1. Lenalidomide : un nuovo farmaco nell’armamentario terapeutico della LLC Alessandra Ferrajoli Department of Leukemia The University of Texas M.D. Anderson Cancer Center Houston, TX, USA

2. Lenalidomide Thalidomide analogue Immunomodulatory drug (IMiD) 3-(4-amino-1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2,6-piperidinedione

3. del(5q) MDS Inhibition of del(5q) erythroid progenitors (↑SPARC and actinin) 1 Multiple Myeloma Changes in BM microenviroment, ↑apoptosis, ↓cell adhesion 2 CLL (??)  immunological synapses formation 3  T-cell and NK-cells function 4 Interference with the microenvironment  pro-survival cytokines (TNF- , VEGF, IL-8, IL-6) Pro-apoptotic effect 1 Pellagatti et al. PNAS 2007; 101:950-68. 2 Mitsiades et al. J Cell Biochem 2007; 104:11406-11. 3 Ramsey G. et al. J. Clin. Invest 2008; 118: 2427-2437 4 Hayashi T et al. Br J. Haem. 2005 128:192-203 Lenalidomide: Mechanisms of Action

4. Lenalidomide in CLL: Background TNF-  and VEGF contribute to CLL proliferation: –High levels of VEGFR2 are associated with decreased survival 1 –TNF-  levels are elevated in the plasma of patients with CLL and TNF-  is a prognostic factor in CLL 2 TNF-  = tumour necrosis factor  ; VEGFR2 = vascular endothelial growth factor receptor 2 (KDR). 1. Ferrajoli A, et al. Clin Cancer Res. 2001;7:795-9. 2. Ferrajoli A, et al. Blood. 2002;100:1215-9.

5. Trial Design: Lenalidomide in Recurrent/Refractory CLL Phase II Previous treatment (purine analogue- based chemotherapy) Lenalidomide 10 mg/day –titrate up by 5 mg every 28 days to 25 mg daily (minimum 5 mg, days 1–21) Treatment continued until progression Ferrajoli A, et al. Blood. 2008;111:5291-7.

6. CharacteristicN = 44 Median age (range), years64 (49–86) Number (range) of prior therapies5 (1–15) Serum  2 -microglobulin (range), g/ml 4.3 (1.6–10.1) Rai disease stage III or IV, %45 Fludarabine refractory, n (%)12 (27) Alkylating-agent refractory, n (%)11 (25) Bulky lymphadenopathy, n (%)17 (39) Unfavourable cytogenetic features, n (%) del(11q23)18 (41) del(17p)8 (18) Unmutated V H, n (%)*29 (66) *Not tested, n = 11. V H = immunoglobulin variable heavy chain. Lenalidomide in Recurrent/Refractory CLL: Patient Characteristics Ferrajoli A, et al. Blood. 2008;111:5291-7.

7. Response Patients, n (%) (N = 44) Complete response3 (7) Nodular partial response1 (2) Partial response10 (23) Stable disease*11 (25) Progressive disease ‡ 19 (43) *Continued lenalidomide treatment. ‡ Including 2 early deaths. ORR 32% Responses evaluated according to 1996 NCI-WG guidelines Lenalidomide in Recurrent/Refractory CLL: Responses Ferrajoli A, et al. Blood. 2008;111:5291-7.

8. Adverse events, n (%) Grade 1 or 2Grade 3 or 4 Hematological Neutropenia35 (11)135 (41) Thrombocytopenia52 (16)51 (15) Anemia63 (19)9 (3) Non-hematological Fatigue71 (22)4 (1) Diarrhea44 (13)6 (2) Rash43 (13)– Tumour flare**31 (10)2 (2) **Occurred in 9 of 17 patients with lymph nodes > 5 cm and in 4 of 27 patients with lymph nodes < 5 Lenalidomide in Recurrent/Refractory CLL: Adverse Events per Course (N=333) Ferrajoli A, et al. Blood. 2008;111:5291-7.

9. Awais A, et al. Clinical Infect Dis. 2008, 46: 69-71. Rapid Resolution of Chronic Skin Infection During Lenalidomide Therapy for CLL Infection with Mycobacterium marinum

10. Lenalidomide in Recurrent/Refractory CLL: T-cell Counts Lymphocytes (× 10 3 /  l) CD3 cells (× 10 3 /  l) 0 10 20 30 40 50 60 BaselineMonth 3Month 6 0 0.5 1.0 1.5 2.0 2.5 3.0 BaselineMonth 3Month 6 Ferrajoli A, et al. Blood. 2008;111:5291-7.

11. Lenalidomide in CLL: Plasma Cytokine Levels (N=12) 0 10 20 30 40 50 Baseline7days 28 days IL-10 0 20 40 60 80 100 Baseline7days 28 days IFN-  0 100 300 500 Baseline7days 28 days Basic FGF 0 5 10 15 20 25 Baseline7days 28 days IL-1  0 50 100 150 Baseline7days 28 days VEGF TNF-  0 10 20 30 Baseline7days 28 days 0 5,000 10,000 15,000 20,000 Baseline7days 28 days IL-2R 0 5 10 15 20 25 Baseline7days 28 days IL-2 0 20 40 60 80 100 Baseline7days 28 days IL-8 SD + NR PR + CR 0 2,000 4,000 6,000 8,000 Baseline7days 28 days TNF-RI IL-6 0 20 40 60 80 Baseline7days 28 days CR = complete response; FGF = fibroblast growth factor; IFN = interferon; IL = interleukin; NR = no response; PR = partial response; SD = stable disease; TNF-R1 = TNF receptor 1. Cytokine concentration (pg/ml) Ferrajoli A, et al. Blood. 2008;111:5291-7.

12. Patients (%) Overall response CR* PR SD 57.5 18 40 14 Lenalidomide in Recurrent/Refractory CLL: Roswell Park Experience Chanan-Khan A, et al. Presented at XII IWCLL, 2007. Chanan-Khan A, et al. J Clin Oncol. 2006;24:5343-49. *Molecular remission occurred in 3 patients with a CR. Total number of patients accrued = 45 Evaluable patients = 32 Non-evaluable patients = 13 Response (intent-to-treat analysis) Lenalidomide 25mg/day Day 1–21

13. Lenalidomide in Recurrent/Refractory CLL: Conclusions Responses seen with an immunomodulatory agent Patients treated with oral therapy on a daily basis Time to response is prolonged, best responses seen after 6-9 months The toxicity profile is manageable and the treatment can be safely given as outpatient Myelosuppression is frequent, requires dose reduction

14. Lenalidomide as Initial Treatment of Elderly CLL: Background Active in relapsed CLL 1,2 Treatment-naïve patients may benefit from an immunostimulatory agent No consensus on best treatment for elderly patients Oral agent 1 Chanan-Khan AA et al. J Clin Oncol 2006 2 Ferrajoli A et al. Blood 2008

15. Lenalidomide in Elderly CLL: Study Design Phase II, 60 patients (11/07-4/09) Untreated and symptomatic Age ≥ 65 yrs Creatinine <2 mg/dL, bilirubin<2 mg/dL Zubrod/WHO performance status 0-2 Response assessment at end of cycle 3, cycle 9 and every 6 cycles

16. Lenalidomide in Elderly CLL: Doses and Schedule Lenalidomide - 5 mg orally daily x 2 cycles (56 days) -  by 5 mg/cycle (28 days)  max 25 mg daily - Treatment continued until progression Response assessment at 3, 9, 12 and 15 months Allopurinol 300 mg d 1 -14 No antibiotic or anti-viral prophylaxis required No DVT prophylaxis required

17. Lenalidomide in Elderly CLL: Patient Characteristics (N=60) CharacteristicValue Median age (range), yrs71 (66-85) Rai stage III/IV, N (%)18 (30) 11q-, N (%) 17p-, N (%) 14 (23) 6 (10) Median β2M (range), mg/dL4.3 (2-10) Unmutated V H, N (%) ZAP-70+, N(%) 33/55 (55) 31/48 (52) Updated IWCLL 2009

18. Lenalidomide in Elderly CLL: Responses Time on therapy N. Evaluable Cycle 3 59 N (%) Cycle 9 58 N (%) Cycle 15 43 N (%) Cycle 21 25 N (%) CR01 (2)2 (5)1 (4) Nod. PR / CRi06 (10)6 (14)4 (16) PR24 (41)25 (41)17 (40)9 (36) Overall Response24 (41)32 (53)25 (58)14 (56) Stable disease26 (44)5 (9)-- Discontinued6 (10)17 (29)17 (40)11 (44) Progression3 (5)4 (7)1 (2)- Updated IWCLL 2009

19. Lenalidomide in Elderly CLL: Absolute and T-cell (CD3+) Lymphocytes p<0.001 N=27 Updated IWCLL 2009

20. Lenalidomide in Elderly CLL: CD4 and CD8 Lymphocytes % peripheral blood lymphocytes N=27 p<0.001 Updated IWCLL 2009

21. Lenalidomide in Elderly CLL: Serum Immunoglobulin G Levels Pre-treatment3915 Cycles of therapy 1400 1200 1000 800 600 400 200 0 Immunoglobulin G (mg/dl) p<0.001 Updated IWCLL 2009

22. Bone marrow lymphocyte populations demonstrate an increase in T cells accompanied by a steady decline in CLL cells during therapy Pre- treatment 391521 p<0.001 % bone marrow aspirate cells N=14 Non-CLL Updated IWCLL 2009

23. Lenalidomide therapy induces a rise in CD5+ T-lymphocytes in the bone marrow % bone marrow aspirate cells p<0.001 N=27 Updated IWCLL 2009

24. Proportions of CD4+ and CD8+ T- lymphocytes and NK-cells in BM of 6 patients after 15 cycles of lenalidomide % bone marrow lymphocytes Updated IWCLL 2009

25. Lenalidomide in Elderly CLL: Regulatory T Cells in Blood Courtesy of B-N Lee

26. Lenalidomide in Elderly CLL: Overall Survival and Time to Treatment Failure

27. Lenalidomide in Elderly Patients: Hematological Toxicities (N=60) Grade > 3 Hematologic N pts% courses Neutropenia4735 Anemia3<1 Thrombopenia114 Updated IWCLL 2009

28. InfectionsN pts (%) Neutropenic sepsis pneumonia2 (3) Pneumonia5 (8) FUO (Gd. 3)3 (5) Neutropenic fever2 (3) Minor infections40 (5*) * N ( %) courses Lenalidomide in Elderly Patients: Infectious Complications Updated IWCLL 2009

29. Lenalidomide in Elderly Patients: Grade 1-2 Toxicities Other Grade < 3N courses (%) Tumour flare38 (5) Rash/itch77 (10) GI – Constipation*40 (5) GI – Diarrhea44 (5) Neuropathy11 pts ‡ Fatigue † 71 (9) * Gd 3 n=1, † Gd 3 n=2, ‡ Median dur’n 287d. Updated IWCLL 2009

30. Lenalidomide in Elderly Patients: Tumor Flare

31. Lenalidomide in Elderly Patients: Conclusions Lenalidomide well tolerated in elderly pts with CLL Rapid decrease in lymphocyte count After 15 cycles: OR 58%; CR 5% Response rate improves with continued treatment (2 flow negative CR) Myelosuppression is common No deaths or increased rate of infections More rapid dose escalation and support with growth factors may improve rate and quality of responses

32. A phase II study of lenalidomide in patients with untreated, symptomatic CLL Patients Chronic lymphocytic leukemia Untreated but symptomatic Treatment Lenalidomide 10mg daily starting dose days 1- 21 on a 28 day cycle Dose escalations by 5 mg weekly Target dose 25mg daily Allopurinol cycle 1, ASA 81 mg daily Endpoints Objective response Design Phase II two-stage design Sample size 25 patients Chen C.. et al. ASH 2008 abstract # 44

33. Lenalidomide Frontline in CLL Study activated in October 2006 - 62 yo male Patient 1 - 62 yo male rapid lymphocyte decrease reached target dose 25mg week 5 tumor lysis with ARF and MI – 64 yo male Patient 2 – 64 yo male Grade 4 neutropenia at 10mg  septic death Modified treatment plan : Cycle 1 (Month 1) 2.5 mg daily x 21/28 days Cycle 2 5.0 mg daily Cycle 3 10 mg daily Cycle 4 and beyond Continued monthly escalation to 25mg allowed for nonresponders Chen C.. et al. ASH 2008 abstract # 44

34. Lenalidomide Frontline in CLL: Toxicities Tumor flare –80% of patients (20/25) –frequently associated with rash, nasal coryza, scalp itching –30% of all cycles –all grade 1-2 –40% required steroids HematologicalGrade 3-4Neutrophils 16 (64%) Platelets 3 (12%) Hemoglobin Chen C.. et al. ASH 2008 abstract # 44

35. Lenalidomide Frontline in CLL: Conclusions Lenalidomide is highly active as a first-line agent in CLL - RR 56%Lenalidomide is highly active as a first-line agent in CLL - RR 56% ToxicitiesToxicities –Myelosuppression, TLS –Tumor flare is mild and manageable Dosing recommendationsDosing recommendations –Start low and go slow –Rebound lymphocytosis suggests continuous dosing may be preferable Chen C.. et al. ASH 2008 abstract # 44

36. Lenalidomide and Rituximab in CLL: Background Different mechanisms of action Non-overlapping toxicity profile Ameliorate the tumor flare reaction

37. Lenalidomide and Rituximab as Salvage Therapy in CLL: Potential Synergism Lenalidomide enhances NK-cell and monocyte- mediated ADCC of rituximab-treated NHL and CLL cells 1 Immunomodulatory drugs stimulate NK-cell function, alter cytokine production by dendritic cells, and inhibit angiogenesis enhancing the anti-tumour activity of rituximab in vivo 2 Lenalidomide and rituximab increase MCL cells apoptosis and rituximab-dependent NK-cell mediated cytotoxicity. 3 1 Wu, L. Blake Bartlett J. et al. Clin Cancer Res 2008;14:4650-4657 2 Reddy N. Br J Haematol. 2008 Jan;140(1):36-45. 3 Zhang L. and Wang M. Am. J. Hematol. E-pub, 2009

38. Lenalidomide and Rituximab as Salvage Therapy in CLL: Potential Antagonism Lenalidomide down-regulates the CD20 antigen and antagonizes direct and ADCC of rituximab on primary CLL cells 1 1 Lapalombella, R., Byrd J. et al. Blood 2008;112:5180-5189

39. Lenalidomide and Rituximab as Salvage Therapy in CLL: Study Design Phase II, 42 patients (10/08-7/09) Prior purine-analogue Creatinine <2 mg/dL, bilirubin<2 mg/dL Zubrod/WHO performance status 0-2 Response assessment at end of cycles 3, 6, 12 and then every 6 cycles

40. Lenalidomide and Rituximab as Salvage Therapy in CLL: Doses and Schedule Rituximab (375 mg/m2) Days 1, 8, 15 and 22 and once every four weeks during cycles 3- 12. Lenalidomide 10mg/day started on Day 9 of cycle 1 and continued daily for twelve cycles (each cycle consists of 28 days) Lenalidomide continued until progression in responders Allopurinol 300 mg d 1 -14 No antibiotic or anti-viral prophylaxis No DVT prophylaxis required

41. CharacteristicN = 37 Median age (range), years59 (44–83) Number (range) of prior therapies2 (1–9) Serum  2 -microglobulin (range), g/ml 3.6 (1.5–9) Rai disease stage III or IV, %41 Fludarabine refractory, n (%)9 (24) Prior rituximab, n (%)37 (100) Unfavourable cytogenetic n (%) del(11q23)10 (37) del(17p)9 (24) Unmutated V H, n (%)*26 (70) V H = immunoglobulin variable heavy chain. Lenalidomide and Rituximab in Recurrent CLL: Patient Characteristics Ferrajoli A, et al. ASH 2009 submitted

42. Lenalidomide and Rituximab in Recurrent CLL: Toxicities (N=37 ) Grade ≥ 3 Adverse EventN. (%) Hematological Neutropenia Thrombopenia 16 (43) 4 (11) Non-hematological FUO Pneumonia Sepsis 6 (16) 2 (5) 1 (3) Tumor lysis syndrome Pain 1 (3) Tumor Flare Reaction Grade 1 Grade 2 8 (22) 1 (3)

43. Lenalidomide: Ongoing Trials Front-line treatment Lenalidomide and rituximab [lenalidomide first] (CLL Research Consortium) Lenalidomide Vs Chlorambucil (age > 65 years, ORIGIN trial, Celgene) Phase I Lenalidomide + Fludarabine + Rituximab (Dana-Farber Cancer Institute) FR Vs FR + lenalidomide cons. Vs FCR (CALG-B) Salvage treatment Bendamustine+ Rituximab+ Lenalidomide ( Univ. Wisconsin) Phase I-II Bendamustine+ Rituximab+ Lenalidomide ( Georgetown Univ.) Phase I-II Lenalidomide+Dasatinib (Scripps Cancer Center) Phase I Lenalidomide + Alvocidib (Ohio State) Phase I-II Lenalidomide + Fludarabine + Cyclophosphamide (GIMEMA) Phase II Lenalidomide + Ofatumumab (MDACC) Consolidation treatment Lenalidomide (MDACC) Lenalidomide after FR as initial therapy (Georgetown Univ.) Lenalidomide after PCR as initial therapy (Mayo Clinic) Lenalidomide Vs placebo after 2 nd line therapy (CONTINUUM trial, Celgene)

44. Thank you!!

45. Studies Exploring Lenalidomide- based Combination Therapies in CLL InvestigatorTrial Name Trial Number or country Chemotherapy combination with Chemotherapy Brown A Phase 1 Dose Escalation Study of Lenalidomide in Combination With Fludarabine-Rituximab for Previously Untreated CLL/SLL NCT00543114 Byrd (CALGB) A Randomized Phase 2 Study of 3 Fludarabine/Antibody Combinations for Patients With Symptomatic, Previously Untreated CLL NCT00602459 Cheson Lenalidomide Following Rituximab and Fludarabine in Untreated CLL NCT00860457 Cheson Phase 1 Clinical Trial of Bendamustine, Lenalidomide and Rituximab in B-Cell Lymphoid Malignancies NCT00864942 Ferrajoli Lenalidomide in Combination With Rituximab as Treatment for Patients With Relapsed CLL NCT00759603 Flinn A Phase 1/2 Study of Fludarabine, Rituximab, and Lenalidomide in Minimally Treated and Untreated Patients With CLL NCT00536341 “Chemo-Free” Regimens Chanan-KhanLenalidomide + RituximabNCT00096044 Ferrajoli Lenalidomide in Combination With Rituximab as Treatment for Patients With Relapsed or Refractory CLL NCT00759603 James CRC and UCSD A Two-Arm, Multi-Center Trial of Lenalidomide and Rituximab, for First-Line Treatment in Patients With B- CLL NCT00628238 Pinilla (MCL/CLL) A Phase 2 Study of Lenalidomide in Combination With Rituximab for Patients With CD5+/CD20+ Hematologic Malignancies Who Relapse or Progress After Rituximab NCT00609869 Sigal A Phase 1/2 Study of Combination Dasatinib and Lenalidomide in Purine Analogue-Failed CLL NCT00829647 Updated June 19, 2009. Clinicaltrials.gov Web site. clinicaltrials.gov. Accessed November 16, 2009 ; Data on file, Celgene.

46. Studies Exploring Lenalidomide- based Combination Therapies in CLL InvestigatorTrial Name Trial Number or country Lenalidomide in maintenance or consolidation setting Ferrajoli A Study of Lenalidomide in Patients With CLL and Residual Disease After Chemotherapy NCT00632359 Shanafelt/Ka y Phase 2 Trial of Pentostatin, Cyclophosphamide, and Rituximab Followed by Lenalidomide Consolidation for Previously Untreated B-CLL/ SLL NCT00602836 Cheson Lenalidomide Following Rituximab and Fludarabine in Untreated Chronic Lymphocytic Leukemia NCT00860457 Fludarabine/Rituximab Combined With Escalating Doses of Lenalidomide Followed by Rituximab/Lenalidomide in Untreated CLL -- a Dose-Finding Study With Concomitant Evaluation of Safety and Efficacy Austria Updated June 19, 2009. Clinicaltrials.gov Web site. clinicaltrials.gov. Accessed November 16, 2009 ; Data on file, Celgene.

47. Open Celgene Sponsored Studies That are Actively Accruing Patients CONTINUUM (NCT 00774345): a phase 3 trial evaluating the role of lenalidomide in the maintenance setting after second-line therapy –To date, there has been no CLL registration study focused on maintenance therapy –Since no CLL patient is “cured,” maintenance presents an opportunity to achieve sustained disease control by extending response rates ORIGIN (NCT 00910910): a phase 3 trial to assess the role of lenalidomide in treating elderly patients (  65 years of age) with CLL –CLL is a disease that mainly impacts the elderly –To date, there has been no registration study focused on patients  65 years of age –Traditional chemotherapy is often poorly tolerated by patients who tend to be infirm and to have comorbidities Clinicaltrials.gov Web site. clinicaltrials.gov. Accessed November 16, 2009 ; Data on file, Celgene.

48. A.Ferrajoli Disclosure Information Consultant: Bayer, Genzyme Grant/Research Support: Bayer, Celgene, Genentech Speaker’s Bureau: none Major Stock Shareholder: none The use of lenalidomide for the treatment of CLL is considered off label

49. Copyright ©2008 American Association for Cancer Research Wu, L. et al. Clin Cancer Res 2008;14:4650-4657 Lenalidomide enhances natural killer cell and monocyte-mediated antibody-dependent cellular cytotoxicity of rituximab-treated CD20+ tumor cells

50. Immunomodulatory drugs stimulate natural killer-cell function, alter cytokine production by dendritic cells, and inhibit angiogenesis enhancing the anti-tumour activity of rituximab in vivo Reddty N.Br J Haematol. 2008 Jan;140(1):36-45.

51. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Lapalombella, R. et al. Blood 2008;112:5180-5189 Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells

52. Copyright ©2008 American Society of Hematology. Copyright restrictions may apply. Lapalombella, R. et al. Blood 2008;112:5180-5189 Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells