1. Scottsdale, Arizona Rochester, Minnesota Jacksonville, Florida Diffuse Large Cell Lymphoma Cell of Origin – Ready for Prime Time? Thomas Witzig, MD Hematology Malignancy Program Mayo Clinic Cancer Center

2. Disclosure  Research funding from Celgene, Novartis, Millenium for clinical trials  Research funding from Millenium for preclinical work  Advisory boards for Spectrum, Celgene, and Bayer (no personal compensation) 2

3. Time for Action? Origins Research in DLBCL Lancet Oncol. 2014 Jun;15(7):674-5

4. Cell of Origin Techniques  Gene expression profiling  Microarrays on RNA expression  Frozen tissue  Thousands of genes  Year 2000 onward  Immunohistochemistry  2004; Many algorithms; Hans still most widely used  2014 - Lymph2Cx Blood. 2014 Feb 20;123(8):1214-7

5. 15 Years Ago Nature. 2000 Feb 3;403(6769):503-11. “And like most overnight successes, it was about twenty years in the making” S. Walton originator of WalMart

6. Nature. 2000 Feb 3;403(6769):503-11.

7. Prognostic in the Pre-RCHOP Era

8. Hans Method – 10 Years Ago Hans et al Blood. 2004;103(1):275-82.

9. OS by TMA Hans et al Blood. 2004;103(1):275-82.

10. Meyer PN et al J Clin Oncol 2011;29(2):200-7. 262 cases of DLBCL; 192 had GEP

11. Meyer P N et al. JCO 2011;29:200-207 2011 by American Society of Clinical Oncology

12. Event-free survival of patients with diffuse large B-cell lymphoma according to immunophenotype by each algorithm. Meyer P N et al. JCO 2011;29:200-207 2011 by American Society of Clinical Oncology Tally Hans

13. Overall survival of patients with diffuse large B-cell lymphoma according to immunophenotype by each algorithm. Meyer P N et al. JCO 2011;29:200-207 ©2011 by American Society of Clinical Oncology Tally Hans

14. Blood. 2014 Feb 20;123(8):1214-7

15. Lymph2Cx

16. Blood. 2014 Feb 20;123(8):1214-7 Lymph2Cx  Matched FFPE and frozen  Traditional GEP; IHC; and Lymph2Cx  Training cohort – 51 cases  20 GCB; 19 ABC; 12 unclassified  Validation cohort – 68 cases  28 GCB; 30 ABC; 10 unclassified  10 micron scrolls; Qiagen AllPrep FFPET kit  Extract RNA from FFPE tissue slice  Digital GEP on 200 ng RNA using Nanostring technology  Sample split and run independently in 2 labs

17. Blood. 2014 Feb 20;123(8):1214-7 Lymph2Cx  Tested 93 genes found by Lenz et al to differentiate GCB from ABC (Lenz NEJM 2008)  20 were all that were needed  15 of the 93 and 5 “housekeeping genes”  NanoString technology on 20 genes was used in these two datasets

18. Blood. 2014 Feb 20;123(8):1214-7 Lymph2Cx Gold Standard GEP PFS OS

19. Prognosis or Helping Choose Therapy? Which drug to add????? Cell of Origin

20. 65 Years of Lymphoma Rx 1949 1963 1997‘02 Rituximab RIT RCHOP 2-CDA 19991975 CHOP ABVD Nitrogen Mustard Vincristine Doxorubicin 1993 CHOP Wins! VP-16 1978 Autologous SCT Cis-platinum 03 Bort 1953 Methotrexate 1983 ‘07 Lenalidomide Everolimus Vorinostat ‘05 Bendamustine ‘09 Pralatrex Romadep Era of Targeted Therapy Era of Chemotherapy 2011 Brentux Ibrutinib Lenalidomide 2013 Idelalisib 2014

21. CP1171726-10 R-CHOP is 15% Better than CHOP Probability 0.0 0.2 0.4 0.6 0.8 1.0 0.00.51.01.52.02.53.03.54.04.55.0 HR=0.64 p=0.003 HR=0.64 p=0.003 R-CHOP CHOP Probability Years from Induction Randomization 0.0 0.2 0.4 0.6 0.8 1.0 0.00.51.01.52.02.53.03.54.04.55.0 HR=0.72 p=0.05 HR=0.72 p=0.05 R-CHOP CHOP Failure-Free Survival Overall Survival Coiffier et al N Engl J Med. 2002; Habermann et al J Clin Oncol 2006

22. R(X)CHOP Era – what is X?  Epratuzumab - ERCHOP  Lenalidomide – R2CHOP  Bortezomib – Bor-RCHOP  Everolimus – ER-CHOP; maintenance E in CRADN2301 (enrolled)  Everolimus – EverRCHOP – N1085  Ibrutinib – IR-CHOP 22 Ann Oncol. 2014. Epub 2014/03/15

23. Lenalidomide-RCHOP (R2CHOP)  Untreated DLBCL eligible for RCHOP  Standard RCHOP-21 x 6 cycles  Lenalidomide d1-10 q 21  Three dose levels tested:  15 mg  20 mg  25 mg  All patients received prophylactic pegfilgrastim d2  All patients received aspirin 81 mg daily 23 Nowakowski G et al Leukemia. 2011;25(12):1877-81

24. MC078E – Phase II Results  Phase I/II trial of R2CHOP for untreated DLBCL  Any age  RCHOP + lenalidomide 25 mg days 1-10 q21 x 6 cycles  ASA daily  Pegfilgrastim day 2  No maintenance  Cell of origin by Hans algorithm Nowakowski G et al J Clin Oncol. 2014. Epub 2014/08/20

25. MC078E  64 patients enrolled; 60 evaluable  87 controls at same time with RCHOP  ORR 98% (59/60)  CR 80% (48/60)  Event-free survival at 24 months - 59%  Overall survival at 24 months - 78%  No difference in GCB vs non-GCB in R2CHOP arm Nowakowski G et al J Clin Oncol. 2014. Epub 2014/08/20

26. MC078E  RCHOP control  EFS/OS at two years for GCB: 46% and 78%  EFS/OS at two years for non-GCB: 28% and 64%  R2CHOP  EFS/OS at two years for GCB: 60% and 83%  EFS/OS at two years for non-GCB: 59% and 75% Nowakowski G et al J Clin Oncol. 2014. Epub 2014/08/20

27. R2CHOP RCHOP

28. Nowakowski G et al J Clin Oncol. 2014. Epub 2014/08/20 R2CHOP RCHOPR2CHOP RCHOP R2CHOP

29. Italian R2CHOP  DLBCL and FL 3b  R2CHOP in 13 centers in Italy  GCB vs non-GCB by IHC (Hans)  Standard RCHOP x 6  Lenalidomide 15 mg days 1-14 q 21  49 patients  92% (45/49) ORR with 86% functional CR 29 Lancet Oncol. 2014;15(7):730-7.

30. Outcome of R2CHOP (Italian) 30 Vitolo et al Lancet Oncol. 2014;15(7):730-7.

31. GCB vs. non-GCB 31 Lancet Oncol. 2014;15(7):730-7.

32. ©2011 MFMER | slide-32

33. ©2011 MFMER | slide-33 ECOG 1412 Randomized phase II of RCHOP vs. R2CHOP First patient in September 19, 2013 GCB and ABC Endpoint is response in ABC as defined by GEP Nanostring on paraffin-embedded tissue 110 patients accrued as of October 2014

34. DLBCL-002  FDA registrational, International Phase III  RCHOP x 6 vs. R2CHOP x 6  Lenalidomide 15 mg days 1-14 vs. placebo  Untreated DLBCL Stage II-IV  Ages 18-80 years  Requires excisional biopsy  ABC by Nanostring GEP on FFPE tissue  Promised 5 day turnaround; steroids allowed  600 patients  Opening Dec 2014 34

35. Furman RR et al Cancer. 2010;116(23):5432-9.

36. L. Staudt

37. Bortezomib R-CHOP  20 patients – 16 DLBCL/4 MCL  Median age 66 years (range, 29-84)  Standard RCHOP-21  Bortezomib - Days 1 and 4 of each cycle  0.7 mg/m2 - 4 patients  1.0 mg/m2 - 9 patients  1.3 mg/m2 - 7 patients  No DLT with any dose; grade 3 neuropathy in 1  95% CR 37 Furman RR et al Cancer. 2010;116(23):5432-9.

38. Bortezomib R-CHOP  At a median follow-up of 56 months  Overall survival at 4 years was 75%  Progression-free survival was 58%  Randomized phase II of RCHOP vs. RBCHOP in progress in US 38 Furman RR et al Cancer. 2010;116(23):5432-9.

39. Phase III Trials  RCHOP vs. Bor-RCHOP in UK 39 Start May 2011 and predicted to end in 2015

40. Ibrutinib with RCHOP 40 Younes A et al Lancet Oncol. 2014 Aug;15(9):1019-26.

41. IR-CHOP  Phase 1/2  DLBCL, MCL, FL  June 2012 – May 2013  No ASA, warfarin, heparin allowed  RCHOP x 6 + ibrutinib daily  No maintenance  No prophylactic G-CSF (allowed but not mandated) 41 Younes A et al Lancet Oncol. 2014;15(9):1019-26

42. IR-CHOP  33 patients  No MTD for ibrutinib; thus 560 mg/d continuously with standard RCHOP-21  18% febrile neutropenia  18 pts with DLBCL in the phase 2 42 Younes A et al Lancet Oncol. 2014;15(9):1019-26

43. IR-CHOP  100% ORR in the DLBCL (18/18)  15 CR and 3 PR  4/4 nonGCB – CR  5/7 GCB – CR  PK not affected for either ibrutinib or vincristine  A randomized phase III is ongoing –placebo controlled (NCT01855750) for non-GCB type 43 Younes A et al Lancet Oncol. 2014;15(9):1019-26

44. Summary about COO in DLBCL  Increases understanding of the biology  Helps predict prognosis but not particularly well  May guide therapy  New treatments with lenalidomide and ibrutinib are focusing on ABC-type  New techniques of GEP will enhance the clinical utility and “bring it to your hospital”  Prediction – you will use COO to “choose X” 44