Novel Strategies for the Treatment of AML: Tailoring Treatment For Specific Genetic Subtypes Martin S. Tallman, M.D. Northwestern University Feinberg School of Medicine Robert H. Lurie Comprehensive Cancer Center Chicago, IL

Topics To Address Introduction and epidemiology Overview of current therapy Distinguishing genetic subtypes Novel strategies for specific genetic subtypes Future Directions

Introduction New patients/deaths in 2004: 12,000/9,000 Median age of AML: 68 years Heterogeneity in genetics and clinical manifestations Outcome varies by prognostic factors

1Douer Blood, 1996, Br J Haematol, 2003; 2Nakase Leukemia, 2000 Epidemiology Therapy-related (alkylating agent or topo II active agents) Evolving from MDS or MPD Congenital chromosomal instability syndromes Higher frequency of APL in Latin Americans1 Higher frequency of t(8;21) in Japan2 1Douer Blood, 1996, Br J Haematol, 2003; 2Nakase Leukemia, 2000

Current Treatment Results In Younger Adults STUDY N CR % ED % OS % (3-5 yr) CALGB 474 72 9 34 GAMLCG HOVON 535 253 74 77 11 7 39 38 ALFA 345 82

Current Treatment Results In Older Adults STUDY N CR % ED % OS % (2-7 yr) CALGB 388 52 25 15 ECOG 348 42 17 10 SWOG 328 43 7 19 MRC 1,314 55

Prognostic Factors Age Intensity of postremission therapy (younger adults) Cytogenetics (distinguish fav- intermed- and unfav-risk groups)

Overall Survival by Cytogenetic Group Years After Entering Study Favorable 121 53 55% Intermediate 278 168 38% Unfavorable 184 162 11% Estimate At Risk Deaths at 5 Years 100 80 60 Cumulative Percent 40 20 Heterogeneity of 3 Groups: p<.0001 2 4 6 8 Years After Entering Study Slovak Blood, 2000

Prognostic Factors Molecular Markers Transmembrane transporter proteins which confer multidrug resistance (MDR1) Mutations in or overexpression of specific genes-unfav. prognosis: WT1, BAX, BCL-2/BAX, BAALC, EVI1, KIT, FLT3, MLL, ERG; fav. prognosis: C/EBP, NMP1 Expressed in cells from pts with normal karyotype

Outcome For AML with Fav-risk Cytogenetics CR % DFS % OS % CBF 3731 3122 86 88 40 47 43 50 1613 96 52 59 APL 4634 91 85 2985 94 90 87 1Castaigne Blood, 2002; 2Appelbaum Proc ASCO, 2005; 3Marcucci J Clin Oncol, 2005; 4Sanz Pro ASCO, 2005; 5Lo Coco Blood, 2004

Outcome For AML in Older Adults with Unfavorable Cytogenetics CR% OS% (5-yr) MRC1 145 26 2.0 ECOG2 61 23 2.5 1Grimwade Blood, 2004; 2Rowe Blood, 2004

Current Therapeutic Strategies Induction with anthracycline 45-60 mg/m2/day for 3 days + cytarabine 100 mg/m2/day for 7 days c.i. Multiple cycles of high-dose ara-C consolidation No maintenance (except APL)

Strategies To Improve Outcome Dose intensification (anthracyclines) Alternative chemotherapy Priming with growth factors New agents

Daunorubicin Dose Intensification Study Dauno Dose CR% FHCRC1 70 mg/m2 80 ALFA2 80 mg/m2 76 CALGB3 90-95 mg/m2 1Appelbaum Ann Int Med, 1984; 2Castaigne Blood, 2003; 3Kolitz Blood, 1998

ECOG Priming Studies Recruiting Leukemic Cells into Cell Cycle GM-CSF Placebo CR 38% 40% Induction mortality 26% 17% OS, median 5.3 mo. 8.5 mo. DFS, median 6.9 mo. 5.1 mo. Rowe Blood, 2004

Priming With Growth Factor (GF) in Younger Patients with Intermediate Cytogenetics Study N CR % DFS % OS % GF/No GF HOVON1 464 87/86 45/33 (p=.006) 45/35 (p=.02) ALFA2 259 91/87 50/35 (p=.05) 56/47 (p=.07) 1Lowenberg NEJM, 2003; 2Thomas Blood, 2005

Specific Genetic Subtypes of AML To Which Therapy Can Be Tailored APL CBF AML CD33 pos AML AML with FLT3 mutatations AML with c-kit mutations AML with MLL PTD

Curative Strategies in APL Induction: ATRA + anthracycline-based chemotherapy Consolidation: Anthracycline-based chemotherapy for 2-3 cycles to molecular negativity Intermediate-dose ara-C for high-risk Maintenance: ATRA +/- low-dose chemo for 1-2 years; ? Role in PCR neg after consol Mol. Monitoring: RT-PCR from PB every 3-6 months for 2-3 years, prob for high-risk only Relapse: Arsenic followed by ASCT (allo if PCR pos) (consider prophylactic IT therapy)

GIMEMA AIDA 2000 Overall Survival N=338 3 years: 87% Survival Years 1.0 .75 N=338 3 years: 87% Survival .50 .25 .5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Years Courtesy of F. Lo Coco

Advances in Therapy in APL Paradigm for tailoring therapy to specific genetic subtype Study Group Contribution No. Am. Intergroup Maintenance PETHEMA Elimination of ara-C PETH/GIMEMA ATRA in consolidation ATO in consolidation GIMEMA Gemtuzumab Shanghai ATRA + ATO Iran ATO single agent Will chemotherapy be eliminated?

Arsenic Trioxide for Rel/Ref APL Pilot and US Multicenter Trial Overall and Relapse-Free Survival 100 80 60 Probability (%) 40 OS - 1st Relapse OS - > 1st Relapse 20 RFS - 1st Relapse RFS - > 1st Relapse 1 2 3 4 Years Soignet J Clin Oncol, 2001; Douer, Tallman J Clin Oncol, 2005

Randomized Trial of Arsenic Trioxide and ATRA in Untreated APL < .02* 11.1 26.3 Relapse (%) (median follow-up 18 months) < .01 119 32 6.7  PML/RARα (fold) < .05 25  5 35  3 40  10 Time to CR (days) NS 95 90 (%) CR — P 21 ATRA + ATO 20 ATO ATRA N Induction/ maintenance therapy Reference Shen ZX, Shi ZZ, Fang J, et al. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci U S A. 2004;101:5328–5335. *All patients also treated with consolidation chemotherapy and 6-MP + MTX as maintenance Shen Proc Natl Acad Sci,U S A, 2004

Disease-Free Survival by Treatment Group 1.0 0.8 0.6 Probability 0.4 ATRA + arsenic trioxide (n = 20) Reference Shen ZX, Shi ZZ, Fang J, et al. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci U S A. 2004;101:5328–5335. 0.2 arsenic trioxide (n = 18) ATRA (n = 19) 4 8 12 16 20 24 28 32 Time of Follow-up (months) Shen PNAS, 2004

Induction With Single-Agent Arsenic Trioxide: Untreated APL Study N CR (%) PCR neg (%) Postremission therapy Zhang – China1 124 88 NR Chemotherapy Ghavamzadeh – Iran2,3,4 111 86 92 ATO  1 Chandy – India5,6,7 References Zhang P. The use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia. J Biol Regul Homeost Agents. 1999;13:195–200. Ghavamzadeh A, Alimoghaddam K, Aghdami N, et al. Treatment of new cases of acute promyelocytic leukaemia by arsenic trioxide. Presented at: 39th Annual Meeting of the American Society of Clinical Oncology; May 31–June 3, 2003; Chicago, Ill. Abstract 2285. Ghavamzadeh G, Ghavamzadeh A, Alimoghaddam K, et al. Treatment of new cases of acute promyelocytic leukaemia by arsenic trioxide. Presented at: 9th Annual Congress of the European Hematology Association; June 10–13, 2004; Geneva, Switzerland. Abstract 093. George B, Mathews L, Balasubramanian P, Shaji RV, Srivastava A, Chandy M. Molecular remission with arsenic trioxide in patients with newly diagnosed acute promyelocytic leukemia. Haematologica. 2004;89:1266–1267. George B, Mathews L, Balasubramanian P, Shaji RV, Srivastava A, Chandy M. Treatment of newly diagnosed patients with acute promyelocytic leukemia using intravenous arsenic trioxide. Presented at: 9th Annual Congress of the European Hematology Association; June 10–13, 2004; Geneva, Switzerland. Abstract 085. 51 80 95 ATO  6 4Ghavamzadeh Ann Oncol, 2005 5George Haematologica, 2004 6George European Hematol Assoc, 2004 7Mathews Blood, 2005 (abstr) 1Zhang J Biol Regul Homeost Agents, 1999 2Ghavamzadeh ASCO, 2003[abst] 3Ghavamzadeh EHA, 2004[abst]

North American Intergroup Trial Induction Consol #1 Consol #2 Maint ATRA 6-MP MTX ATRA Dauno Ara-C ATO ATRA Dauno Untreated APL CR No ATO ATRA 7 days, QOW Tests arsenic as early consolidation and 2 maint regimens

ATRA Daunorubicin Ara-C Gemtuzumab Ozogamicin Proposed North American Intergroup Study-Phase III Low- and Intermediate-Risk Induction Consol #1 Consol #2 Maint ATRA 6-MP MTX neg ATRA Daunorubicin Ara-C ATRA Daunorubicin PCR ATO OBS pos Gemtuzumab Ozogamicin HSCT Tests benefit of maint in PCR neg patients

Proposed North American Intergroup Study-Phase II High-Risk Induction Consol #1 Consol #2 Consol #3 Maint ATRA ATO Gemtuzumab Ozogamicin Gemtuzumab Ozogamicin ATRA 6-MP MTX ATRA Daunorubicin ATO Tests benefits of 3 nonchemotherapy agents in induction

Incidence of CBF AML by Age Total inv(16) t(8;21) P-value 16-30 27% 43% 57% 0.056 31-40 26% 55% 45% 41-55 59% 41% 56-65 9% 50% 66-83 10% 62% 38% Appelbaum FR, Kopecky KJ, Tallman MS, et al (submitted)

Outcome of t(8;21) AML Study N CR OS Postrem Palmeri 17 82% 79% HiDACx3 Nishii 85 95% 52% No I-or HDAC Baer 29 90% 45% >/=1 HiDAC Marcucci 139 89% 46% S-, I-, HiDAC Appelbaum Schlenk 174 191 85% 87% 65% Variable HiDAC Palmeri Leuk Res, 2002; Nishii Leukemia, 2003; Baer Blood, 1997; Marcucci J Clin Oncol, 2005; Appelbaum (submitted); Schlenk J Clin Oncol, 2004

Outcome of inv16/t(16;16) AML Study N CR OS Postrem Marcucci 164 87% 54% S-, I-HiDAC Delaunay 110 93% 58% variable Appelbaum 196 89% 50% Schlenk 201 60% HiDAC Marcucci J Clin Oncol, 2005; Delaunay Blood 2003; Appelbaum (submitted); Schlenk J Clin Oncol 2004

Overall Survival by Abnormality Disease-Free Survival by Treatement Outcome for CBF Leukemias 100 Overall Survival 80 5Year N Deaths Estimate All Patients 370 197 48% 60 Percent 40 20 5 10 15 20 25 Years Overall Survival by Abnormality Disease-Free Survival by Treatement 5Year N Events Estimate FA 53 20 61% HCT 31 12 61% HDAC 44 23 50% Other 136 96 31% Years 100 Percent 40 60 80 20 5Year N Events Estimate inv 16 196 97 50% t(8;21) 174 100 45% 10 25 5 15 100 80 60 Percent 40 20 5 10 15 20 25 Years After Start of Post-CR Regimen Appelbaum et al.(submitted)

CALGB Protocol 19808: Induction +/- MDR Modulation Followed by Cytogenetic Risk-Adapted Intensification in Younger Adults HiDAC Consolidation Therapy x 3 ADE Favorable Cytogenetics Obs. HiDAC G-CSF VP-16 If able to Receive PSCT BU/VP-16 PSCT Stem Cell Mobilize Unfavorable Cytogenetics IL-2 If unable to Receive PSCT HiDAC Consolidation Therapy x 2 ADEP VP-16 HiDAC G-CSF Tests HiDAC for fav. Cyto and IL-2 post-ASCT

Specific Genetic Subtypes Are Heterogeneous: t(8;21) CD56 expression may confer poor prognosis Associated trisomy 4 is a distinct subtype with poor prognosis Receptor tyrosine kinase pathway mutations in 49% and confer poor prognosis Baer Blood, 1997; Nishii Leukemia 2003; Nanri Leukemia 2005

Valproic acid, SAHA, depsipeptide Deoxyadenosine analogs New Agents Class Agent Target Antibodies Gemtuzumab CD33 MDR inhibitors PSC833, Zosuquidar P-gp FT inhibitors Tipifamib Lamin A, HJJ-2 FLT3 inhibitors PKC-412, CEP-701, MLN518, SU11248 FLT3 ITD HDAC inhibitors Valproic acid, SAHA, depsipeptide HDAC Antiangio agents Bevacizumab VEGF Apoptosis inhibitors Genasense BCL-2 Deoxyadenosine analogs Nucleoside analogs Clofarabine Tiazofurin DNA IMPDH

Gemtuzumab Ozogamicin Structure hP67.6 - humanized anti-CD33 antibody Blue- linker Periwinkle - calicheamicin hP67.6 HN S O NHN Me Approved for adults > 60 in first relapse Induces CR + CRp in ~ 30%1,2 Rare VOD/SOS if allo < 3.5 mo.3 S H HO O OCH 3 NH N Et OH CH HN I 1Sievers J Clin Oncol, 2001; 2Larson Leukemia, 2002; 3Wadleigh Blood, 2003

Gemtuzumab Ozogamicin CR MD Anderson1 Single Agent 8% With IL-11 36% Northwestern2 27% EORTC3 23% Followed by Chemo 35% 1Estey Blood, 2002; 2Nabhan Leukemia Res, 2004; 3Amadori Blood, 2004 [abstr]

Gemtuzumab Ozogamicin in Induction Study Chemotherapy GO Dose CR DeAngelo Dauno/Ara-C 6 mg/m2 d4 83% Kell DAT or FLAG-ida 3 mg/m2 d1 85% Prompts SWOG Phase III trial of chemotherapy +/- GO d4 DeAngelo Blood, 2003 (abstr); Kell Blood, 2003

Daunorubicin 45 mg/m2/day Daunorubicin 90 mg/m2/day ECOG Protocol E1900: Dose Intensification in Induction and Gemtuzumab Ozogamicin (GO) pre-ASCT in Younger Adults with Untreated AML Daunorubicin 45 mg/m2/day + Cytarabine High Risk Allogeneic HSCT CR Autologous ASCT HiDAC x 2 PBSH after 2nd course Daunorubicin 90 mg/m2/day + Cytarabine GO 6 mg/m2 IV day 1 Tests anthracycline dose intens and GO as in vitro purge pre- ASCT

Inhibition of Multidrug Resistance P-gp-mediated MDR plays major role in clinical resistance to chemotherapy P-gp correlates inversely with CR 71% of patients > age 60 express moderate to high P-gp1 Major limitation is alteration in pK of concomitant chemotherapy 1Leith Blood, 1994

MDR Modulation Studies in AML De novo > 60 years Group Modulator Regimen Outcome CALGB1 PSC-833 ADE+PSC-833 Closed due to toxicity HOVON2 DA+PSC-833 DFS, OS not improved 1Baer Blood, 1999; 2 Van der Holt Blood, 2004 (abstr)

Inhibition of Multidrug Resistance LY335979 (Zosuquidar) Selective P-gp inhibitor with high affinity1 In vitro conc of 50-100nM circumvent P-gp-mediated resistance 2,3 Does not alter PKs of co-administered drugs2 Phase II trial in poor-risk AML CR or mCR 42%4 ECOG phase III trial 1Sato Cancer Res, 1991; 2Dantzig Cancer Res, 1996; 3Green Biochem Pharmacol, 2001; 4Cripe Blood, 2001 (abstr)

ECOG Protocol E3999: Dauno + Cytarabine +/- Zosuquidar in Older Adults Induction Consolidation I Daunorubicin Cytarabine Zosuquidar E V A L U T E CR or MR Cytarabine Consolidation II Daunorubicin Cytarabine Placebo Daunorubicin Cytarabine Zosuquidar Daunorubicin Cytarabine Placebo Tests novel MDR modulator

Ras Ras mutations Activating mutations in 10-30% of AML1,2 Frequent in t(3;5) and inv(16)3 Active inhibition of farnesyl transferase (FT), inhibits ras protein Inhibitors of FT active in AML4,5 Gene expression profiling may predict response to Tipifarnib6 1Radich Blood, 1990; 2Neubauer Blood, 1999; 3Bowen Blood, 2005; 4Karp Blood, 2001; 5Lancet Blood, 2002; 6Raponi Blood, 2005

Tipifarnib-Phase II Trial in Untreated High-risk AML/MDS Med age 74 yrs (46-85) CR in 21% Med CR dur 5-8 mo. (1.5-11+) Gr. 4 neutropenia 13% Inhibition of FT in 74% of samples Encouraged US Intergr Phase II trial of 2 different doses/schedules in older adults Lancet Blood, 2003 (abstr)

S0432: US Intergroup Phase II Study Zarnestra for Previously Untreated AML in Patients > Age 70 Randomization Arm 1 Arm 2 Arm 3 Arm 4 Zarnestra 600 mg bid x 21 days q 28 days Zarnestra 600 mg bid x 7 days every other week q 28 days Zarnestra 300 mg bid x 7 days every other week q 28 days

AML With Mutant RAS and Cytarabine Intensification wtRAS mutRAS LoDAC HDAC Yrs to relapse 0.8 1.1 0.6 NR RR 82% 71% 100% 45% Neubauer ASCO, 2005

Prognostic Significance of FLT3 ITD 100 80 60 Survival (%) ITD- n=627 44% 40 ITD+ n=227 32% 20 P<0.001 1 2 Years 3 4 5 Kottaridis Blood, 2001

Tyrosine Kinase Inhibitors PKC412 Staurosporine-derived, targets PKC, KDR, VEGF-R2, PDGFR, c-KIT, FLT3 Phase II study1 – 28 pts with FLT3 mutation, HI in 50%, but no CR or PR CEP-701 Indolcarbazole alkyloid-targets TrkA, VEGFR, FLT3 Phase I/II study2-14 pts with FLT3 mutation, biologic activity in 35%, but no CR or PR 1Estey Blood, 2003; Smith Blood, 2004

FLT3 Inhibitors and Chemotherapy in AML CEP-7011 N=34 in first relapse MEC or HiDAC +/- CEP-701 10/17 CR with CEP-701 PKC4122 N=19 de novo Dauno + Ara-C + PKC412 CR 71-75% 100% FLT3 mut 62% FLT3 wt 1Lewis, Blood, 2005 (abstr); 2 Stone, Blood, 2005 (abstr)

1Stone Blood, 2005; Levis, Blood 2005 FLT3 Inhibitors In vitro data strong Modest clinical activity-few, if any, CRs How best to develop? Focus on combinations with chemotherapy1, 2; Phase III US Intergroup trial planned (dauno + ara-C +/- PKC412) Will combining agents with in vitro activity, but modest clinical activity, be effective? 1Stone Blood, 2005; Levis, Blood 2005

Genasense (Oblimersen Sodium) Bcl-2 associated with poor outcome in AML Phosphorothioate 18-mer antisense oligonucleotide directed at first 6 codons of Bcl-2 Genasense + dauno/cytarabine in high-risk de novo pts 26 pts, med. age 67 CR in 45.4%; no unexpected toxicity CALGB phase III trial underway Marcucci Blood, 2003

CALGB 10201: Daunorubicin + Cytarabine +/- G3139 (Genasense) in Older Adults High-Dose Cytarabine CR CR Cytarabine + Daunorubicin High-Dose Cytarabine Remission Induction Consol I & II Tests bcl-2 antisense strategy

Clofarabine Intentionally designed to incorporate the favorable properties of fludarabine/cladribine Multiple mechanisms of action Inhibits DNA replication and repair Disrupts mitochondrial function leading to apoptosis Active in both dividing and non-dividing cells

Clofarabine Trials in AML Regimen N Population CR/OR % Clo 31 Rel/Ref 01 28 Untreated 59/752 Clo +IDAC 25 22/383 Clo + IDAC 60 52/604 Clo + LoDAC 32 59/625 1Kantarjian Blood, 2003;2Faderl Blood, 2005; 3Burnett Blood, 2005; 4Faderl Blood, 2005; 5Faderl Blood, 2005

Proposed ECOG Trial HLA-iden sibling Mini-MUD alloSCT CClofarabine Low-dose cytarabine CClofarabine Low-dose cytarabine Age 60-69 R CCytarabine 1.5 mg/m2 IV Q12 d1-6 DDaunorubicin Cytarabine C R RESPONSE CCytarabine 1.5 g/m2 IV Q12 d1-3 Age 70-79 R CClofarabine Clofarabine Can we eliminate conventional chemotherapy in older adults? No CR Off Study

Specific Genetic Subtypes Of AML To Target In The Very Near Future C-KIT AML: Imatinib, Dasatinib, FLT3 inhibitors MLL PTD AML: HDAC and DMT inhibitors Bcr-abl pos AML: IMP dehydrogenous inhibitor (Tiazofurin ) Whitman Blood, 2005; Malek Leuk Res, 2004

Other Active Agents Awaiting Assignement To Specific Genetic Subtypes Choretazine (VNP40101M): sulfonylhydrazine alkylating agent1 Tandutinib: small molecule inhibitor or type II receptor tyrosine kinases2 XL99: inhibits multiple receptor kinases(VEGFR-2, PDGFR-alpha, PDGFR-beta, c-KIT, SRC, FGFR1, FLT4, FLT3) Low-dose decitabine3 Arsenic plus low-dose ara-C4 1Giles Blood 2005 (abstr); 2Deangelo Blood, 2004 (abstr); 3Lubbat Blood, 2005 (abtsr); 4Robosz Blood, 2005(abstr)

Future Directions Gene expression profiling to determine signatures characteristic of specific genetic subtypes, identify cooperating mutations and perturbed pathways and predict treatment response Further characterization of normal karyotype AML and adverse karyotype AML Increased collaboration among cooperative groups to study myriad of new agents to tailor to specific genetic subtypes