Making Sense of Novel Prognostics: NOTCH1, SF3B1 Jennifer R Brown, MD PhD Director, CLL Center

1. Making Sense of Novel Prognostics: NOTCH1, SF3B1 Jennifer R Brown, MD PhD Director, CLL Center Dana-Farber Cancer Institute October 24, 2014

2. What is High Risk CLL? Historically defined solely by clinical features: Stage, lymphocyte doubling time, b2m Therapy resistance Biologic prognostic factors are increasingly important: IGHV, FISH, somatic mutation profile

3. Immunoglobulin VH Gene Mutation Med Surv >24 yrs Med Surv 9 yrs Years from Diagnosis Blood 94: 1840, 1999

4. 100 80 60 40 20 0 0 12 24 36 48 60 72 84 96 120 144 168 Overall Survival by FISH 17p- 11q- 12q trisomy Normal 13q deletion as sole abnormality Patients surviving (%) Del 11q: 79 m Del 17p: 32 m Months NEJM 2000;343:1910

5. IGHV Mutation and Cytogenetics: Independent Predictors Blood 100: 1410, 2002

6. Insights from Sequencing: NOTCH Mutations NOTCH1: recurrent mutation (2 bp deletion; P2515fs) -Fabbri et al: -15.1% overall, assoc with UM IGHV and TP53 disruption -21% in chemorefractory -31% in Richter’s transformation -predictor poor OS in MVA -Puente et al: -12.2% overall, assoc with UM IGHV, ZAP70, CD38 -23% in RT; poor OS 21% at 10 yrs • Fabbri et al. J Exp Med. 2011 Jul 4;208(7):1389-401. • Puente et al. Nature. 2011 Jun 5;475(7354):101-5.

7. NOTCH Mutations: Short TFS and Higher Risk RT Blood 119: 521, 2012

8. Mutation Discovery Through Sequencing a Large Initial Sample Set • Increases detection of the full range of mutated genes • Enables reconstruction of gene pathways underlying disease pathogenesis • Reveals associations between genetic events and clinically important disease features • NEJM 365:2497, 2011

9. Established Recently associated Novel TP53 SF3B1 MYD88 ATM FBXW7 Significantly mutated genes NOTCH1 ZMYM3 DDX3X MAPK1 0 4 8 12 16 # mutations / 91 CLLs Sequencing CLL Reveals 9 Significantly Mutated Genes NEJM 365:2497, 2011

10. K700E (7) G742D (2) G740E K741N N626H Q903R R625L 1 1304 aa 1 22 PP2A repeats 22 SF3B1 Mutated in 15% of CLLs SF3B1 chromosome 2 q33.1 • At the catalytic core of U2 snRNP • 14 mutations in 14 CLL patients in a restricted region of the C-terminal domain • - K700E was recurrent • Mutations in SF3B1 also seen in myelodysplasia Yoshida et al, Nature 2011; Papaemmanuil et al, NEJM 2011

11. 1 91 del(13q) FISH Cytogenetic Features Trisomy 12 del(11q) del(17p) P<0.001 TP53 Cell cycle or DNA damage ATM P=0.004 RNA processing SF3B1 DDX3X Significantly mutated genes P=0.009 NOTCH1 Notch1 signaling FBXW7 P=0.009 MYD88 Inflammatory pathway MAPK1 P=0.001 ZMYM3 IGHV mutational Status The Significantly Mutated Genes Associate with Known Prognostic Markers • NEJM 365:2497, 2011

12. Months from diagnosis to first therapy SF3B1 Mutation Independently Predicts Poor Prognosis • NEJM 365:2497, 2011

13. SF3B1 Mutations Confer Poor Prognosis Rossi et al Blood 2011;118:6904-8

14. BIRC3 Mutation Associated with Poor OS Blood 119:2854

15. New NOTCH1, SF3B1 and BIRC3 Lesions are Developed During the CLL Clinical Course High riskclonalevolution (TP53, NOTCH1, SF3B1, BIRC3) • Inclusion criteria: • >2 years of follow-up • >2 sequential samples collected at the following time points: • Diagnosis • Progression requiring treatment in progressive cases • Last follow-up in non-progressive/untreated cases 24% at 10 years N=36 18% N=202 cases N=469 samples Median interval between sampling: 62 months Frequency of CE (%) N=13 6% N=10 5% N=10 5% N=10 5% N=9 4% N=8 4% N=6 3% N=5 2% N=5 2% N=4 2% N=1 0.5% N=0 N=0 Blood 2013;121(8):1403–1412

16. 25 Recurrent Drivers in CLL (n=160 Patients) 16 previously reported CLL drivers Wang et al., NEJM, 2011 Quesada et al., Nat Gen, 2011 Fabbri et al., JEM, 2011 Brown et al., ClinCan Res, 2011 Edelmann et al., Blood 2012 * 9 novel putative CLL drivers identified 82/160 WES used in Wang et al. NEJM 2011 Landau et al Cell 2013

17. NOTCH1Mutation Status: High Risk Patient Characteristics

18. Inferring Earlier and Later Drivers in CLL 100 % of affected samples that are clonal 0 Number affected *Higher rate of clonal frequencies, q<0.1 Landau et al Cell 2013;152:714–726

19. Biology of High Risk CLL Clinical significance of del17p/TP53 mutation > del11q > UM IGHV is well established Genomic complexity (FISH, karyotype or CN) associates with prognosis and appears quite adverse– but has not been routinely studied Not analyzed in multivariable analyses Sequencing data suggest SF3B1, NOTCH1 and subclonal driver mutations associate with poor prognosis Increasing data on TP53, SF3B1 and NOTCH1 in clinical cohorts

20. Recurrent Mutations Refine Prognosis Balakas et al. Leukemia 2014: 1-8.

21. Recurrent Mutations Refine Prognosis Balakas et al. Leukemia 2014: 1-8.

22. Recurrent Mutations Correlated to Cytogenetics Jeromin et al. Leukemia 2014: 108-117

23. Recurrent Mutations Refine Prognosis Balakas et al. Leukemia 2014: 1-8.

24. Integrated Mutational and Cytogenetic Modelfor CLL Prognostication Treatment OS del13q14 del13q14 Normal/+12 Normal/+12 NOTCH1 M/SF3B1 M/del11q22-q23 NOTCH1 M/SF3B1 M/del11q22-q23 TP53 DIS/BIRC3 DIS TP53 DIS/BIRC3 DIS p<0.0001 p<0.0001 Rossi et al. Blood 2013;121(8):1403

25. By Integrating Mutations, ~20% Low Cytogenetic Risk CLL are Reclassified into High Risk Subgroups del13q14 only by FISH Normal by FISH +12 by FISH Rossi et al

26. What About After Therapy?

27. CLL8 Study Design 817 patients with untreated, active CLL and good physical fitness (CIRS ≤ 6, creatinine clearance ≥ 70 mL/min) 6 courses FCR Follow up R FC C1 C2 C3 C4 C5 C6 Demographics similar between two treatment arms Median observation time 5.9 years Hallek M, et al: Lancet. 376:1164, 2010

28. CLL8: Addition of Rituximab to FC

29. CLL8: Addition of Rituximab to Fludarabine and Cyclophosphamide

30. CLL8: Survival after FCR by FISH +12q 13q-single 11q- Not 17p-/11q-/+12q/13q- 17p- 17p deletion Lancet 2010: 376: 1164

31. MDACC: TTF after FCR Based on FISH (2004-2010) Proportion Courtesy of M Keating

32. MDACC: TTP for FCR Responders by IGHV and 11q Proportion

33. Incidence of Genetic Lesions CLL8:CLL3X:*CLL2H:# 1st Line High-Risk F-refractory (FC vs. FCR) (Allo-SCT) (Alemtuzumab) n=635 n=80 n=97 TP53mut11.5 30.0 37.4 NOTCH1mut10.0 13.8 13.4 SF3B1mut18.4 26.3 17.5 IGHVUM 63.0 95.6 76.3 17p- 8.2 18.1 30.1 11q- 24.6 36.1 19.4 *Dreger et al. abstract 966, Tue 8:45, #Schnaiter et al. abstract 710, Mo 4:45

34. CLL8 Multivariable Analysis: PFS Cox regression including: FC, FCR, age, sex, stage, ECOG, B-symptoms, WBC, TK, β2MG, 11q-, +12, 13q-, 17p-, IGHV, TP53, NOTCH1, SF3B1 PFS: HR p-value FCR 0.510 <.001 TK>10 1.367 0.019 IGHVUM 1.727 <.001 11q- 1.536 <.001 17p- 2.949 <.001 TP53mut 2.113 <.001 SF3B1mut 1.348 0.024

35. CLL8 Multivariable Analysis: OS OS: HR p-value FCR 0.652 0.006 Age>65 y. 1.467 0.018 ECOG>0 1.609 0.002 ß2MG>3.5 1.493 0.014 TK>10 1.961 0.003 IGHV U 2.125 <.001 17p- 3.175 <.001 TP53mut 2.852 <.001

36. CLL8: Impact of TP53 Mutation on OS TP53: wild type mutated Therapy: FCR FC

37. CLL8 Multivariable Analysis: Predictive Factors Cox regression including: FC, FCR, TP53, NOTCH1, SF3B1, and treatment interaction PFS: HR p-value FCR 0.544 <.001 TP53mut 3.607 <.001 SF3B1mut 1.355 0.012 NOTCH1mut 1.652 0.022 Interaction OS: HR p-value FCR 0.654 0.002 TP53mut 4.470 <.001 NOTCH1mut 1.331 0.344 Interaction

38. CLL8: NOTCH1 as Predictive Marker NOTCH1: wild type mutated Therapy: FCR FC

39. Mutation Frequency in Fludarabine Refractory CLL • Rossi et al. Blood 2013;121:1403

40. TP53, SF3B1, and NOTCH1 Mutations and Outcome of Allotransplantation Dreger et al. Blood 2013: 121 (16); 3284-3286

41. Summary SF3B1 mutation: associated with 11q deletion, UM IGHV, and shorter TTFT and PFS, ?OS NOTCH1 mutation: associated with trisomy 12, UM IGHV, shorter TTFT and Richter’s transformation ? No benefit of anti-CD20 antibody Genomic complexity is adverse but requires further analysis in large trials

42. Summary Deletion of 17p or TP53 mutation predict highest risk CLL, followed by 11q deletion (NOTCH1, SF3B1) and Unmutated IGHV Early data suggest high response but shorter PFS with 17p or 11q deletion even with BCR pathway inhibitors No good data with BCR inhibitors for NOTCH1, SF3B1 AlloSCT may overcome poor prognosis of these somatic mutations

43. What is Actionable? 17p / TP53: prognosis; therapy selection (TP53 independent: BCR, ?SCT) 11q: prognosis; FC-based CIT Mutated IGHV (esp trisomy 12): FCR SF3B1, NOTCH1, BIRC3: prognosis

44. Acknowledgments Brown Lab, DFCI Bethany Tesar Stacey Fernandes Sasha Vartanov Reina Improgo Josephine Klitgaard Clinical Research Karen Francoeur Karen Campbell Shannon Milillo Hazel Reynolds Wu Lab, DFCI Catherine Wu Dan-Avi Landau LiliWang YouzhongWan Broad Institute Eric Lander Gaddy Getz Carrie Sougnez NirHacohen Stacey Gabriel Mike Lawrence PetarStojanov AndreySivachenko KristianCibulskis David Deluca Lymphoma Program, DFCI Arnold S Freedman David C Fisher Ann S LaCasce Eric Jacobsen Philippe Armand Matthew Davids Okonow-Lipton Fund Melton Fund Rosenbach Fund Center for Cancer Genome Discovery, DFCI Megan Hanna Laura Macconaill NIH, NHGRI CLL Research Consortium DFCI Biostatistics Donna Neuberg Lillian Werner Haesook Kim Kristen Stevenson