2. Profiles in CML: Overview of Practice Challenges Moshe Talpaz, MD Professor Department of Internal Medicine, Hematology-Oncology University of Michigan Comprehensive Cancer Center Ann Arbor, Michigan

3. Chronic Myelogenous Leukemia (CML) Abnormal clonal hematopoietic stem cell disorder, increased proliferation, decreased apoptosis and adhesion Chronic, accelerated, and blastic phases Ph t(9;22) (q34;q11) cytogenetic and BCR-ABL molecular abnormalities

4. CML—A Myeloproliferative Disorder Courtesy of John K. Choi, MD, PhD. NormalCML Chronic-Phase

5. CML Is Linked to a Single Cytogenetic Abnormality—The Philadelphia Chromosome 12345 6781011912 131415161718 19202122 XY Stoll C, et al. Blood. 1978;52:828-838.

6. The Philadelphia Chromosome and BCR-ABL BCR-ABL ABL FUSION PROTEIN WITH TYROSINE KINASE ACTIVITY 22 BCR Ph (or 22q-) 9 9 q+ 1 p210BCR-ABL p190BCR-ABL 2-11 Chromosome 9 c-BCR Chromosome 22 c-ABL 2-11 Exons Introns CML breakpoints ALL breakpoints t(9;22) translocation BCR-ABL gene structure Faderl S, et al. N Engl J Med. 1999;341:164-172. Melo JV. Blood. 1996;88:2375-2384.

7. ABL Chromosome 9 Chromosome 22 wwwwwww BCR wwwww BCR-ABL wwwwwwww q34 wwwwwwwww q11 t(9;22) (q34;q11) BCRABL 210 KD protein Faderl S, et al. N Engl J Med. 1999;341:164-172. Melo JV. Blood. 1996;88:2375-2384.

8. Chronic Phase Accelerated Phase Blast Crisis Advanced Phases The Clinical Course of Untreated CML Faderl S, et al. Ann Intern Med. 1999;131:207-219. Pasternak, G et al. J Cancer Res Clin Oncol. 1998;124:643-660. Median duration 5–6 years Median duration 6–9 months Median survival 3–6 months

9. Incidence and Mortality Of CML Based on current data, median survival is expected to exceed 15–20 years. YearNumber of CasesNumber of Deaths 1997 1 43002400 2007 2 4570 490 1.Parker SL, et al. CA Cancer J Clin. 1997;47:5-27. 2.Jemal A, et al. CA Cancer J Clin. 2007;57:43-66..

10. Survival in Early Chronic Phase CML With permission from Quintas-Cardama A, et al. Mayo Clin Proc. 2006;81:973-988.

11. IRIS Study in Chronic Phase CML—7-Year Update 1106 patients originally enrolled, 553 per arm Estimated overall survival at 7 years: 86% Late-progression events –Kaplan-Meier estimate of event-free survival at 7 years: 81% –Kaplan-Meier estimated rate without accelerated phase/blast crisis at 7 years: 93% Safety –Grade 3/4 events decreased in incidence after years 1–2 –No unique, previously unreported adverse events emerged O’Brien SG, et al. 50th ASH Annual Meeting; December 6-9, 2008. Abstract 186.

12. IRIS Overall Survival (ITT Principle) Imatinib Arm With permission from O’Brien SG, et al. 50th ASH Annual Meeting; December 6-9, 2008. Abstract 186. Abbreviation: ITT, intent to treat. Months Since Randomization Probability of Survival Survival: deaths associated with CML Overall survival Estimated overall survival at 7 years is 86% (94% considering only CML-related deaths)

13. Annual Event Rates—Imatinib Arm KM estimated EFS at 7 years = 81% KM estimated rate without AP/BC at 7 years = 93% Year % with Event Event Loss of CHR, Loss of MCR, AP/BC, Death during treatment AP/BC a Total events (n = 5), including loss of MCR (n = 3) and deaths (n = 2, one of which was coded a progression to AP/BC in a patient in CHR 6 months prior to death). Abbreviations: AP/BC, accelerated phase/blast crisis; CHR, complete hematologic response; EFS, event-free survival; MCR, major cytogenic response; KM, Kaplan-Meier. With permission from O’Brien SG, et al. 50th Annual ASH Meeting; December 6-9, 2008. Abstract 186. a

14. IRIS 7-Year Update—Key Findings Overall survival: 86% Event-free survival: 81% –7% progression to accelerated phase/blast crisis (AP/BC) Complete cytogenetic response (CCR) achieved by 456/553 (82%) patients –17% subsequently lost CCR –3% progressed to AP/BC –2% died from CML –Time to CCR did not correlate with the rate of progression to AP/BC Major molecular response rates and depth of molecular response increased over time While imatinib is efficacious, it does not work for all patients O’Brien SG, et al. 50th ASH Annual Meeting; December 6-9, 2008. Abstract 186.

15. Imatinib Resistance BCR-ABL specific –Mutations  >50 described with variable degrees of impact  ~50% of patients –Amplification or overexpression  ~7-10 % BCR-ABL independent –Cellular pharmacology  Drug import/export –Other pathways  Wnt, notch  Autocrine factors  Lyn (other Src-family kinases) 50%–60% 40%–50% Courtesy of M. Talpaz, MD.

16. 24 Mutations in 19 Amino Acids P-LoopKDA-Loop M244V Q252H Y253F/H E255K/V T315IM351T E355G L387F/M H396R CCAACCAA BCCCBCCC C A A‘ M CCCCMMMCCCCMMM CCCAAALCCCAAAL C A A‘ M A A‘ C A M‘ C C‘ A A‘ M L248V C A‘ C‘ A F317L ACAC F311L A F359C/V C A A‘ CACA A397P T277A M‘ D267G AM V379I G250E/R L324Q C 5 patients had 2 or more mutations (‘). Gorre ME, et al. Science. 2001;293:876-880. von Bubnoff N, et al. Lancet. 2002;359:487-491. Branford S, et al. Blood. 2002;99:3472-3475. Hofmann W-K, et al. Blood. 2002;99:1860-1862. Roche-Lestienne C, et al. Blood. 2002;100:1014-1018. Shah NP, et al. Cancer Cell. 2002;117-125. Hochhaus A, et al. Leukemia. 2002;16:2190-2196. Al-Ali HK, et al. Hematol J. 2004;5:55-60.

17. Role of Kinase Conformation in Imatinib Resistance Point mutations in BCR-ABL kinase domain restricts its ability to adopt an inactive conformation With permission from Schindler T, et al. Science. 2000;289:1938-1942. With permission from Lombardo LJ, et al. J Med Chem. 2004;47:6658-6661. Mutations that directly affect imatinib binding Mutations that affect the conformation required to bind imatinib

18. Dasatinib Inhibits the Growth of Most Imatinib Mesylate—Resistant BCR- ABL—Expressing Ba/F3 Cell Lines In Vitro With permission from Shah NP, et al. Science. 2004;305:399-401. Ba/F3 Bcr-Abl E255K M351T M244V G250E Q252H Q252R Y253F Y253H E255V F317L E355G F359V H396R F486S T315I Parental Ba/F3 cells M351T 0 0.2 0.4 0.6 0.8 1.0 1.2 02.552550 Relative Growth After 48 h of Drug Exposure Concentration of Dasatinib (nM) Wild-type BCR-ABL E255K T315I 0.5

19. With permission from Cortes J, et al. Blood. 2007; 110:4005-4011. Spectrum and Frequency of BCR-ABL Kinase Domain Mutations Developing During Treatment with Imatinib, Dasatinib, and Nilotinib The solid color corresponds to the 1st amino acid change; the broken color corresponds to the 2nd amino acid change if applicable. 0 5 10 15 20 Imatinib Dasatinib Nilotinib G250E Y253H/F E255K V299L F3111 T315I F317L M351T E355G/A F359C/V H396R/P % BCR-ABL Mutation

20. Case 1: AK Neil P. Shah, MD, PhD Assistant Professor Division of Hematology/Oncology UCSF School of Medicine San Francisco, California

21. AK 33-Year-Old Male Referred for recently discovered leukocytosis of 253K noted in blood work performed after he presented to his primary care physician with left shoulder pain and ongoing night sweats Palpable splenomegaly Differential: 3% basophils, immature granulocytes, and 2% blasts Bone marrow biopsy revealed: hypercellular marrow with 4% blasts and an M:E ratio of 10:1, consistent with a myeloproliferative disorder Cytogenetics: t(9;22) in all 20 metaphases analyzed AK has 2 siblings and no other significant medical history

22. Decision Point 1 What is the appropriate first-line treatment for this patient? Hematopoietic stem cell transplantation Imatinib Dasatinib Nilotinib Interferon alpha

23. AK Imatinib 400 mg daily is initiated AK tolerates therapy well, with the exception of peripheral edema, mild nausea, and muscle cramps 1 month later, CBC reveals a complete hematologic response (CHR) 6 months after initiating therapy, AK continues to have a CHR. Bone marrow aspiration is performed, and the t(9;22) translocation is detected in 5/20 metaphases 12 months after initiating therapy, only 2/20 bone marrow metaphases contain the t(9;22) translocation 6 months later, despite continuing to have a CHR, marrow metaphase analysis reveals the t(9;22) translocation in 18/20 metaphases

24. Decision Point 2 What is your next step? Assess patient compliance Do a mutational analysis Increase imatinib dosage Switch to dasatinib or nilotinib Refer for allogeneic stem cell transplantation All of the above

25. AK AK states that he has been very compliant with therapy BCR-ABL kinase domain mutation test is ordered; the imatinib dose is increased to 800 mg daily AK experiences increased fatigue, nausea, and edema on this dose 3 months after imatinib dose escalation –CBC: WBC of 18K with immature forms and 3% basophils –Mutation analysis: E255K mutation in a large proportion of cells

26. (Incomplete) Map of BCR-ABL Kinase Domain Mutations Associated with Clinical Resistance to Imatinib Gorre ME, et al. Science. 2001;293:876-880. von Bubnoff N, et al. Lancet. 2002;359:487-491. Branford S, et al. Blood. 2002;99:3472-3475. Hofmann W-K, et al. Blood. 2002;99:1860-1862. Roche-Lestienne C, et al. Blood. 2002;100:1014-1018. Shah NP, et al. Cancer Cell. 2002;117-125. Hochhaus A, et al. Leukemia. 2002;16:2190-2196. Al-Ali HK, et al. Hematol J. 2004;5:55-60. E255K/V M244V M351T Y253H/F E279K F317L E355G/D F359C/V/D/I H396R/P Q252H/R S417Y E459K/Q F486S E450G/Q M388L G250E/A/F D276G T277A L387F/M V379I A397P E453G/K T315I/N F311L/I V289A L298V L248V E281A L364I G383D E292V x P C A Abbreviations: P, P-loop; C, catalytic domain; A, activation loop. Courtesy of Tim Hughes, MD.

27. Role of Kinase Conformation in Imatinib Binding Imatinib binds to an inactive conformation of BCR-ABL, and is stabilized by a H-bond with Thr315 Imatinib With permission from O’Hare T, et al. Cancer Res. 2005;652:4500-4505. Helix  C Activation loop P-loop

28. Differential Binding of Dasatinib and Imatinib to ABL Kinase With permission from Schindler T, et al. Science. 2000;289:1938-1942. With permission from Lombardo LJ, et al. J Med Chem. 2004;47:6658-6661.

29. How Resistant is the E255K Mutation to Imatinib in Vitro?

30. Resistance to Imatinib of Cells Bearing BCR-ABL Mutations MutationBiologic IC 50 (µM) E355G2.38 M351T4.38 F317L7.50 Y253F8.94 Q252H3.12 G250E>10 T315I>10 E255K>10 WT P2100.60 Measured by determining concentration dependence of normalized viable cell counts. Shah NP, et al. Cancer Cell. 2002;2:117-125.

31. Decision Point 3 How would you treat this imatinib-resistant patient with a E255K mutation? Hematopoietic stem cell transplantation Switch to dasatinib Switch to nilotinib

32. How Resistant is the E255K Mutation to Dasatinib in Vitro?

33. Efficacy of Dasatinib Against Imatinib- Resistant Kinase Domain Mutations in Vitro With permission from Shah NP, et al. Science. 2004;305:399-401. Ba/F3 Bcr-Abl E255K M351T M244V G250E Q252H Q252R Y253F Y253H E255V F317L E355G F359V H396R F486S T315I Parental Ba/F3 cells 0 0.2 0.4 0.6 0.8 1.0 1.2 02.552550 Relative Growth After 48 h of Drug Exposure Concentration of Dasatinib (nM) unmutated BCR-ABL E255K T315I 0.5

34. How Responsive is the E255K Mutation to Dasatinib in Patients?

35. With permission from Stone R, et al. 49th ASH; December 8-11, 2007. Abstract 734. Dasatinib 70 mg Twice Daily in CML-CP Response by Individual Baseline BCR-ABL Mutation Cellular IC 50 (nM) DasatinibImatinibn M244V1.3200017 L248V15009 G250E/V1.81350–390023 Y253F/H/K1.3–10>10,00014 E255K/V5.6–134400–840010 D276G15003 T315I>1000>100003 F317L7.410504 M351T1.193015 E355G4006 F359C/I/V2.212008 L387M2.010002 H396P/R0.6–1.13850–420017 Other30 Complete CyR Partial CyR Complete HR No response Abbreviations: CML-CP, chronic myeloid leukemia-chronic phase; CyR, cytogenetic response; HR, hematologic response.

36. How Is Longer-Term Survival Impacted by Dasatinib in Chronic Phase CML Patients?

37. Dasatinib 100 mg in Imatinib-Resistant and -Intolerant CML-CP Overall Survival Months % Alive 100 80 60 0 100 mg once daily 24-month overall survival = 91% 036912151821242730 Overall survival n12 Months24 Months 100 mg once daily16796%91% 70 mg BID16894%88% 140 mg once daily16796%94% 50 mg BID16896%90% With permission from Shah NP, et al. 50th ASH; December 6-9, 2008. Abstract 3225. Abbreviation: CML-CP, chronic myeloid leukemia-chronic phase.

38. Survival of Patients Who Discontinued Imatinib Study Therapy % Survival (all deaths) 0 10 20 30 40 50 60 70 80 90 100 Months After Stopping Imatinib Study Therapy 0122436 48 60728496 Safety (n = 30) Efficacy (n = 82) Bone marrow transplant (n = 16) Other reason (n = 80) Survival 85% at 5 years after discontinuing study Survival approximately 50% at 5 years after stopping imatinib study drug With permission from O’Brien SG, et al. 50th ASH; December 6-9, 2008. Abstract 186.

39. How Resistant is the E255K Mutation to Nilotinib in Vitro?

40. Activity of Nilotinib on Imatinib-Resistant BCR-ABL Mutants in Vitro Ba/F3 cell proliferation 72-hour proliferation assay with BCR-ABL–expressing Ba/F3 cells Weisberg E, et al. Br J Cancer. 2006;94:1765-1769. O’Hare T, et al. Cancer Res. 2005;65:4500-4505. Weisberg E, et al. Cancer Cell. 2005;7:129-141. Maternal + IL3 BCR-ABL wt M237I M244V L248V G250A G250EG250V Q252H Y253HE255DE255K E255V E255RE275K E276G E281K K285N E292K F311V T315I F317C F317L F317V D325N S348L M351T E355A E355G F359C F359V A380S L387F M388L F468S 0 500 1000 1500 2000 2500 3000 IC50 (nM) on Proliferation Imatinib Nilotinib

41. How Responsive is the E255K Mutation to Nilotinib in Patients?

42. Phase II Nilotinib in CML-CP Response and Progression Based on Mutation IC 50 Patients, n/N (%) MutationCCyRProgressed No mutation35/83 (42)19/83 (23) IC 50 ≤150 nM18/45 (40)14/45 (31) IC 50 >150 nM Y253H0/8 (0)3/8 (38) E255K/V0/8 (0)6/8 (75) F359C/V0/10 (0)9/10 (90) Others14/33 (42)13/33 (39) Response rates and progression rates were similar in patients without baseline mutations and in patients with mutations with IC 50 ≤150 nM for nilotinib Less favorable responses seen in patients with Y253H, E255K/V, and F359C/V –8 of 26 patients were dose escalated to 600 mg BID –Highest rates of progression for E255K/V and F359C/V With permission from Hughes TP, et al. Blood. 2007;110(11). Abstract 320. Blood. 2007;110(11). Abstract 320. Abbreviations: CCyR, complete cytogenetic response; CML-CP, chronic myeloid leukemia-chronic phase.

43. AK Dasatinib 100 mg daily is initiated Therapy is tolerated well except for an occasional mild headache 2 weeks later, AK once again has a normal CBC After 6 months, bone marrow aspiration reveals no karyotypically abnormal cells 12 months later, AK continues to have a complete cytogenetic response on dasatinib

44. Conclusions Loss of response to imatinib is frequently associated with the evolution of resistance-conferring BCR-ABL kinase domain mutations Mutations confer varying degrees of resistance to imatinib, and many are not likely to respond to imatinib dose escalation Dasatinib and nilotinib are effective against most imatinib-resistant BCR-ABL kinase domain mutants in vitro and in patients –Survival data with dasatinib compare favorably with transplant after 2 years Patients with select imatinib-resistant BCR-ABL kinase domain mutations should be preferentially treated with either dasatinib or nilotinib –Consultation with a chronic myeloid leukemia expert is indicated when treating patients with imatinib resistance

45. Case 2: WL Michael J. Mauro, MD Associate Professor Center for Hematologic Malignancies, Knight Cancer Institute Oregon Health & Science University Portland, Oregon

46. WL 45-Year-Old Female Diagnosed with chronic myeloid leukemia (CML) WBC 150k –Left shift, 3% blasts, 3% basophils, platelets 700k Splenomegaly 8 cm below left costal margin Bone marrow shows typical CML chronic phase (CML-CP) –95% cellular, myeloid hyperplasia, 5% blasts –Karyotype: 100% classic t(9:22) Matched unrelated donor option identified; no sibling donor Patient asks about initial treatment options

47. Decision Point 1 What should be the initial therapy for this patient? –Imatinib 400 mg/day –Imatinib 600 mg/day –Imatinib 800 mg/day –Immediate matched unrelated donor stem cell transplantation

48. Imatinib 400 mg/day is the Indicated Dose for Initial Therapy in This Patient Imatinib 400 mg/day Imatinib 600 mg/day Imatinib 800 mg/day Immediate matched unrelated donor stem cell transplantation

49. WL Follow-Up at Month 3 Imatinib 400 mg/day was advised At month 3 –Complete hematologic response with mild leukopenia (WBC 2.5–4k, ANC >1500) –Periorbital edema and myalgias present Peripheral blood qPCR is performed –Results: 1.0 log reduction from baseline How is she doing? Any recommendations, changes?

50. Decision Point 2 Response to 3-month results? –Decrease imatinib dose to 300 mg/day due to reduced WBC –Continue imatinib at 400 mg/day –Increase imatinib dose due to failure –Change to nilotinib or dasatinib due to failure –Move to matched unrelated donor stem cell transplantation

51. CHR at 3 Months is an Adequate Response, Although the Transcript Reduction is Marginal; the Toxicity (Including Myelosuppression) Does Not Warrant Dose Interruption or Reduction Decrease imatinib dose to 300 mg/day due to reduced WBC Continue at 400 mg/day imatinib Increase imatinib dose due to failure Change to nilotinib or dasatinib due to failure Move to matched unrelated donor stem cell transplantation

52. % Achieving MMR 50 60 70 80 90 100 20 30 40 10 0 P <.001 69% 100% 36912151824302127 Months on Imatinib 13% >2 log reduction 1–2 log reduction 0–1 log reduction Hughes T, Branford S. Blood Rev. 2006;20:29-41. Abbreviations: MMR, major molecular response; qPCR, quantitative polymerase chain reaction. 3-Month qPCR Predictive of Ability to Achieve Subsequent MMR—IRIS Trial

53. WL Follow-Up at Month 6 Bone marrow at 6 months shows 80% Ph+ by karyotype She feels well; edema and myalgias manageable CBCs have shown fairly consistent minimal leukopenia (total WBC 3–4k) with ANCs >1500 How is she doing now? Would you change anything at this point? What do you tell her about her response?

54. Decision Point 3 Response to 6-month results? –Continue imatinib at 400 mg/day –Increase imatinib based on failure –Change to dasatinib or nilotinib trial because of failure –Move to matched unrelated donor stem cell transplantation

55. ABL kinase domain mutation testing should be considered Imatinib 800 mg/day Could be Considered Given that the Cytogenetic Response is Adequate (<95% Ph+) but Considered “Suboptimal” (36%–95% Ph+) at 6 Months Continue imatinib at 400 mg/day Increase imatinib to 800 mg/day based on suboptimal response Change to dasatinib or nilotinib trial because of failure Move to matched unrelated donor stem cell transplantation

56. With permission from Baccarani M, et al. Blood. 2006;108:1809-1820. Kinase mutations: high degree = IM resistance, failure; low degree = suboptimal response. Abbreviations: CCyR, complete cytogenetic response; CHR, complete hematological response; CyR, cytogenetic response; HR, hematologic response; MMR, major molecular response; PCyR, partial cytogenetic response; qPCR, quantitative polymerase chain reaction. Failure, Suboptimal Response— European LeukemiaNet Consensus TimeFailureSuboptimal ResponseWarnings Diagnosis——Sokal High; del9q+; clonal evolution 3 moNo HRLess than CHR— 6 moNo CyR (Ph+ >95%) Less than PCyR (Ph+ >35%) — 12 moLess than PCyR (Ph+ >35%) Less than CCyR (0% Ph+) Less than MMR 18 moLess than CCyRLess than MMR— AnytimeLoss of CHR; loss of CCyR Confirmed loss of MMR; evidence of clonal evolution Change in qPCR; Ph (-) clonal cytogenetic abnormalities

57. CyR at 6 months (Ph+): 1%–35%36%–65% 66%–95%>95% (n = 81) (n = 16) (n = 19) Graph: with permission from Druker B, et al. Blood. 2003.102;182a. Abstract 634. Table: with permission from Baccarani M, et al. Blood. 2006;108:1809-1820. Probability of CCyR, EFS by Cytogenetic Response at 6 Months—IRIS Trial % Ph+ at 6 mo CCyR at 12 mo EFS at 42 mo 0% 1%–35% N/A 80% 95% 36%–90% >95% 50% 15% 75% % of Patients with Subsequent CCyR

58. Dasatinib 70 mg BID (n = 101) Imatinib 800 mg (n = 49) CML-CP resistant to imatinib 400–600 mg/day Randomization 2:1 Cytogenetics at 12 weeks Continue therapy or crossover for: Progression Lack of MCyR Intolerance ENDPOINTS: Primary: MCyR and CCyR at 3 months Secondary: rates of MCyR, CCyR, major molecular response; time to treatment failure; progression-free survival Abbreviations: CCyR, complete cytogenetic response; IM, imatinib; MCyR, major cytogenetic response. Kantarjian H, et al. Blood. 2007;110: abstract 736. What If This Was Imatinib Failure? Dasatinib vs Higher Dose IM in CML-CP for Imatinib Failure—Study Schema

59. 53 44 29 33 18 12 0 10 20 30 40 50 60 MCyRCCyRMMR Dasatinib Imatinib Percent P =.017 P =.0025 P =.028 Abbreviations: CCyR, complete cytogenetic response; IM, imatinib; MCyR, major cytogenetic response; MMR, major molecular response. Kantarjian H, et al. Blood. 2007;110: abstract 735. Dasatinib vs High-Dose IM in CML-CP Best Response (Prior to Cross-Over)

60. P =.0033 P =.0562 % PFS 100 80 60 40 20 0 Imatinib 400 mg Dasatinib 03691215182124273033 Months Imatinib 400 mg Imatinib 800 mg Imatinib 600 mg Dasatinib Imatinib 600 mg Imatinib 800 mg Abbreviation: PFS, progression-free survival. With permission from Kantarjian H, et al. Blood. 2007;110: abstract 736. Start-R—PFS by Prior Imatinib Dose (400 & 600 mg) and Intervention (High- Dose Imatinib or Switch to Dasatinib)

61. Suboptimal Response and Failure Early in the Course of Imatinib (6–12 Months) Have Similar Outcomes At 6 months: failure = no cytogenetic response (>95% Ph+) At 6 months: suboptimal response = minor/minimal reduction (35%–95% Ph+) Suboptimal and failure category patients both have significantly inferior likelihood of gaining remission and remaining progression free –Likelihood of complete cytogenetic response  Failure at 6 months (Y/N): 19% vs 92%  Suboptimal at 6 months (Y/N): 64% vs 97% –Progression-free survival:  Failure at 6 months (Y/N): 73% vs 87%  Suboptimal at 6 months (Y/N): 62% vs 91% Marin D, et al. Blood. 2008;112:4437-4444.

62. Suboptimal Response and Failure Early in the Course of Imatinib (6–12 months) Have Similar Outcomes Combine failure and suboptimal into “nonresponder” group –Likelihood of complete cytogenetic response: 39% “nonresponders” vs 96% “responders” –Overall survival: 87% vs 98% –Progression-free survival: 70% vs 92% Similar data for “lumping” suboptimal and failure together at 12 months At 18 months, no statistical difference in overall and progression-free survival: failure vs suboptimal –Note: 18-month response based on molecular findings (MMR or no MMR) Marin D, et al. Blood. 2008;112:4437-4444.

63. WL 1-Year Mark At 6 months, imatinib was increased to 800 mg/d –Bone marrow studies performed at 1 year  Karyotype: 60% Ph+, 40% normal XX  Fish: 55% of cells with fusion signal c/w Ph+ –Blood counts and side effects remain similar How is she doing at this time? Would you change anything at this point? How do you counsel her regarding the results?

64. Decision Point 4 What is your reaction to 12-month results? –Optimal response; no change in therapy –Suboptimal but adequate, no change in therapy –Suboptimal, change therapy –Failure, change to alternate therapy

65. Lack of Major Cytogenetic Response (ie, >35% Ph+) at 12 Months is Considered “Failure” and Therapy Change is Warranted Optimal response; no change in therapy Suboptimal but adequate, no change in therapy Suboptimal, change therapy Failure, change to alternate therapy ABL kinase domain mutation testing should be performed (if not done previously and repeated, if done previously)

66. Rate of Progression to the Accelerated Phase or Blast Crisis on the Basis of Cytogenetic Response After 12 Months or Molecular Response After 18 Months of Imatinib Therapy With permission from Druker BJ, et al. N Engl J Med. 2006;355:2408-2417. B Patients Without Progression (%) A

67. Mechanisms of Resistance to Imatinib BCR-ABL kinase domain mutations 1 –Most common cause for clinical resistance to imatinib (≥50%) –~100 identified, occur at various regions and convey variable degrees (activation loop <phosphorylation loop <<kinase-binding pocket) of imatinib insensitivity –Mutation at position 315 conveys resistance to imatinib as well as dasatinib and nilotinib BCR-ABL amplification/increased expression Ph+ clonal evolution 1. O’Hare T, et al. Blood. 2007;110:2242-2249.

68. Mechanisms of Resistance to Imatinib Decreased drug exposure –Decreased amount/activity of influx protein OCT-1 –Increased P-glycoprotein (ABCB1/MDR1) efflux –  1 acid glycoprotein sequestration Other mechanisms –Src-related (Lyn) kinase overexpression? 1 –Others: HSP70 overexpression; p53 mutations; PI3K/Akt/mTor activation; autocrine GM-CSF based JAK- 2/STAT-5 activation 1. Donato NJ, et al. Blood. 2003;101:690-698.

69. Suggested Common and Differentiating Factors in Choosing Salvage Therapy (Dasatinib or Nilotinib) After Imatinib FactorPotential Issue for DasatinibPotential Issue for Nilotinib Clinical Myelosuppression (for both dasatinib and nilotinib) ? Select cardiac diseases (hypertension, hypercholesterolemia) ? Pre-existing pleural/pericardial disease ? Autoimmune disease ? Rash on prior imatinib or after starting dasatinib Ongoing need for anticoagulation, antiplatelet therapy Pre-existing bleeding disorders ? Prior pancreatic disease and liver disease ? Poorly controlled diabetes Known QTc prolongation a Required concomitant therapy with risk of prolonging the QTc Molecular ABL mutation T315I (for both dasatinib and nilotinib) ABL mutation at positions 317 and 299ABL mutation at positions 253, 255, 359 a Black box warning regarding QT prolongation and sudden death.

70. WL: Month 18 ABL kinase mutation analysis done: NO MUTATION Based on imatinib failure, prior myelosuppression issues, etc, nilotinib chosen, 400 mg BID Marrow at 15 months still shows rising burden of Ph+ cells –Now 80% Ph+ by karyotype, FISH concurs at 80% Repeat ABL kinase domain mutation now shows a T315I mutation, dominant over wild-type Patient asks how she is doing and if any change is needed

71. Nilotinib Phase II CML-CP—Efficacy (19 Month Follow-Up) With permission from Kantarjian H, et al. Blood. 2008;112(11): abstract 3238. # of Pts = Median time to CHR 1 month; MCyR 2.8 months a Patients without CHR at baseline. 207 CHR a 76% 95 Imatinib Intolerant 65% 321 Overall 44% Imatinib Resistant 41% 0 100 80 60 40 20 321 Overall 59% 226 Imatinib Resistant 56% MCyR Imatinib Intolerant 226 95 51% CCyR % of Patients

72. Nilotinib Efficacy According to Baseline BCR-ABL Mutations in CML-CP 3% 4% 5% 15% 24% 45% No Mutation IC 50 ≤ 150 nM Y253H E255K/V F359C/V T315I Others a IC 50 -based grouping b IC 50 ≤150 nM M244V, L248V, G250E, Q252H, E275K, D276G, F317L, M351T, E355A, E355G, L387F, F486S IC 50 >150 nM Y253H, E255K/V, F359C/V IC 50 >10,000 nM T315I a Mutations without available IC 50 data. b Nilotinib IC 50 data cited were established in Ba/F3 in vitro proliferation assay (Weisberg E. Br J Cancer. 2006;94:1765-1769.). With permission from Hochhaus A, et al. Blood. 2008;112(11): abstract 3216.

73. Decision Point 5 What is your response to 15-month results? –Failure, change therapy –Failure, proceed to matched unrelated donor stem cell transplantation

74. Identification of Resistant Disease Harboring a Dominant T315I Mutation Is Grounds for Proceeding to Stem Cell Transplantation and/or a Clinical Trial to Stabilize Disease/Gain Response Failure, change therapy Failure, proceed to matched unrelated donor stem cell transplantation

75. Other Novel Alternative ABL Inhibitors Bosutinib –Spectrum similar to dasatinib, different toxicity profile –Less myelosuppression, pleural effusions; GI toxicity, rash –Activity looks quite promising; being studied in other tyrosine kinase inhibitor resistance and front-line studies INNO-406 –ABL/Lyn inhibitor, expected to have CNS penetration –Spectrum and toxicity appear similar to nilotinib MK0457 –First “t315i” inhibitor; in phase II studies PHA 739358, AP 24534, XL 228, SGX 393 –The next wave of “T315i” inhibitors; in preclinical or phase I

76. Conclusions Moshe Talpaz, MD Professor Department of Internal Medicine, Hematology-Oncology University of Michigan Comprehensive Cancer Center Ann Arbor, Michigan

77. Conclusions The development of imatinib has revolutionized the treatment of CML Imatinib is a specific inhibitor of the BCR- ABL tyrosine kinase

78. Conclusions Some patients may be resistant to or intolerant of imatinib or develop resistance during treatment In many of these patients, resistance is due to mutations in the BCR-ABL gene

79. Conclusions Early monitoring, by cytogenetic and molecular methods, may help define treatment BCR-ABL mutational analysis should be focused on imatinib-resistant patients Determination of BCR-ABL mutation may help define specific treatment

80. Conclusions Nilotinib and dasatinib are second-line BCR-ABL TKIs that have been shown effective in most patients resistant to imatinib TKIs that inhibit BCR-ABL with mutations conferring resistance to all 3 agents are in development

81. Conclusions Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for patients resistant to TKIs