Médecine de précision en oncologie pédiatrique - biologie moléculaire et

décisions thérapeutiques

Gudrun Schleiermacher, MD PhD

Département d’Oncologie Pédiatrique

Laboratoire RTOP « Recherche Translationelle en

Oncologie Pédiatrique »

INSERM U830

Institut Curie, Paris

Les cancers pédiatriques

• Incidence : 1700 – 2000 nouveaux patients par an en France (0 – 15 ans) (Rapport INCa, 2012)

• caractères histopathologiques et biologiques différents des cancers de l’adulte:

• extrême rareté des carcinomes,

• leucémies (29 % des cas, dont 80 % de leucémies aiguës lymphoblastiques),

• les tumeurs du système nerveux central (24 %) et les lymphomes (11 %).

• tumeurs embryonnaires : néphroblastomes, neuroblastomes, rétinoblastomes…,

• Taux de survie 82% à 5 ans

• Décès suite à un cancer :

• 1 % des décès avant 1 an

• 21 % entre 1 et 14 ans (la deuxième cause de décès après les accidents (26 %) dans cette classe d’âge)

Connaître les altérations moléculaires

Outil diagnostic :

confirmation d’un diagnostic anatomopathologique

Biomarqueurs pronostiques :

définir des groupes de risque

Biomarqueurs prédictifs

prédire la réponse à un traitement

Amplification de MYCN

(FISH)

Recherche d’un transcrit de fusion

EWS-Ets (qRT-PCR)

Les questions

• De plus en plus de médicaments sont développés sur la base d’une altération moléculaire (mutation BrafV600E)

• Il est de plus en plus fréquent de devoir caractériser les altérations moléculaires d’une tumeur

Identifier certaines anomalies moléculaires dans le cadre de protocoles de recherche clinique

Critères d’éligibilité pour un essai

Intérêt majeur à connaître les altérations moléculaires d’une tumeur:

Orienter le patient d’emblée vers un essai adapté

Éviter toute perte de temps pour le patient : le

screening pour un essai particulier peut durer de 2 à 4semaines

Bcp d’altérations moléculaires sont d’une incidence faibleen oncologie pédiatrique

Intérêt pour les promoteurs d’essai car garantie derecrutement de patients

Augmenter le nombre de patients entrant dans des essais cliniques précoces basées sur d’identification de biomarqueurs moléculaires

Objectifs secondaires : Objectifs cognitifsRecherche translationelle

Mise en place d’une RCP moléculaire :

RCP moléculaire à l’Institut Curie : Organisation

Patients avec une tumeur

de haut risqueen récidive

ousans

traitementefficace connu

RCP moléculaire

Pathologie

Prélèvementtumoral/sanguin

Plateformes

Proposition thérapeutique

Génétique

Integration des données clinico-

biologiques & analysemoléculaire

Bioinformatique

Base de données

Suivi du patient

Prescription moléculaire

RCP moléculaire à l’I CurieTechniques moléculaires

Sur du matériel tumoral archivé (biopsie faite dans le cadre des soins;

consentement institutionnel):

CGH array: analyse du profil du nombre de copies

NGS panel : recherche des mutations

Unité de PharmacogénomiqueUnité de Génétique Somatique

Service de GénétiqueCeline Callens, Ivan Bieche, Etienne Rouleau, Gaëlle Pierron, Olivier Delattre

Profil Génomique MoléculaireAltération du Nombre de copiesAmplifications focales

HER2, EGFR, ALK, MDM2, CDK4/6, FGFRx ….Délétions Homozygote

PTEN, CDKN2A (p16), CTNNB1 (bCaténine)…Statut Allélique

Perte d’hétérozygotie (LOH)Isodisomie

Olivier Delattre, Gaëlle Pierron

Analyse par CGH array

100 % tumor cell contentDeletion homozygote de CDKN2A

Homozygous deletion of CDKN2A

NGS Panel; Technology : Ion torrent®

Design120kb1200 amplicons46 genes

→ 500X expected

PGM , « Personal genome machine »Life technologies

Panel design used in a « diagnostic » setting

How it works : Validation et performances

- Limite de détection : 2 à 5%

- PGM 318 : série de 10 à 15

échantillons / semaine

- Échantillons en réplicat

- avec contrôle interne

- Résultat en moins de 15 jours

NGS panel : quelques alertes

Contamination d’un échantillon?Identité des échantillons?

Identitology

Clustering of samples based on polymorphisms: AAR analysis

Clustered samples highlights their polymorphisms : similar AAR Expected with tumor & constit sequencing

Might highlight a sample that has been sequenced twice by error

Contamiation analysisAAR distribution

0 20 40 60 80 100polymorphism AARs

Nu

mb

er o

f p

oly

mo

rph

ism

s0

5

1

0

1

5

AAR distribution of sample without any contamination must highlight : A peak for RefRef close to 0% A peak for RefAlt close to 50% A peak for AltAlt close to 100%Example above highlight a small contamination Some RefRef and AltAlt are « far » from 0% or 100%. Statistical analysis allow sto highlight such inexpected distribution.

To dig further on AAR may highlight whether contaminant is within the run (same polymorphisms & samples side by side) or an unknown DNA (contamination during DNA extraction for example)

NGS panel : SNP ou mutation?

Comment identifier les « vraies « mutations?

SNP : single nucleotide polymorphismSNV : single nucleotide variationMutation : une variation avec un impacte fonctionel

Identification et analyse de mutations ponctuelles somatiques: NGS Panel

Estimation du caractère pathogène du variant et prédiction

des conséquences fonctionnellesBases de données internationnales

COSMIC (Catalogue of Somatic Mutations in Cancer), et littérature (PubMed)

SIFT, POLYPHEN, Cbioportal, Tumorportal…Et intégration de l’ensemble des informations

Elimination des Polymorphismesconnus et référencés

Interogations des outils spécialisés (USA)dbSNP, 1000 genomes, ESP (Exome sequencing Project)…

RCP moléculaire (Jan – Dec 2015): Molecular analysis to search for targetable genes (DOPAJA, I Curie)

Number of patients: 67 (4 patients with analysis in progress) : 61 samples analysed=> analysis on 37 samples at diagnosis=> analysis on 31 samples at relapse

Diagnosis: Ewing sarcoma (5)Glioma (10)Medulloblastoma (4)Osteosarcoma (4)Rhabdomyosarcoma (4)Neuroblastoma (8)Other tumors (32) such as hepatic tumor, brain tumor, sarcoma

nephroblastoma, rhabdoid tumor etc.

Median age: 11, range: 0 – 21 years

Molecular analysis to search for targetable genes - RESULTS

Alterations of targetable genesidentified by a CGH:- Amplicon CDK4/MDM2 (3

patients)- Amplicon KIT/PDGFRA/KDR and

amplicon ERBB3/CDK4- Homozygous deletion of CDKN2A

(4 patients)- Loss of genes such as PTEN,

mTOR, VHL, STK11, BRAF- Isodisomy of genes such as NF1- ALK alteration

Mutations of targetable genes(pathogenic variant) identified by NGS: - NOTCH2 p.V2075M- FGFR3 p.K650R- PIK3CA p.E545K- NF1 p.S2601X- PTEN p.R233X- MTOR p.L1460P- NOTCH4 p.C1191R- PDFGRA p.N659K

63 patients with available results for the 2 techniques- 55 aCGH done (including 22 done at diagnosis)- 58 NGS sequencing done (including 11 NGS on circulating DNA in paralleleto NGS on tumoral DNA)

22 patients with alterations of targetable genes ie 34 % of the patients

- TSC1 p.V178I- PIK3R1 p.N564D- KRAS p.G13D- KRAS p.K12V- BRAF p.V600E- NRAS p.Q61R- NRAS p.Q61L

Patients ayant reçu un traitement « ciblé » selon des ces résultats : 5

Permettre une analyse plus “globale” : Projet National

MAPPYACTS

PI : Birgit Geoerger, GR

Co PI : Gudrun Schleiermacher, IC

MoleculAr Profiling for Pediatric and Young Adult Cancer Treatment Stratification

CSET 2015/2244EudraCT n°2015-A00464-45

MAPPYACTS

ON-PURPOSE FRESH TUMOR BIOPSY/SURGERY & PATHOLOGY CONTROL

AT RELAPSE

MOLECULAR PROFILINGWES, RNAseq

MOLECULARTUMOR BOARD

Within 4 weeks

TREATMENTPhase 1 /2

OrCompassionate

use

PI : B Geoerger,GRCo PI: Gudrun Schleiermacher, IC

Inclusion at the time of progression or relapse

MAPPYACTS A multicentric, prospective proof-of-concept study

MoleculAr Profiling for Pediatric and Young Adult Cancer Treatment Stratification

Recruitment period June 2015 during 3 years

Patient population ~ 300 children and adolescents with solid tumors and leukemia

Main objective To screen the maximum of relapsed or refractory pediatric patients to provide them with their individual molecular tumor profile and treat them with matched innovative targeted agents

Secondary objectives OR, TTP, OS, detection of new targets, percentage of therapysuggestions, etc.

Primary MolecularAnalysis

WES and RNA Seq

Secondary MolecularAnalyses

Methylation array, miRNA expression"The Immune Contexture of Pediatric Cancers”

Ancillary Studies Circulating DNA, preclinical models and patient-derived xenografts

Operationals and Contacts

Center of analysis

Coordinating Investigator Dr Birgit GEOERGER (Co-PI) : Dr Gudrun SCHLEIERMACHER

Molecular Profiling Platform Dr Ludovic LACROIX Dr Olivier DELATTRE

Bioinformatics Guillaume MEURICE Virginie BERNARD

Biostatistics Stefan MICHELS / Mohamed Amine BAYAR

Data manager Nathalie OLLIVIER

Pharmacovigilance Dr Salim LAGHOUATI

Sponsor CRA Audrey DUSSUD

Sponsor : Gustave Roussy

Présentation MAPPYACTS – Réunion SFCE

Procedures Step 1: Main Inclusion/Exclusion Criteria

Informed Consent

Confirmed solid tumor or leukemia which is recurrent or refractory to standard treatment

In case of solid tumor, lesion must be accessible for biopsy or surgical resection or cytological puncture

Age: ≥ 6 months at relapse and ≤ 18 years at initial diagnosis*

Performance Status: ≥ 70% / Life expectancy ≥ 3 months

Adequate hematopoietic (leukemia excluded*), hepatic and renal functions:

- Neutrophils >1.0 x 109/l (if bone marrow involvement: ≥0.75 x 109/l)* (unsupported)

- Platelets >100 x 109/l (if bone marrow involvement: ≥ 75 x 109/l)* (unsupported)

- Hemoglobin >80 g/l (transfusion allowed)

- ALAT/ASAT <2.5 x ULN

- Bilirubin ≤1.5 x ULN (in case of tumor involvement of the liver ALAT/ASAT<5 x ULN)

- Serum creatinemia <1.5 x ULN for age (if >1,5 x ULN, creatinine clearance > 70mL/min/1,73m2 or GFR >70%)

× Symptomatic or progressive CNS metastases

× Malignant disease other than that being treated in this study

× Coagulation disorder that prevents the accomplishment of a biopsy or surgery

× Uncontrolled infections

17/03/2016 Présentation MAPPYACTS – Réunion SFCE*Amendment submitted

Procedures Step 3: Molecular Analysis WES and RNAseq

• WES and RNAseq of tumor DNA/RNA and paired constitutional DNA (blood sample)– Solid tumors: Blood sample – Leukemia: Blood/bone marrow sample during MRD– WES: mean depth of coverage: 100 – 120x in tumor samples, 80-100x in normal tissue, 95%

at 20x

• Bioinformatics analysis of WES based on Illumina Pipeline consisting in:– alignment script– allele counting – variant detection based on hg19 human genome– Calling: SNV (mutations), SV, CNA (aCGH-like)– Secondary bioinformatics analysis

• Bioinformatic analysis of RNAseq:– sequences alignments on genome– detection, assembling and quantification of genes and transcripts – annotation of transcripts by comparison with refseq and identification of novel isoforms – DeFuse, TopHat 2, Chimera Scan, Fusion Map, … – Calling of:– Detection of fusion transcripts– Detection of splice variants– Secondary analysis: miRNA, long non coding RNA etc

ConstitDNA raw

sequences

TumorDNA raw

sequences

Sequence alignment against

hg19

Alignment recalibration

Quality & stat report

CNA & LOH detection

profils

Variantcalling

Variant processingFilter-out variants without

interest for project

Variant annotationLocation, exonic function,

deleterious effect...

MAPPYACTS BioInformatics Pipeline (WESeq & RNAseq)

Sequence alignment against

hg19

Alignment recalibration

Quality & stat report

Variantcalling

Variant processingFilter-out variants without

interest for project

Variant annotationLocation, exonic function,

deleterious effect...

Sequence alignment against

hg19

Alignment recalibration

RNA validationHighlight variants

expressed at RNA level

Tables+Images+Plot+circosTables+view

+Plot

TumorRNA raw

sequences

Gene Fusiondetection

Fusion processingFilter-out false positives

Table+

view

IGV view of aligned sequences

Filtered false positive variants

Tumor

control

RNA

IGV view of aligned sequences

Validated mutations

Tumor

control

RNA

Output : Identification of actionable targets / point mutations

Validation of pE545K PIK3CA Necessity of a decisional algorithm/ MBB Virginie Bernard

Circos representation of all detected alterations

Mutations

Coverage T/C

Copy number

LOH

Gene fusion

Pluridisciplinary therapeutic molecular biology meeting: MTB

Basic and translational researchers/ biologists, Pathologists,

Bioinformaticians, Technical platform

experts, Pediatric oncologists

International level:

INFORM, etc

Clinical relapse staff RCPPI/II

CMTB (Canpedif):

With treating physician

Data analysis and Therapeutic recommendation

Data interpretation:

High grade amplification

Translocations

Homozygous deletion of genes previously implicated on oncogenesis

Mutation/deletion of a gene previously implicated on oncogenesis

Therapeutic strategy

Targeted cancer treatment approved for the pathology

Targeted cancer treatment in the context of a clinical trial phase I-II

Non targeted cancer treatment in the context of clinical trial phase I-II

Non targeted cancer treatment not in a clinical trial

Children and adolescents with

refractory or recurrent tumors

or leukemia

Signed informed consent

BIOPSY/Surgery

Molecular analysis:− WES − RNAseq (RT-PCR confirmatory)− IHC, FISH (confirmatory)

Exploratory:- Methylation array- miRNA- Immunmarker/modulators

ITCC European Precision Medicine in Pediatric Oncology and Hematology – 2016

Data interpretation &Therapy suggestion

MAPPYACTSin ITCC centers If actionable targets:

If no actionable targets:

Clinical Trials

INCa-NGS Platforms: IGR, IC

MultidisciplinaryTherapeutic Molecular Biology Tumor Board(with signed report from

biologists)

Bioinformatics

Ongoing targeted Phase I/II

trials :• ALK: LDK378, Create, AcSé

• MET, ROS: CREATE, AcSé

• Pan-ERB: Afatinib

• BRAFv600: Dabrafenib

• MEK: Trametinib, Cobimetinib

• VEGF: BEACON (NB)

• Multi-TKI VEGFR-FGFR-PDGFR:

Regorafenib, Lenvatinib

Ongoing non-targeted Phase

I/II trials:• Abraxane: RMS, NB, Ewing

• VIT (RMS)

• CTL-A4: Ipilimumab (melanoma)

• PD-1: Pembrolizumab,

Atezolizumab

Clinical Relapse Tumor Board

(RCPPI)

Children and adolescents with

refractory or recurrent tumors or

leukemia

Signed informed consent

BIOPSY/Surgery

Molecular analysis:− WES − RNAseq (RT-PCR confirmatory)− IHC, FISH (confirmatory)

Exploratory:- Methylation array- miRNA- Immunmarker/modulators

- CtDNA- Preclincial models

European Precision Medicine Program: Pediatric Oncology-Hematology 2016

Data interpretation &Therapy suggestion

If actionable targets:

If no actionable targets:

Clinical Trials

INCa-NGS Platforms: IGR, IC

MultidisciplinaryTherapeutic Molecular

Tumor Board(with signed report from

biologists)Bioinformatics

Ongoing targeted Phase I/II trials or Acseprogram

Ongoing non-targeted Phase I/II trials or“non-targeted” arms of ESMART

Clinical Relapse Tumor Board

(RCPPI)

Registered targeted agents inpediatric/adult cancer with pediatric doserecommendation

ESMART prospective PoC trial(AcSe):IST multi-agent from pluri-companyEuropean Proof–of-Concept TherapeuticStratification Trial of MolecularAnomalies in Relapsed or RefractoryTumors in children

Matrix TrialGenentech Roche

MAPPYACTSin F, SP, I, Ir, Is

D: INFORM; NL: iTHER; UK: COMET

INFORM

Institut Curie – IC 2014-01 NGSKids

NGSKids IC2014 – 01

Utilisation des altérations génétiques spécifiques des cellules tumorales

identifiées par NGS pour le suivi des échantillons périphériques chez des

enfants atteints de tumeurs solides métastatiques et/ou de haut risque

Main objective:

Feasibility study identification of genetic alterations of tumoral cells identified by NGS techniques todetect circulating tumoral DNA (ctDNA) in peripheral samples collected before, during and aftertreatment

To evaluate prematurely response to treatment, To anticipate relapse To evaluate minimal residual disease To study clonal evolution during treatment To identify genetic alterations which might be considered for targeted therapy

Funding:Barletta Foundation, private gift, AO Siric ( with international project review)

Institut Curie

Results of ct DNA

Example of 2 NB patients:=> Analysis of tumoral +constitutional DNA and ctDNAat diagnosis, during treatmentor at relapse by WES

Patient 1:Patient 2:

After HD Chemo

Mathieu Chicard

17 patients included to date: Median age: 7 years• 6 metastatic neuroblastoma• 3 Ewing sarcoma• 5 RMS (alveolar and embryonal)• 1 hepatoblastoma• 1 medulloblastoma• 1 SMARCB1-deficient tumor (chordoma)Per patient : ~ 6 blood samples (1 each at diagnosis, D3, D10, M2, M4, and end of treatment)Tumoral and constitutional DNA are under analysis by WESBioinformatical analyses ongoing

Conclusion

Programme national pour la caractérisation moléculaires des cancers pédiatrique en rechute

Pour orienter au mieux les traitements lors de la rechute

Importance d’intégrer les aspects « évolution »

Acknowledgements

Unité de Bio-Informatique

INSERM U830Olivier Delattre

Emmanuel BarillotValentina Boeva

Unité de Génétique Somatique

Gaelle PierronEve LapoubleDavid Darmon

Département de PédiatrieJean MichonFrançois Doz

Département de TransfertDavid Gentien

Sergio Roman Roman

Clinicians and pathologists of the SFCE

Plateforme de SéquençageSylvain BaulandeVirginie Raynal

Virginie Bernard

Equipe SiRIC RTOP« Recherche translationelle en

Oncologie Pédiatrique »Gudrun Schleiermacher

Leo Colmet DaageAngela Bellini

Nathalie ClémentCéline Chauvin

Zhiyan HanWilfrid RicherPaul Deveau

Amaury LerusteIrene JImenez

Franck Bourdeaut

Lyon, CLB

Valerie Combaret

and colleagues