0.51)
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$
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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)
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer
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
Neaodjuvant Treatment in Triple Negative Breast Cancer