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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
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