1. Subcutaneous Cladribine for HCL in the BRAF era Francesco Forconi (MD, DM, PhD, FRCPath) Cancer Research UK Centre and Haematology Department Southampton University Hospital Trust email: F.Forconi@soton.ac.ukF.Forconi@soton.ac.uk

2. Intravenous Cladribine-iv2CdA – 0.7 mg/kg single cycle Continuous 0.1 mg/kg daily over 7 days 2-hour 0.1 mg/kg daily over 7 days Subcutaneous Cladribine -sc2CdA –0.7 mg/kg single cycle –100% bioavailability CLADRIBINE I.V. AND S.C. IN HCL Juliusson et al, Lancet 1993, J Clin Oncol 1996 Juliusson, NIH, Bethesda, 2010

3. INFECTIONS WITH CLADRIBINE Intravenous Cladribine (iv2CdA) –0.7 mg/kg single cycle –42% febrile episodes –13% documented infections Subcutaneous Cladribine (sc2CdA) In indolent non-Hodgkin lymphomas other than HCL: –at the dose of 0.7 mg/kg/cycle, efficacy of sc2CdA is similar to iv2CdA –reduction to 0.5 mg/kg/cycle determines equivalent efficacy and lower toxicity 55% p=.0001 p=.003 p=.72 p=.21 Betticher (SAKK), J Clin Oncol 1998 Saven (Scripps), Blood, 1998

4. ICGHCL2004 protocol EudraCT code: ICGHCL2004 2CdA s.c. 0.1 mg/kg/day Arm A 5 days Arm B 7 days

5. PATIENTS EudraCT code: ICGHCL2004 Central revision ARM A WHO 2001 criteria morphology HCL phenotype CD11c + CD19 + CD20 + CD25 + CD27 – CD38 – CD103 + FMC7 + WHO 2001 criteria morphology HCL phenotype CD11c + CD19 + CD20 + CD25 + CD27 – CD38 – CD103 + FMC7 + Median follow-up 62 months Classic HCL ARM B WHO 2008 criteria ANXA1 + (IHC) - HC>10 10 /L - Spleen > 10 cm - Refractory WHO 2008 criteria ANXA1 + (IHC) - HC>10 10 /L - Spleen > 10 cm - Refractory 156 patients (randomised) 24 centers 156 patients (randomised) 24 centers

6. NON HEMATOLOGICAL GRADE 3-4 TOXICITY P=,017 P=,012 Infections or FUO Hospitalization days 7-30 NO YES Higher infection rate and hospitalization in standard dose (Arm B) than in reduce dose (Arm A)

7. RESPONSES TO SUBCUTANEOUS 2CDA ■ CR/PR ■ mR/NR ~50% patients investigated for BRAF V600E mutation: no differences in response rates p=0.7

8. TFI: Treatment free interval (2° treatment) OS: Overall survival ENDPOINTS OF SURVIVAL AFTER TREATMENT

9. TFI and OS Arm B Arm A p=0.58 Time to 2° tratment (months) % Treatment free Arm B Arm A p=0.9 Overall survival (months) % surviving ~50% patients investigated for BRAF V600E mutation: no differences in survivals

10. 10 MOLECULAR PROGNOSTIC MARKERS OF TFI IN BRAF+ HCL P<.001 M-IG UM-IG IgA IgM IgD IgG AID + Ongoing SHM Multiple Ig isotype HCL has a very stabe genomic profile

11. BCR Signaling Pathway PIP3 PI3K PDK AKT PLC  2 SYK BLNK BTK LYN survivalapoptosisproliferationmigration IP 3 DAG PKC NFκB ERK JNKp38 Ca 2+ NFAT RAS RAF1 IKK MYC JUN ATF2 CD79A CD79B P P P P P P P Dasatinib Fostamatinib Ibrutinib GS-1101 RAS MEK ERK RAF RTK survival proliferation transformation V600E RAF-MEK Pathway Vemurafenib

12. Slide 12 Conclusions Cladribine at reduced doses has lower toxicity and similar efficacy BRAF mutational status: critical to identify patients non benefiting from Cladribine? New effective treatments (if/when required): HCL express high levels of multiple Ig isotypes that are functional Surface Ig and signaling is irrespective of BRAF V600E BCR-inhibitors act on the tumor component that signals through BCR BCR-inhibitors as an alternative strategy? Markers to identify poor outcome in BRAF+ HCL