Malattia Minima Residua nelle Mieloproliferative Croniche Corso Nazionale di Aggiornamento in Ematologia Clinica, SIE 2008 Catania, 6-7 Novembre 2008 Malattia Minima Residua nelle Mieloproliferative Croniche Giovanni Martinelli, MD Institute of Hematology and Medical Oncology “ L. e A. Seragnoli” Bologna Italy

Network of tyrosine kinase fusions in MPDs PCM1 8p21 9q33 ABL 9q34 PDGFRA 4q12 SYK 9q22 JAK2 9p34 PDGFRA 4q12 ETV6 12p13 BCR 22q11 ABL 9q34 FIP1L1 4q12 ABL 9q34 FGFR1OP1 6q27 FGFR1OP2 12p11 CEV14 14q32 H4 10q21 HIP1 7q11 HCMOGT1 17p11 FGFR1 8p11 MYO18A 17q11 HERV-K 19q13 RABEP1 17p13 11p13 PDE4PIP 1q22 NIN 14q24 PDGFRB 5q33 PDGFRB 5q33 ZNF198 13q12 CEP1 9q33 12q13 12q15 TIF1 7q34 3p22a 3p22b TP53BP1 15q22 KIAA1509 14q32

METHODS TO MONITOR CLINICAL RESPONSE and TO DETECT MRD Based on the leukemia phenotype Suitable for 100% of the cases(?) Based on the presence of genetic markers Suitable only for a fraction of the cases Morfology Immunologic Phenotype Diagnosis 10-1 10-2 10-3 10-4 10-5 10-6 10-7 Cytogenetics FISH P C R

MINIMAL RESIDUAL DISEASE Leukemic Burden 1013 1012 1011 1010 109 108 107 106 105 104 103 102 10 4 logs reduction Hematological remission Degree of molecular remission PCR positivity PCR negativity 4 logs reduction GIMEMA CML Working Party

Why measure BCR-ABL transcript level? Provides a convenient and accurate measure of the fall and rise of leukaemia Insights into CML biology Clinical relevance: Track individual response and relapse Surrogate marker of response for trial purposes (FDA, EMEA approved) GIMEMA CML Working Party

Track Individual Response 8% 0.8% 0.08% 80% 0.008% Bologna Base line 1.0 Log reduction of BCR-ABL 2.0 Imatinib ceased 3.0 4.0 Pre 3 6 9 12 15 18 Months from the start of Imatinib GIMEMA CML Working Party

Track Individual Response Restarted 8% 0.8% 0.08% 80% 0.008% Adelaide Base line 1.0 Log reduction of BCR-ABL 2.0 Imatinib ceased 3.0 4.0 Pre 3 6 9 12 15 18 Months from the start of Imatinib GIMEMA CML Working Party

Track Individual Response W M E453G Standardised baseline 80 8 1 0.8 BCR-ABL/BCR% Log Reduction 2 0.08 3 .0008 There were 2 patients with a mutation who have maintained a CCR and both received an increased dose. One of these patients has maintained CCR for 11 months after the mutation was detected. 453 mutation is a rare mutation that is located in the carboxy terminal of the kinase domain beyond the activation loop in a region that does not make direct contact with imatinib 2.1-fold rise 4 Pre 3 6 9 12 15 18 21 24 27 30 33 36 39 Months from the start of Imatinib GIMEMA CML Working Party

Track Individual Response W M E453G Standardised baseline 80 8 1 0.8 BCR-ABL/BCR% Log Reduction 2 0.08 3 .0008 800mg 4 Pre 3 6 9 12 15 18 21 24 27 30 33 36 39 Months from the start of Imatinib GIMEMA CML Working Party

2. Surrogate marker of PFS 100 90 80 p<0.001 70 60 % without progression 50 42 months 40 No CCR (<2 log reduction) 75% (67-84) 30 CCR and 2 to 3 log reduction 90% (84-97) 20 10 98% (96-100) MMR (3 log reduction) 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 Months on imatinib

2a. Surrogate marker of early molecular response 1 0.1 No CgR P=0,01 100 2 / · m b abl bcr 0.01 PCgR 0.001 P=0,01 CCgR 0.0001 1 2 3 6 12 months Iacobucci et Al 2006 Blood

KINETICS OF MOLECULAR RESPONSEIS (ALL PATIENTS) GIMEMA Protocol CML 0307 KINETICS OF MOLECULAR RESPONSEIS (ALL PATIENTS) (MMR <0.1%IS ) 3% 22% 59% 74% HERE ALL PATIENTS PRESENTED. RIC, IS IT POSSIBLE TO CONVERT THIS SLIDE IN A MORE ATTRACTIVE WAY? A BAR GRAPH?? CAN YOU GET SOME SUPPORT FOR THIS? Kantarjian H, Giles F, Wunderle L, et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome–positive ALL. N Engl J Med. 2006;354:2542-2551. Months Pts Evaluated 67 67 61 43 GIMEMA CML Working Party

GIMEMA CML WP – STUDI APERTI ALL’ARRUOLAMENTO INTERMITTENT IMATINIB STUDIO TKI SETTING N. Arruolati / Target CML 0206 (Vaccino – b3a2) IMA CCgR ≥ 18 mesi MRD detectabile 42/69 SI0207 (Vaccino – b2a2) 0/68 INTERIM INTERMITTENT IMATINIB ≥ 65 anni CCgR ≥ 24 mesi 22/78 CML 0408 ROTAZIONE NILOTINIB – IMATINIB ECP 0/114 GIMEMA CML Working Party

3. How to express results in a cooperative network. 4 3. How to express results in a cooperative network? 4. How to compare results from different labs? GIMEMA CML Working Party GIMEMA CML Working Party

How to express RT-PCR results How to express RT-PCR results? Italian experience sa national experience Before : BCR-ABL/ Beta2 M x100 = es. 0,1 Now: BCR-ABL/ABL x100 = es. 10 Undetectable results: Before: BCR-ABL/ Beta2 M x100 = es. 0,00001 now: BCR-ABL/ABL x100 = es. 0,001- 0,0001 GIMEMA CML Working Party

Real Case Diagnostic samples Same patients at the follow-up Results (copie di P210 / copie di ABL)*100 = 120,955 Sens(BM)= - log10 (21592/6901) - log10 (6901) = -4,3 Same patients at the follow-up Result (copie di P210 / copie di ABL)*100 = 1,86 Sens(BM)= - log10 (21592/6901) - log10 (2282) = -3,9 100 Nessuna risposta molecolare 78,955 1,860 1 Risposta subottimale 0,1 0,086 Risposta molecolare maggiore 0,001 Undetectable Diagnosi I Follow up II Follow up GIMEMA CML Working Party

GIMEMA CML Working Party

Laboratori che hanno già acquisito il CF Bergamo Milano Monza Pavia Verona Orbassano Genova Bologna Ravenna Firenze Pesaro Perugia Roma Bari Roma Tor Vergata Nuoro Napoli 06/08 Per Gianantonio: Numero e città dei laboratori sono stati aggiornati sulla base degli ultimi dati in ns possesso. Palermo Reggio Calabria Catania Laboratori che hanno già acquisito il CF GIMEMA CML Working Party

Laboratori che hanno fatto richiesta di acquisire il CF Brescia Vicenza Torino (Osp. Maggiore) Parma Rimini Pisa Pescara Potenza 06/08 Per Gianantonio: Numero e città dei laboratori sono stati aggiornati sulla base degli ultimi dati in ns possesso. Laboratori che hanno fatto richiesta di acquisire il CF GIMEMA CML Working Party

When? MRD monitoring in pts with CML Every 3 mths during first year and until CMR will be optained Every six months furthermore.

Evoluzione Prospettica della Risposta Molecolare 100% 0.001% 0.01% 0,1% 1% 10% Ratio BCR-ABL (IS) Pre 3 6 9 12 15 18 24 Mesi dall’inizio della terapia

Risposta subottimale? Compliance scarsa? Variabile ed individuale assorbimento del farmaco?

Mutazioni che conferiscono totale insensibilità ad imatinib BCR-ABL Imatinib IC50 (nM) biochemical cellular WT 300 260-500 M244V 380 2000 L248V n.a. 1500 G250E 3900 Q252H 1200-2800 Y253F >5000 3475 Y253H >10000 E255K 2800 4400-8400 E255V D276G F311L 775 480 T315I F317L 900 810-1500 M351T 820 930 E355G 400 F359V 4700 1200 V379I 800 1630 A380T 340 2450 L387M 1100 L387F H396P 340-800 850-4200 H396R 1950 1750 F486S 1230 Mutazioni che conferiscono totale insensibilità ad imatinib P-LOOP CATALYTIC DOMAIN ACTIVATION LOOP

Mutazioni che potrebbero essere bypassate con una dose escalation? BCR-ABL Imatinib IC50 (nM) biochemical cellular WT 300 260-500 M244V 380 2000 L248V n.a. 1500 G250E 3900 Q252H 1200-2800 Y253F >5000 3475 Y253H >10000 E255K 2800 4400-8400 E255V D276G F311L 775 480 T315I F317L 900 810-1500 M351T 820 930 E355G 400 F359V 4700 1200 V379I 800 1630 A380T 340 2450 L387M 1100 L387F H396P 340-800 850-4200 H396R 1950 1750 F486S 1230 P-LOOP Mutazioni che potrebbero essere bypassate con una dose escalation? CATALYTIC DOMAIN ACTIVATION LOOP

Quando effettuare il blood level testing ? In caso di resistenza secondaria: - perdita di CCgR con incremento dei livelli di - perdita di MCgR - perdita di HR - progressione ad AP o BC trascritto >1 log

Blood Level testing 06/08 Per Gianantonio: Ho provato a integrare la narrativa come da tua richiesta: può funzionare? BENE Per Gianantonio/Pedrizzi: Vale il commento sulla lingua: se il BLT deve essere tutto in italiano, ecco la traduzione dei testi: ANDIAMO SEMPRE IN INGLESE!

Dove effettuare il blood level testing? Orbassano Napoli 06/08 Per Gianantonio: Numero e città dei laboratori sono stati aggiornati sulla base degli ultimi dati in ns possesso. GIMEMA CML Working Party

Mutational analysis

Imatinib Imatinib La resistenza e’ acquisita Ph+ PhR PhR PhR PhR PhR

The persistence of MRD detectable by PCR may reflect the presence of subclones resistant to imatinib Imatinib Imatinib Ph+ BCR/ABL reduction PhR PhR The MRD amount measures the burden of the Ph-positive cells resistant to imatinib and potentially prone to progression. Higher the amount, higher the risk! GIMEMA CML Working Party

Loss of inhibition of BCR-ABL TK activity by Imatinib can be due to: Mutations of BCR/ABL BCR-ABL amplification Insufficient Tissue Levels Insufficient Intracellular levels

AID can trigger mutations at many locations in the genome AID wt Genes were divided into three groups: group I, consisting of 23 (19%) of the most highly mutated genes (q,0.05;see Methods); group II, consisting of 25 (21%) genes with intermediate mutation frequencies (0.05#q,0.2); and group III, consisting of 70 (60%) infrequently mutated genes (q$0.2). AID-/- Somatic hypermutation targets include Bcl6, Cd83, Btg1, the tyrosine kinases Blk and Syk, the B cell transcription factor Ocab, and Ung

Relevance of mutation monitoring of pts Secondary resistance is due to a critical point mutation on bcr-abl gene RELAPSE ON IMATINIB RELAPSE ON 2nd-LINE TKI 90% RELAPSE ON 3rd-LINE TKI 97% 55% At least, this is what we observe.. While the average incidence of mutations in patients who relapse on imatinib is approximately 50%, almost all of the patients who relapse after 2° or 3° line TKI therapy harbour one or more mutations. Resistant pts harbouring Abl KD mutations Relevance of mutation monitoring of pts Soverini et Al 2006 JCO

Perche’ ricercare le mutazioni? Perche’ non tutte le mutazioni sono ugualmente sensibili ai farmaci TKI Perche’ abbiamo farmaci attivi di 2° o 3° generazione sui mutati. Per ottimizzare la terapia con TKI. Mettere dia da Ilaria

Incidence at baseline,% Mutational Analysis CML-CP Best Responses to Nilotinib by 12 Months in Imatinib-Resistant Patients by Baseline Mutation Status Mutation* IC50 (nM) Incidence at baseline,% MCyR n/N (%) CCyR MMR No mutation 45 50/87 (60) 35/87 (40) 21/76 (28) Any mutation 52 51/100 (51) 33/103 (32) 18/91 (20) Unknown IC50 15 19/30 (63) 15/30 (50) 5/20 (25) IC50 ≤ 150 nM 23 26/44 (59) 18/44 (41) 12/40 (30) IC50 > 150 nM Y253H 700 4 1/8 (13) 0/8 (0) 0/7 (0) E255K/V 548/791 3/7 (43) 1/7 (14) F359C/V 258/161 6 2/11 (18) 0/11 (0) 0/10 (0) Comparable rate of responses were observed in patients with or without baseline mutations:1,2 Less favorable responses were seen in patients with mutations exhibiting IC50 >150 nM (Y253H, E255K/V, and F359C/V). These mutations rarely occur in imatinib-resistant patients. 1,2 Patients with T315I were excluded from efficacy analyses. 1,2 References Saglio G, Radich J, Kim D-W, et al. Response to nilotinib in chronic myelogenous leukemia patients in chronic phase (CML-CP) according to BCR-ABL mutations at baseline. Presented at: 44th ASCO Annual Meeting; May 30-June 3, 2008; Chicago, Ill. Abstract 7060. Radich J, Kim D-W, Martinelli G, et al. Response to nilotinib in chronic myelogenous leukemia patients in chronic phase (CML-CP) according to BCR-ABL mutations at baseline. Presented at: 13th Congress of the European Hematology Association; June 12-15, 2008; Copenhagen, Denmark. Abstract 0137. *Patients with T315I were excluded (3%); patients with multiple mutations grouped to the known and higher IC50 group Saglio G, et al. ASCO 2008. Abstract 7060. Radich J, et al. European Hematology Association 2008. Abstract 0137.

GIMEMA LAL1205 Protocol R. Foa et Al. EHA 2008 BMS at 70 mg twice a day (total planned treatment is 12 weeks, i.e. 84 days) R. Foa et Al. EHA 2008

Philadelphia-Positive Acute Lymphoblastic Leukemia Patients Already Harbour Bcr-Abl Kinase Domain Mutations at Low Levels at the Time of Diagnosis Mostly of the Philadelphia-Positive Acute Lymphoblastic Leukemia patients who relapsed harbored a T315I mutation on resistant clone at the time of relapse 100 CHR Pt no.1, Ph+ ALL LLM= Low level of mutations T315I LLM T315I Relapsed CHR Pt no.2, Ph+ ALL T315I Relapsed CHR Pt no.3, Ph+ ALL T315I Relapsed CHR Pt no.4, ALL Ph+ T315I Relapsed CHR CCgR Pt no.5, Ph+ ALL T315I Relapsed CHR RTC PCgR Pt no.6, Ph+ ALL T315I Relapsed 100 Stop D CHR Glivec 600 Pt no.7, Ph+ ALL T315I E255 K Relapsed CHR Pt no.8, Ph+ ALL WT Relapsed To assess whether mutations are already present at the time of diagnosis, we analyzed RNA samples from 13 pts with newly diagnosed Ph+ ALL enrolled on the GIMEMA LAL1205. The study pts (males/ females: 6/7; median age: 56 years, range 30-73) included 6 pts who subsequently relapsed with dasatinib-resistant mutations, 2 pts who relapsed without evidence of mutations as assessed by direct sequencing and 5 pts who responded. Sequencing 150 to 200 independent clones for each pt. All pts, irrespective of their subsequent response to dasatinib, we had evidence of aberrant KD sequences due to point mutations (13/13 pts), small insertions (2/13 pts) or small deletions (1/13 pts). CHR Pt no.9, Ph+ ALL CHR Pt no.10, Ph+ ALL 0 1 2 3 6 9 12 15 Months on dasatinib (70 mg BID) Soverini et Al for GIMEMA WP. Personal comunication

Nuovi inibitori della BCR-ABL T315I TK inhibitor Company Phase Target(s) MK-0457 Merck Hold Aurora, Jak2, Abl PHA-739358 Nerviano II Aurora , Abl, XL-228 Exelixis I Aurora, Abl, Src, IGF1R KW-2449 Kyowa Aurora MSerono Merck Serono AZD0530 Astra-Zeneca Aurora, Abl, Src SGX-70430 Sgx/ Novartis Abl Deciphera Decifera Pre clin.-I

Quando effettuare lo screening mutazionale? In caso di resistenza secondaria: - perdita di CCgR con incremento dei livelli di - perdita di MCgR - perdita di HR - progressione ad AP o BC trascritto >1 log

Dove effettuare lo screening mutazionale? Bologna 06/08 Per Gianantonio: Numero e città dei laboratori sono stati aggiornati sulla base degli ultimi dati in ns possesso. GIMEMA CML Working Party

Take home message 1 Point mutation are present on resistant clones 2) Point Mutations, are dependent form AID expression 3) AID+ mutations rate is dependent from DNA repair status 4) Point mutation analysis interferes with TKI selection. by Gimema Network

SNPs relevant to response/resistance to imatinib 400 mg/d What’s new? ….for individual therapy… Imatinib DMEs (CYP3A4, CYP3A5, CYP1A1, CYP1A2, CYP1B1) Imatinib transporters (hOCT1, ABCG2, ABCB1, ABCC3) Imatinib targets (Abl, ARG, PDGFRs, c-kit, c-fms)… L-asparaginasi polimorphisms MTX metabolism polymorphisms De-toxifications gene polymorphisms SNPs relevant to response/resistance to imatinib 400 mg/d

Single nucleotide polymorphisms To identify one or more SNPs in key genes which may serve as predictors of imatinib efficacy in CML pts: 1. response/resistance 2. toxicity to high doses 3. likelihood of mutation selection IM IM IM transporter IM targets CYPs (Ph+ cell) IM metabolites IM CYPs (liver) transporter imatinib metabolites transporter

Genome-wide analysis of genetic alterations and Single-nucleotide polimorphism (SNP) analysis by chip assay… Identificazione di numerose alterazioni del contenuto genico Ph+ ALL samples (n=20) Esperimenti di genotyping mediante SNP array hanno evidenziato come le leucemie acute linfoblastiche ed in questo caso le LAL Ph+ siano caratterizzate da numerose alterazioni del contenuto genico, in particolare le delezioni sono circa il triplo delle amplificazioni. Chromosomes Genetic losses Genetic gains Iacobucci et al. Blood 2008 Diploid genome

Microfluid card or DDchip Gimema WP Italy GIMEMA WP Bologna SNPs Array Service Microfluid card or DDchip 06/08 Per Gianantonio: Numero e città dei laboratori sono stati aggiornati sulla base degli ultimi dati in ns possesso.

Microfluid card or DID chip Fluidigm empty device

Microfluid card or DID chip Fluidigm: Architecture Sample input Control

DID Chip - Fluidigm 9600 Single cell RT-PCR Sample Negative control CD34+ CML Sample Negative control Positive control

Accurate Digital PCR-based Target Quantification 5.2 3.5 1.7 0.9 0.5 0.4 0.2 0.1 Number of target molecules loaded per partition (2500 x 90 picoliter partitions/sample)

T315I Bcr-Abl Positive Detection Experiment by Fluidigm Ddchip 9600 single cell PCR 10 fg T315I plasmid 10 fg T315I plasmid 10 fg T315I plasmid + 10 fg WT 10 fg T315I plasmid + 10 fg WT 10 fg T315I plasmid + 100 fg WT 10 fg T315I plasmid + 100 fg WT 10 fg T315I plasmid + 1 pg WT 10 fg T315I plasmid + 1 pg WT 1 pg WT plasmid only 1 pg WT plasmid only NTC NTC CRR by ARIC@unibo.it

Applicazioni delle “microfliud card” PCR su singola cellula nel liquido cefalorachidiano PCR su singole celllule (9600) da biopsie agomirate T315I Abl mutazione o “point mutations assay” Multiplex of other Abl mutations Studio delle staminali midollari (es. CD34+ CD38 neg, Lin neg) N-Ras- NPM- FLT3- per AML Jak2 mutazione in singole cellula Multiplex per tutte le “leukemia translocations” ……

GIMEMA CML WP

ACKNOWLEDGMENTS Michele Baccarani GIMEMA-CML WP Molecular biology: Simona Soverini Ilaria Iacobucci Sabrina Colarossi Alessandra Gnani Sandra Durante Angela Poerio Marilina Amabile Cytogenetics: Simona Luatti Giulia Marzocchi Nicoletta Testoni Clinical team: Gianantonio Rosti Fausto Castagnetti Francesca Palandri Stefania Paolini Panagiota Giannoulia Giuseppe Saglio Fabrizio Pane Michele Baccarani