1. John Gribben Professor of Experimental Cancer Medicine, Director of the Experimental Cancer Medicine Centre and Director of Stem Cell Transplantation at St Bartholomew’s Hospital, Queen Mary’s School of Medicine, University of London, UK Former Research Fellow in Haematology at University College London, UK Former Associate Professor of Medicine at Harvard Medical School, Boston, Massachusetts, USA Former Attending Physician at the Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Boston, Massachusetts, USA Author of more than 300 manuscripts and book chapters Founding member of the CLL Research Consortium Associate Editor of Blood St Bartholomew’s Hospital

2. Management of relapsed CLL: why rituximab? John Gribben St Bartholomew’s Hospital, Queen Mary’s School of Medicine University of London, UK

3. Introduction Fludarabine-based therapy is the standard first-line treatment for fit patients Ultimately, all patients treated for CLL progress and require subsequent therapy A significant proportion of patients become fludarabine resistant Clonal evolution is observed with accumulation of negative molecular biology features The majority of patients are aged >65 years Allogeneic SCT is effective in some patients with relapsed CLL –use is limited in patients with advanced age and/or comorbidities CLL = chronic lymphocytic leukaemia; SCT = stem cell transplantation

4. Relapsed versus refractory CLL Relapse or progression –response to therapy: PR at least, lasting more than 6 months Fludarabine-refractory disease –no response to therapy –or progression on therapy –or progression within 6 months of completion of fludarabine therapy PR = partial response

5. Fludarabine-based regimens for relapsed/refractory CLL StudyRegimenn Refractory patients (%) OR/CR non-refractory (%) OR/CR refractory (%) Keating et al. 1 F7864 93/57 38/28 O’Brien et al. 2 FC7431 80/1239/3 Bosch et al. 3 FCM6058 72/3234/6 Mauro et al. 4 FAND2361100/78 50/29 OR = overall response; CR = complete response; A = ara-C; C = cyclophosphamide; D = dexamethasone; F = fludarabine; M = mitoxantrone; N = novantrone; 1 Keating M, et al. Blood 1993;81:2878–84 2 O’Brien SM, et al. J Clin Oncol 2001;19:1414–20 3 Bosch F, et al. Br J Haematol 2002;119:976–84 4 Mauro FR, et al. Haematologica 2002;87:926–33

6. Poor prognosis of advanced, relapsed CLL Survival (years) Responsive to both alkylating agents and fludarabine2–3 Alkylator refractory1–2 Fludarabine refractory (17p-)<1 Thomas D, et al. In: Cheson B, editor. Chronic lymphoid leukemias. M Dekker, 2001. p. 275–335 There is therefore an outstanding need for more effective, feasible therapies in relapsed/refractory CLL

7. Rituximab in relapsed/refractory CLL: rationale Rituximab is an active targeted therapy that acts via CD20 to eradicate lymphoma cells Rituximab is generally well tolerated Rituximab demonstrates good clinical activity Rituximab demonstrates synergistic activity with fludarabine

8. R-FC for previously treated CLL: regimen and patient characteristics 179 patients with pretreated CLL –rituximab (R) 375mg/m 2 cycle 1, 500mg/m 2 cycles 2–6 –F 25mg/m 2 days 1–3 every 4 weeks x 6 –C 250mg/m 2 days 1–3 every 4 weeks x 6 –infectious prophylaxis during treatment and 2 months after Patient characteristics –mean age: 59 years (36–81 years) –Rai stage III/IV: 49% –median number of prior treatments: 2 (1–10) –46% of patients completed 6 cycles; 37%  3 cycles Wierda W, et al. J Clin Oncol 2005;23:4070–8

9. R-FC for previously treated CLL: median survival over 3 years Outcomen Patients at t=0 (n) Died80177 Relapsed60129 Months Overall survival (OS) Time to progression (TTP) Median OS: 42 months Median TTP: 28 months 061218243036424854 Probability 1.0 0.8 0.6 0.4 0.2 0 Rituximab dose Cycle 1: 375mg/m 2 Cycles 2–6: 500mg/m 2

10. R-FC for previously treated CLL: TTP by response status Probability 0 612 18 24 30 36 42 48 54 1.0 0.8 0.6 0.4 0.2 0 Months n Response Median (months) 45 CR39 28 Nodular CR33 56 PR15 Wierda W, et al. J Clin Oncol 2005;23:4070–8

11. NR = no response; ED = early death n Response Median (months) 51 CR 45+ 28 Nodular CR 30+ 56 PR39 42 NR11 6 ED 2 R-FC for previously treated CLL: OS by response status Wierda W, et al. J Clin Oncol 2005;23:4070–8 Probability 0 612 18 24 30 36 42 48 54 1.0 0.8 0.6 0.4 0.2 0 Months

12. R-FC is well tolerated in patients with previously treated CLL Tolerability –no serious infusion-related adverse events –grade 3/4 neutropenia 62% of courses –grade 3/4 thrombocytopenia 17% of courses –major infection 5% of courses Wierda W, et al. J Clin Oncol 2005;23:4070–8

13. R-FC in relapsed/refractory CLL: response by treatment regimen Patients (%) F ± P* (n=251) FC* (n=111) R-FC (n=143) CR131228 Nodular PR251614 PR213930 NR2824 Early death11 7 4 Wierda W, et al. Cancer 2006;106:337–45 *Historical controls P = prednisone 596772

14. R-FC in relapsed/refractory CLL: OS by treatment regimen Wierda W, et al. Cancer 2006;106:337–45 1.0 0.8 0.6 0.4 0.2 0 024487296120144168192216 Proportion surviving PatientsDied Median survival (months) Protocolp value 25124120F ± P 111 8731FC 14366949R-FC <0.01 0.05 Months

15. Increasing response rates in relapsed/refractory CLL ORR (%)CR (%) Chlorambucil/CAP22–350–6 Fludarabine32–59 3–37 FC40–80 3–15 R-FC7325 Gribben J. Hematology (ASH Education Book) 2005;292–8 O’Brien S, et al. J Clin Oncol 2001;19:1414–20 Wierda W, et al. J Clin Oncol 2005;23:4070–8 ORR = overall response rate CAP = cyclophosphamide, doxorubicin, prednisone

16. The REACH trial: R-FC versus FC in relapsed CLL CLL Binet B or C >18 years Relapsed disease, excluding fludarabine- refractory RANDOMISERANDOMISE R-FC every 3 weeks x 3 FC every 3 weeks x 3 RESTAGERESTAGE R-FC every 3 weeks x 3 FC every 3 weeks x 3 SD, PD off study CR, PR Rituximab dose Cycle 1: 375mg/m 2 Cycles 2–6: 500mg/m 2 SD = stable disease; PD = progressive disease

17. Other rituximab combinations in relapsed/refractory CLL

18. Rituximab plus alemtuzumab Regimen –rituximab 375mg/m 2 (day 1) and 500mg/m 2 on days 8, 15, and 22 –alemtuzumab 15mg (days 2–7) and 30mg on days 3 and 5 of weeks 2–4 20 patients with relapsed/refractory CLL –six (30%) were refractory to both fludarabine and alkylators –all had prior exposure to rituximab and two (10%) had prior exposure to alemtuzumab ORR 55%, CRR 30% Faderl S, et al. Blood 2005;106 (Abstract 2963) CRR = complete response rate

19. R-FCA in relapsed CLL: treatment regimen R 500mg/m 2 on day 2* F 25mg/m 2 on days 3–5 C 250mg/m 2 on days 3–5 A 30mg on days 1, 3, and 5 ARCFACFCFA Day Every 28 days for 6 cycles *375mg/m² dose adjustment for cycle 1 only A = alemtuzumab 1234512345 Wierda WG, et al. Blood 2006;108:31 (Abstract 2839)

20. R-FCA in relapsed CLL: response by previous treatment No. of patients (n=78) CR after R-FCA (%) ORR after R-FCA (%) FC First line Salvage 3 7 0 14 100 43 R-FC First line Salvage 20 25 30 16 65 48 SCT 425 75 Wierda W, et al. Blood 2006;108:14a (Abstract 31)

21. R-FCA in relapsed CLL: progression-free survival Proportion 1.0 0.8 0.6 0.4 0.2 0 061218243036 Months 27 months 10 months Patients RelapsedResponse 19 5CR 3214PR p<0.001 CR PR Wierda W, et al. Blood 2006;108:14a (Abstract 31)

22. R-FCA in relapsed CLL: OS Proportion 1.0 0.8 0.6 0.4 0.2 0 06121824303642 Months Patients DiedResponse 19 2CR 3221PR 2722SD/PD 0.007 0.008 p value Wierda W, et al. Blood 2006;108:14a (Abstract 31)

23. R-PC in relapsed/refractory CLL Regimen (all drugs administered on day 1 of a 3-week cycle for a total of 6 cycles) –R 375mg/m 2 –P 4mg/m 2 –C 600mg/m 2 32 patients with relapsed/refractory CLL –eight patients fludarabine-refractory ORR 75%, CRR 25% –six of eight fludarabine-refractory patients responded with one CR Of three patients with 17p deletion, two achieved a CR and one a PR Lamanna N, et al. J Clin Oncol 2006;24:1575–81 R-PC = rituximab, pentostatin, cyclophosphamide

24. Case study 56-year-old woman presents in 1997 with –cervical lymphadenopathy –no B symptoms –WBC WNL CT scan –cervical and inguinal lymphadenopathy –no splenomegaly LN biopsy: small lymphocytic lymphoma BM biopsy: diffuse infiltrate with CD19+CD5+CD23+ small B cells FISH of BM sample reveals del 13q WBC = white blood cell; WNL = within normal limits; CT = computed tomography LN = lymph node; BM = bone marrow; FISH = fluorescence in-situ hybridisation

25. Case study (cont’d) Watch and wait approach explained to patient Over the next 3 years, slowly progressive disease –watch and wait November 2000 presents with increased cervical, axillary, and inguinal adenopathy –largest nodes increased to 7cm Peripheral blood –WBC 1,4000/µL with lymphocytosis –Hgb 9.5g/dL –platelets 8,9000/µL No B symptoms, but uncomfortable with degree of adenopathy

26. Case study (cont’d) Commenced on therapy with FCR Tolerated first cycle well –notes marked decrease in lymph nodes Returns for third cycle –notes increasing lymphadenopathy Restaging after three cycles shows no response

27. Autologous and allogeneic SCT for poor-risk CLL Gribben J, et al. Blood 2005;106:4389–96 1.0 0.8 0.6 0.4 0.2 0 Probability 1.0 0.8 0.6 0.4 0.2 0 Probability OSProgression-free survival 0123456789101112131415 Years Allogeneic Autologous Allogeneic Autologous p=0.96 p=0.04

28. Sorror M, et al. J Clin Oncol 2005;23:3819–29 Outcome after reduced-intensity conditioning transplantations for CLL Related recipientsUnrelated recipients Months after HCT Percentage 100 80 60 40 20 0 Months after HCT Percentage OS DFS NRM Relapse-related mortality OS DFS NRM Relapse-related mortality 100 80 60 40 20 0 0612182430364248 NRM = non-relapse mortality DFS = disease-free survival

29. FK506; short-course MTX FC versus R-FC for non-myeloblative SCT conditioning in CLL: MD Anderson experience Cohort 1 (1996–1999): FC Day –13 –5 –4 –30 +8 Rituximab 1,000mg/m 2 Fludarabine 30mg/m 2 Cyclophosphamide 750mg/m 2 Cohort 2 (2000–2002): R-FC Day –13–6 –5 –4 –30 +1+8 FK506; short-course MTX Khouri IF, et al. Exp Hematol 2004;32:28–35 MTX = methotrexate

30. FC versus R-FC for NST conditioning in CLL: outcome (MD Anderson) FCR-FCp value n 7 10 2-year REL (%)14 20ns 2-year OS (%)571000.03 ext. chr. GVHD (%)81 360.04 2-year TRM (%)43 00.03 Khouri IF, et al. Exp Hematol 2004;32:28–35 REL =relapse rate; ns = not significant ext. chr. = extensive chronic GVHD = graft versus host disease TRM = treatment-related mortality

31. Allogeneic transplantation for relapsed/refractory CLL CLL transplant consensus Allogeneic SCT is a reasonable treatment option for younger patients with –non-response or early relapse (<12 months) after purine analogue-based therapy –relapse <24 months after purine analogue combinations or auto-SCT (plus high-risk genetics) –p53 mutation with treatment indication Dreger P, et al. Leukemia 2007;21:12–17

32. Case study: outcome Underwent RIC allogeneic transplant from HLA-matched unrelated donor Early withdrawal of immunosuppressive therapy because of disease –concomitant with development of GVHD – good evidence of GVL effect Remains in CR –now 5 years post transplant RIC = reduced-intensity conditioning GVL = graft versus leukaemia

33. Case study: tumour burden after non-myeloablative allogeneic SCT 1E+07 1E+06 1E+05 1E+04 1E+03 1E+02 1E+01 1E+00 GVHD grade 3 skin IgH/GAPDH copy number 12 months post SCT Limit of detection 036948

34. Conclusions Immunochemotherapy is becoming standard treatment for relapsed/refractory CLL –the REACH trial will provide further information Allogeneic SCT is an option for selected patients Best supportive care and the testing of novel therapies remain very important for relapsed/ refractory CLL patients