Oncogénétique des LAL-B Emmanuelle Clappier Laboratoire d Hématologie, hôpital Saint-Louis et Département de Génétique, hôpital Robert Debré Faculté de Médecine, Université Paris-Diderot
Contenu du cours Notion d oncogenèse muti-étape Groupes oncogéniques définis par altérations «classantes» Epidémiologie (fréquence, âge) et pronostic des principaux groupes oncogéniques Actualités: - nouveaux groupes oncogéniques: ERGdel, BCR-ABL1-like - anomalies additionnelles et nouveaux marqueurs pronostiques: IKZF1del, CRLF2, JAK2, PDGFRB Projet biologique dans le cadre du GRAALL 2013
Genome-wide analysis of genetic alterations in ALL 1960: chromosome Philadelphia 2007: CGH/SNP-array analysis 2012: Massive parallel sequencing Nowell, 1960 Mullighan and Downing, Nature 2007 Mullighan, Nature 2012 Multiplicité des lésions oncogéniques additionnelles: fin du schéma 1 leucémie = 1 translocation
Multi-step oncogenesis Normal hematopoiesis Homeostasis
Multi-step oncogenesis Normal hematopoiesis Homeostasis
Multi-step oncogenesis
Multi-step oncogenesis From 1 to more than 10 driver lesions are necessary to lead to overt leukemia
Multi-step oncogenesis «Neo-Darwinian» tumoral development Greaves, 2009
Multi-step oncogenesis From 1 to more than 10 driver lesions are necessary to lead to overt leukemia Leukemia at diagnosis
Conséquences cellulaires des anomalies génétiques: acquisition du phénotype tumoral Non différenciation Prolifération (accumulation) Autorenouvellement Non apoptose Non sénéscence D après Hanahan et Weinberg, Cell 2000
Que peut-on attendre de la caractérisation génomique des leucémies? Comprendre les mécanismes de leucémogenèse Aider à prédire la réponse au traitement et la rechute Guider la stratification thérapeutique Mettre au point des thérapies ciblées et/ou personnalisées
LAL avec t(9;22)(q34;q11) / Ph+ / BCR-ABL1 ~ 30-40% LAL-B adultes, <5% chez l enfant Imatinib ib (Glivec) Yanada, JCO 2006
Genetic subtypes in BCP ALL in children EORTC-CLG 58951 N= 1253 BCP-ALL from children aged 1-1717 Hyperdiploidy 30 35% «B other» 25 30% Hypo/haploidy MLLt ETV6 RUNX1 20 25% BCR ABL1 TCF3 PBX1 < 5% each
Analysis of gene-expression profiling Yeoh, Cancer Cell 2002
Hyperdiploïdie standard High Hyperdiploidy: 51 à 60 chr environ avec gains chromosomiques non aléatoire: 1 2 3 4 classiquement, +4, +10, +17, +18, +21, +21 1 2 3 4 5 30-35% LAL-B chez enfants pic 2-5 ans, F>M < 10% adultes Très bon pronostic Détection: - Caryotype - Index ADN 1.16 - CGH-array 6 7 8 9 10 11 12 X 13 14 15 16 17 18 19 20 21 22 Y
t(12;21)(p12;q22) / TEL-AML1 (ETV6-RUNX1) ~ 20-25% LAL-B enfants Pic 2-5 ans Très bon pronostic (parfois rechutes tardives) Détection: - FISH (cryptique au caryotype) - Bio mol: RQ-PCR transcrit de fusion TEL TEL fusion Split AML1 AML1 AML1 12 21 Fusion + sonde AML1 splitée +/- délétion 2 éme allèle de TEL
Incidence by age of BCP ALL genetic subtypes in children 250 Other 200 Low hypo/near haploidy MLLt 150 iamp21 BCR ABL1 ERGdel 100 TCF3 PBX1 ETV6 RUNX1 50 High hyperdiploidy 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
t(1;19)(q23;p13) / E2A-PBX1 (TCF3-PBX1) ~ 4% LAL enfant et jeune adulte, M > F Pronostic plutôt bon chez l enfant (bon dans EORTC 58951, variable selon protocoles) Pré-B cig+ sig- typiquement (20-30% des LAL pré-b) Souvent translocation déséquilibrée avec tri1q et 19pter- Fusion E2A-PBX1 tri1q Détection: - Caryotype - Bio mol (RQ-PCR)
t(4;11)(q21;q23) / MLL-AF4 et autres translocations MLL MLL-AF4: ~ 60% LAL nourrisson ~ 2% enfant, 5-10% adulte LAL typiquement pro-b CD10-, souvent My+ Mauvais pronostic Autre partenaires fréquents dans les LAL: AF9, ENL, AF10 Au total ~ 80% LAL-B < 1an (rare après 1 an) Détection: - Caryotype + FISH MLL - Bio mol (RQ-PCR) principaux transcrits de fusion Chromosome 11 normal MLL réarrangé
Analysis of gene-expression profiling Yeoh, Cancer Cell 2002
Genetic subtypes in BCP ALL in children EORTC-CLG 58951 N= 1253 BCP-ALL from children aged 1-17 «B other» 25 30% Hyperdiploidy 30 35% Hypo/haploidy MLLt iamp21 ERGdel BCR ABL1 TCF3 PBX1 ETV6 RUNX1 20 25% < 5% each
Amplification en tandem de la région 21q22 : iamp21 Enfants et adolescent :âge median 11 ans Pauciblastiques Mauvais pronostic Détection: - FISH (avec sonde AML1) - CGH-array TEL AML1 AML1 12 21 TEL Soulier, Leukemia 2003 Harewood, Leukemia 2003
Genetic landscape of hypodiploid ALL Near-haploid ALL Low-hypodiploid ALL 24-31 chromosomes 32-39 chromosomes RTK and and Ras signaling (71%) IKZF3 (13%) TP53 (91%), commonly present in nontumor cells in children IKZF2 (53%) RB1 (41%) Activation of Ras and PI3K-signaling g -> therapeutic strategies to be explored Holmfeldt, Nat Genetics 2013
Un nouveau groupe caractérisé par une délétion intragénique de ERG Sls #01 Sls #02 Sls #03 Identification par CGH-array d une microdélétion récurrente ERG del No ERG del kda ERG 50 kb ERG normal isoforms 50 40 ERG short form Vinculin 25 Chr. 21 1M Agilent Expression d une protéine ERG tronqué - Expression aberrante du CD2, CD56 - morphologie particulière - ~3% des LAL-B pédiatriques - 40% de délétions d IKZF1 (4-7) Clappier, Leukemia 2013
(%) -free survival Event Un nouveau groupe caractérisé par une délétion intragénique de ERG n = 876 patients (LAL BCR ABL1 exclues) n=46 patients avec IKZF1 4 7 Good risk genetic subtypes (hyperdiploidy et ETV6 RUNX1) Poor risk risk genetic subtypes (MLL translocation, hypo/near haploidy) haploidy) ERGdel (n=11) Autres No ERGdel (n=35) ERGdel EFS EFS 100 90 80 70 60 50 40 30 20 10 0 years 0 2 4 6 8 10 12 14 O N No. of patients at risk : 51 526 512 402 263 148 52 4 Good-risk 20 47 35 25 14 8 6 0 Poor-risk 60 274 243 186 125 74 29 2 Other 3 29 27 20 14 7 1 0 ERGdel t-free surviva al (%) Event 100 90 80 70 60 50 40 30 20 10 HR 0.16 (95% CI 0.02-1.20) p=0.041 85.7% 51.3% 0 years 0 2 4 6 8 10 12 O N No. of patients at risk : 16 35 28 19 18 10 4 1 11 11 10 7 2 0 No ERGdel ERGdel Bon pronostic, sans impact péjoratif des délétions d IKZF1 Clappier, Leukemia 2013
BCR ABL+ALL IKZF1 genomic alterations 75% of IKZF1 deletions in children (Mullighan et al, 2008) and adults (Iacobucci et al, 2009) Poor prognosis in adult Ph+ ALL (Martinelli etal al, 2009) Other BCP ALL 10% of IKZF1 dlti deletions in pediatric i BCP ALL poor prognosis (Mullighan et al., NEJM 2008; Kuiper et al., Leukemia 2010, etc ) Medium risk ALL
Frequency of IKZF1 deletions in distinct BCP ALL subtypes in children EORTC CLG 58951 n=1253 BCR ABL1 negative BCP ALL IKZF1 15% % of IKZF1 del in each genetic subtype 100 90 80 70 60 50 40 30 20 10 0 BCR-ABL1 MLL / 11q23 low hypo / iamp21 TCF3-PBX1 ERGdel ETV6-RUNX1 High B-other abn. near-haploidy hyperdiploidy
Prognostic impact of IKZF1 deletions in BCR-ABL1-negative BCP-ALL in children EORTC-CLG 58951 n = 1223 patients EFS OS 100 90 80 86.5% 100 90 80 92.4% 86.7% 70 60 67.7% 7% 70 60 50 40 30 20 HR = 2.41 (1.75, 3.32) p<0.0001 50 40 30 20 HR = 1.67 (1.03, 2.72) p<0.035 10 10 0 (years) 0 2 4 6 8 10 12 14 0 (years) 0 2 4 6 8 10 12 14 O N Number of patients at risk : IKZF1 deletion O N Number of patients at risk : IKZF1 deletion 134 1044 984 771 511 303 129 10 Negative 73 1044 1010 833 567 339 152 15 Negative 52 179 159 118 84 49 13 1 Positive 21 179 172 137 105 69 21 1 Positive
Prognostic impact of IKZF1 deletions according to genetic subtypes
Prognostic impact of IKZF1 deletions in BCR ABL1 negative BCP ALL in adults GRAALL 2005
Distribution by age of BCP ALL genetic subtypes 100% 90% 80% 70% 60% 50% 40% 30% 20% to be explored in adult 10% 0% 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 25 30 30 40 40 50 50 60 ERGdel TCF3 PBX1 ETV6 RUNX1 High hyperdiploidy Other Low hypo/near haploidy MLLt iamp21 BCR ABL1 to be explored in adult
Un nouveau groupe de LAL B de mauvais pronostic: «BCR ABL1 like» or «Ph like» ProB signature Frequent IKZF1 deletions (40 90%) Den Boer, Lancet Oncology 2009 Harvey, Blood 2010
CRLF2 deregulation Incidence ~7% (50% in Down syndrome associated ALL) Translocation involving the IGH@ locus : t(x;14)(p22;q32) or t(y;14)(p11;q32) Interstitial (PAR1) deletion centromeric to CRLF2 juxtaposing CRLF2 with the P2RY8 promoter FISH P2RY8 CRLF2 chimeric transcript Point mutation in the CRLF2 gene Sequence Prognosis: rather poor, but depends on series RT PCR MLPA Russel, Blood 2009 Mullighan, Nat Genet 2009
JAK mutations (mostly JAK2 R683, rarely JAK1, JAK3) Incidence ~5%, nearlyalwaysassociatedalways associated with CRLF2 deregulation JAK mutation + CRLF2 deregulation + IKZF1 deletion > «Ph like» profile (in US pediatric series) Mullighan, PNAS 2009
RNA seq in Ph like ALL cases identifies multiple gene fusions involving kinases (ABL1, PDGFRB, JAK2, ) > in vitro response to inhibitors Roberts, Cancer Cell 2012
Case report: successful TKI therapy in a refractory BCP ALL with EBF1 PDGFRB rearrangement 16 y old male diagnosed with ALL at AYA unit, Saint Louis hospital, Paris, with: hyperleukocytosis (167 G/L) Lymph nodes, liver and spleen enlargement No CNS involvement Biology: Immunophenotype: EGIL II BCP ALL with aberrant CD33+ et CD36+ Normal karyotype, yp DNA index=1, common fusion transcripts negative Treatment and repsonse: Induction according to FRALLE 2000 group B (aged >10 years, >50G/L) Resistance to prephase at day 8 (26 000 blastes/µl) Persistent blasts in peripheral blood at day 21 > pursuing induction according to FRALLE 2000 group B2 BM aspiration at day 43: cytologic remission but MRD Ig/TCR >> 10 2 (1,7.10 1 ) Lengline, Haematologica 2013
Case report: successful TKI therapy in a refractory BCP ALL with EBF1 PDGFRB rearrangement A B RT PCR 300 bp 100 bp EBF1-PDGFRB fusion transcript 174 bp C EBF1 exon 15 PDGFRB exon 11 8.6 Mb deletion Sequence Array CGH Lengline, Haematologica 2013
Case report: successful TKI therapy in a refractory BCP ALL with EBF1 PDGFRB rearrangement MRD follow up al disease Minimal residu 10-1 10-2 induction consolidation imatinib BMT Imatinib introduced in combination with GRAAPH consolidation blocks 10-0 BMT with HLA identical sibling donor 10-3 10-4 10-5 Time from diagnosis 50 100 150 200 250 (days) Lengline, Haematologica 2013 Second similar case: 17 year old male with induction failure: ~80% blasts in BM after FRALLE 2000 B2 induction and 1st consolidation block > same EBF1 PDGFRB fusion > after 1 month imatinib treatment with EsPhALL consolidation block: MRD = 2.10 4
Plan for GRAAL 2013: genomic landscape of adult BCP ALL BCR-ABL1 35-40% «B-other» ~40% MLL translocations ~8% Hyperdiploidy ~6% ETV6-RUNX1 ERGdel? TCF3-PBX1 iamp21 Hypo/haploidy <5% each Prospectively identify druggable, BCR-ABL1-like cases for TKI treatment implementation: Evaluate prognostic significance of various oncogenic alterations Investigate the B-other group -> systematic search for alterations in ABL1, PDGFR, CRLF2, JAK2 -> RNA-seq, exome sequencing, GEP
t(12;21)(p12;q22) / TEL-AML1 (ETV6-RUNX1) Protéine chimérique avec activité de répresseur transcriptionnel constitutif
Prognosis associated with genetic subtypes in EORTC-CLG 58951 n = 1223 BCP-ALL 100 90 80 70 60 50 40 30 20 10 0 EFS BCR-ABL1-negative 1-17 years years 0 2 4 6 8 10 100 90 80 70 60 50 40 30 20 10 0 OS 0 2 4 6 O N Number of patients at risk : O N Number of patients at risk : 42 419 406 317 210 125 15 419 413 341 236 24 305 297 246 170 93 12 305 300 259 181 3 38 36 27 18 10 1 38 38 27 19 7 49 44 36 21 15 3 49 46 39 23 87 354 314 231 159 99 51 354 333 262 184 7 20 15 10 5 5 6 20 17 11 6 11 27 24 16 8 3 2 27 27 24 18 5 11 7 6 4 2 4 11 8 7 5
Prognostic impact of IKZF1 deletions in BCR-ABL1-negative BCP-ALL in children EORTC-CLG 58951 n = 1223 patients EFS 30 Aug 2013 14:38 100 90 80 86.5% 100 90 80 70 60 50 40 67.7% HR = 2.41 (1.75, 3.32) 30 30 p<0.035035 20 20 p<0.0001 10 10 70 60 50 40 HR = 1.67 (1.03, 2.72) 0 (years) 0 2 4 6 8 10 12 14 0 0 2 4 6 8 10 O N Number of patients at risk : IKZF1 deletion 134 1044 984 771 511 303 129 10 Negative 52 179 159 118 84 49 13 1 Positive O N Number of patients at risk : 73 1044 1010 833 567 339 15 21 179 172 137 105 69 21