Fludarabine-refractory Chronic Lymphocytic Leukemia Research Articles

High CD52 mRNA Expression Is An Adverse Prognostic Factor In Fludarabine-Refractory CLL Treated With Alemtuzumab On The Gcllsg CLL2H Trial

Alemtuzumab is a humanized mAb that targets CD52 and has been widely used for the treatment of chronic lymphocytic leukemia (CLL) patients with refractory disease or loss / mutation of TP53. The cell-surface marker CD52 is variably expressed on peripheral blood mononuclear cells and shows relatively high expression levels on the surface of both healthy B lymphocytes and on CLL cells, but the biologic function of CD52 has not been sufficiently clarified yet.To elucidate the prognostic value of CD52 expression, we analyzed a large gene expression dataset. Gene expression profiling was performed on Affymetrix Exon 1.0 ST Arrays. Data sets were generated for 75 fludarabine-refractory CLL patients treated within the multicenter phase II CLL2H trial of the GCLLSG (3x30 mg Alemtuzumab s.c. per week, max. 12 weeks). Normalization of the arrays was conducted by using the RMA algorithm. Statistical analysis was performed with the R software version 2.15.1.Patients were grouped into quartiles (Q1-Q4) according to CD52 expression levels (CD52 low: Q1, n=19; CD52 intermediate: Q2-Q3, n=37; CD52 high: Q4, n=19).Progression free survival (PFS) and overall survival (OS) were markedly different for patients from Q1 and Q4 (median PFS: Q1=413 days vs. Q4=145 days (p=0.0102); median OS: Q1= 879 days vs. Q4=413 days (p=0.0164)). Patients with progressive disease were overrepresented in Q4 (PD 7/18 (38.8%); ORR 5/18 (27.7%)) while rare in Q1 (PD 2/19 (10.5%); ORR 9/19 (47.3%)). Patients with low expression of CD52 had less frequently genetic/biologic and clinical characteristics of aggressive disease (Q1 vs. Q4; TP53 mutation/17p deletion: 47.36% vs. 36.84%; IGHV unmutated: 61.11% vs. 73.68%; leukocyte counts (median): 45.8 G/L vs. 55.4 G/L; thymidine kinase: 22.2 U/L vs. 30.4 U/L; β2-microglobulin: 3.3 mg/l vs. 5.2 mg/l; no palpable lymph nodes: 47.36 % vs. 10.52 %).Mutations of SF3B1 and NOTCH1 were present in 4/17 (23.5%) and in 5/17 (29.4%) cases in Q1. In contrast, cases from Q4 harbored mutations of SF3B1 and NOTCH1 in 2/18 (11.1%) and 1/18 (5.5%).Cases with high CD52 levels showed overexpression (FDR<1e-07;FC>2) of genes predominantly involved in signaling of the B-cell-, toll-like- and interleukin-receptor (BTK, SYK, CD79B; TLR7, TLR10, IL27RA, IL2RG, TRAF3IP3, TRAF5).The corresponding gene set overrepresentation analysis on KEGG pathways was significantly enriched for gene sets related to cell-extracellular matrix interaction, focal adhesion, regulation of actin cytoskeleton and cell adhesion molecules as well as associated signaling pathways like WNT- and MAPK-signaling.In conclusion, high CD52 expression is an adverse prognostic factor in refractory CLL treated with Alemtuzumab. Although suggestive, the level of CD52 expression and subsequent antibody binding capacity appear not to determine the effectivity of treatment. The association with genetic and biological characteristics indicates an independent role in the biology of aggressive CLL and highlights a potential function in cell-microenvironmental interactions for CLL cells. [Display omitted] Disclosures:Stilgenbauer:Genzyme: Honoraria, Research Funding.

Five-Year Follow-up of Safety and Efficacy of a Phase III Randomized Trial of Ofatumumab Therapy Versus Physicians' Choice in Patients with Bulky Fludarabine Refractory Chronic Lymphocytic Leukemia

Five-Year Follow-up of Safety and Efficacy of a Phase III Randomized Trial of Ofatumumab Therapy Versus Physicians' Choice in Patients with Bulky Fludarabine Refractory Chronic Lymphocytic Leukemia

Phase III, randomized study of ofatumumab versus physicians’ choice of therapy and standard versus extended-length ofatumumab in patients with bulky fludarabine-refractory chronic lymphocytic leukemia

We report results of a randomized, phase III study of ofatumumab versus physicians’ choice treatment in patients with bulky fludarabine-refractory chronic lymphocytic leukemia and explore extended versus standard-length ofatumumab treatment. Patients (79 ofatumumab, 43 physicians’ choice) completed a median 6 (ofatumumab) or 3 (physicians’ choice) months’ therapy. Ofatumumab-treated patients with stable disease or better were randomized (2:1) to 6 months’ extended ofatumumab treatment or observation. Although the study did not meet the primary endpoint of progression-free survival (PFS) by independent review committee (ofatumumab: 5.4 months, physicians’ choice: 3.6 months; p = 0.27), median PFS by investigators was significantly longer for ofatumumab versus physicians’ choice (7.0 versus 4.5 months; p = 0.003) as was time to next therapy (median 11.5 versus 6.5 months; p = 0.0004). PFS and time to next therapy were significantly longer with ofatumumab extended treatment than observation (p = 0.026 and p = 0.002, respectively; n = 37). The adverse-event profile of long-term ofatumumab administration showed no unexpected findings (Clinicaltrials.gov identifier: NCT01313689).

Complex karyotype is a stronger predictor than del(17p) for an inferior outcome in relapsed or refractory chronic lymphocytic leukemia patients treated with ibrutinib-based regimens.

Ibrutinib is active in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). In patients treated with ibrutinib for R/R CLL, del(17p), identified by interphase fluorescence in situ hybridization (FISH), is associated with inferior progression-free survival despite equivalent initial response rates. Del(17p) is frequently associated with a complex metaphase karyotype (CKT); the prognostic significance of CKT in ibrutinib-treated patients has not been reported. This study reviewed 88 patients treated for R/R CLL at The University of Texas MD Anderson Cancer Center with investigational ibrutinib-based regimens from 2010 to 2013. Pretreatment FISH and lipopolysaccharide-stimulated metaphase cytogenetic analysis were performed on bone marrow. An adequate pretreatment metaphase karyotype was available for 56 of the 88 patients. The karyotype was complex in 21 of the 56 cases; 17 of the 21 had del(17p) according to FISH. The overall response rate, including partial remission with persistent lymphocytosis, was 94%; 18% had complete responses. In a multivariate analysis (MVA), only CKT was significantly associated with event-free survival (EFS; hazard ratio [HR], 6.6 [95% CI 1.7-25.6]; P = .006). Fludarabine-refractory CLL (HR, 6.9 [95% CI 1.8-27.1], P = .005) and CKT (HR 5.9 [95% CI 1.6-22.2], P = .008) were independently associated with inferior overall survival (OS) in MVA. Del(17p) by FISH was not significantly associated with EFS or OS in MVA. CKT is a powerful predictor of outcomes for ibrutinib-treated patients with R/R CLL and may be a stronger predictor of biological behavior than del(17p) by FISH. Because of their relatively poor outcomes, patients with CKT are ideal candidates for studies of consolidative treatment strategies or novel treatment combinations.

Induction of TAp73 by platinum-based compounds to overcome drug resistance in p53 dysfunctional chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), strategies to overcome drug resistance due to p53 dysfunction are highly needed. Platinum-based compounds such as cisplatinum (CDDP) are active in fludarabine-refractory CLL through a largely unknown mechanism. We analyzed the mechanism of action of CDDP in the context of p53 dysfunctionality. In vitro treatment with CDDP did not induce death in quiescent CLL cells, but did induce apoptosis in CD40-ligand (and CpG) stimulated and proliferating cells, irrespective of p53 function. In the p53 dysfunctional prolymphocytic cell-line MEC1, CDDP treatment resulted in apoptosis, cell cycle arrest and ABL1-dependent expression of TAp73, CDKN1A, PUMA and BID. TAp73 RNA-interference decreased sensitivity to CDDP. Finally, both in vitro stimulated CLL cells and lymph node (LN) derived CLL cells showed increased TAp73 expression in comparison with quiescent peripheral blood derived cells. Activity of CDDP may therefore be mediated by TAp73, especially in the context of activation such as occurs in the LN microenvironment.

Ofatumumab (OFA) Vs. Physician’s Choice (PC) of Therapy in Patients (pts) with Bulky Fludarabine Refractory (BFR) Chronic Lymphocytic Leukaemia (CLL): Results of the Phase III Study OMB114242

OFA was approved for the treatment of fludarabine and alemtuzumab refractory CLL on the basis of a single-arm study which also included pts with BFR CLL. This open label, randomized, Phase III study of OFA or PC treatment in pts with CLL refractory to fludarabine and with bulky lymphadenopathy (at least one lymph node > 5 cm) was conducted to confirm the results of OFA in BFR pts and also through a 2nd randomization to compare extended OFA (12 months) vs. approved regimen of OFA (6 months). PC was considered an appropriate comparator for these pts given the lack of a consensus around standard of care treatment for this difficult-to-treat population.Pts were randomized 2:1 to receive either the approved regimen of 8 weekly infusions of OFA followed by 4 monthly infusions (Dose 1, 300 mg; Doses 2–12, 2000 mg) or PC of non-OFA containing therapy, including treatments approved for CLL and well established standards of care for up to 6 months. Pts in the OFA arm who had an investigator-assessed CR, PR or SD underwent a 2nd 2:1 randomization to 6 additional OFA infusions 2000 mg every 4 weeks (OFA Ext), or observation (Obs). Pts in the PC arm who developed disease progression during the study could receive OFA salvage therapy for up to 12 months of treatment. Premedication for OFA infusions consisted of glucocorticoid, paracetamol, and antihistamine.The primary objective of this study was to evaluate progression-free survival (PFS) with OFA treatment versus PC as assessed by an Independent Review Committee (IRC). Response assessments were performed using the 2008 Update of the NCI-WG CLL Guidelines [Hallek, 2008]. Secondary endpoints included overall response rate (ORR), time to next therapy (TNT), overall survival (OS), and safety.122 pts were randomized (79 OFA, 43 PC). Pts completed a median of 12 OFA infusions and 3 months PC therapy. The median PFS (mPFS) as measured by IRC was 5.4 months for OFA and 3.6 months for PC (hazard ratio [HR] 0.79, p=0.27, stratified log rank test; Fig 1). Median PFS as assessed by the investigator (INV) was 7.0 months for OFA and 4.5 months for PC (HR 0.56, p=0.003). The median time to start of next anti-CLL treatment was 11.5 months for OFA and 6.5 months for PC (p=0.0004, stratified log rank test). The ORR (95% CI) by IRC evaluation was 38% (27%, 50%) for OFA and 16% (7%, 31%) for PC (p=0.02). 37 pts underwent a 2nd randomization to either OFA Ext (n=24) or Obs (n=13). From 2nd randomization, mPFS (INV) was 5.6 months for OFA Ext and 3.5 months for Obs (HR: 0.49, p=0.026, stratified log rank test) (Fig 2). Grade ³3 infusion reactions occurred in 5% of pts in the OFA arm with no fatal reactions in either arm. Grade ≥3 cytopenias in the OFA arm included neutropenia (24%, no prolonged neutropenia and 1 patient with late onset neutropenia), thrombocytopenia (8%), anemia (8%). Of the 43 PC pts, 22 received OFA Salvage with an ORR of 50% and mPFS of 5.4 months.Table of Response and Time to event EndpointsOFA (N=79)PC (N=43)CharacteristicsMedian (range)Age, years61.5 (46 – 82)63 (40 – 76)No. prior therapies4 (2 – 16)3 (2 – 11)No. of infusions12 (1 – 18)3 (1 – 6)IRCInvestigatorEfficacy endpointsOFAPCp-valueOFAPCp-valueResponders, %38%16%0.01949%37%0.415mPFS, months5.363.610.26774.50.003OFAPCTNT, months11.56.5p=0.0004OS, months19.214.5p=0.13 [Display omitted] [Display omitted] Conclusions: Although the study did not meet its primary endpoint of demonstrating statistically significant superior PFS by IRC, the ORR, PFS by investigator, TNT, and OS favor OFA and were consistent with previously reported results in BFR pts. There was also a longer PFS in pts who underwent the 2nd randomization to receive OFA Ext vs. the approved OFA treatment regimen of 6 months treatment. DisclosuresÖsterborg:GSK: Research Funding. Off Label Use: Discussion of extended ofatumumab dosing (12 months) in addition to the approved 6 month dosing of ofatumumab in refractory CLL. Zaritskey:Novartis: Consultancy, Honoraria. Kaplan:GSK: Honoraria. Steurer:GSK: Consultancy, Honoraria, Research Funding. Schuh:GSK: Honoraria; Roche: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Napp: Honoraria. Montillo:GSK: Honoraria. Kulyaba:GSK: Honoraria. Gorczyca:GSK: Employment. Daly:GSK: Employment. Chai-Ni:GSK: Employment. Lisby:Genmab: Employment. Gupta:GSK: Employment.

52IN - Targeting the Cyclins

ABSTRACT Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the western countries. The disease can be quite heterogeneous in terms of clinical behavior with a portion of patients experiencing an indolent disorder with no or late need of therapy, while others more rapidly become symptomatic and require therapy. These patients will eventually relapse and some of them will show no response to the most effective treatments including immunochemotherapeutic combinations. Patients refractory to purine-nucleoside analogue based chemotherapy (“fludarabine refractory CLL”) have a particularly dismal prognosis, with an anticipated survival of 10 months. A number of novel treatments are now becoming available targeting relevant pahogenetic pathways in CLL and showing a promising level of activty in relapsed/refractory patients. Cyclin-dependent kinases (CDKs) act as “master regulators” of the cell cycle and are often overactive in human cancer, including lymphoid malignancies, leading to a loss of checkpoint integrity resulting in uncontrolled proliferation. Selective inhibition of CDKs has become of interest in CLL as it might limit the progression of leukemic cells through the cell cycle and facilitate induction of apoptosis, through a mechanism that is independent of TP53, the major genetic determinant of refractoriness in the disease. A number of inhibitors has been produced and tested in clinical studies showing significant responses in patients with relapsed CLL, including those with bulky lymphadenopathy or high risk genetic features such as TP53 abnormalities (i.e. del(17p). Characteristic dose limiting toxicity for all compounds tested is hyperacute tumor lysis syndrome (TLS). Though these compounds have probably created less enthusiasm as compared to other novel classes of inhibitors, until CLL remains uncurable, agents with novel mechanisms of action such as CDK inhibitors might well be part of the armamentarium of onco-hematologists when patients undergo multiple relapses. For this reason, while the future of these compunds appeared to be rather uncertain, more recently clinical interest in this class of highly-effective agents has been reinvigorated, with the appearance of novel compounds and the opening of new clinical trials. Disclosure: P. Ghia: Advisory board: Abbvie, Boehringer Igelheim, Gilead, Medimmune, Pharmacyclics, Roche Italia Research grant; GSK, Roche.

Outcomes of patients with fludarabine-refractory chronic lymphocytic leukemia: a population-based study from a well-defined geographic region

Patients with fludarabine-refractory (FR) chronic lymphocytic leukemia (CLL) receive novel agents in pivotal, non-randomised phase-2 trials. Understanding outcome of FR-CLL in health-care may provide important contextual information. Records from 1301 patients (Stockholm-Cancer-Registry 1991–2010) identified 92 FR-patients; bulky lymph-nodes (BFR-group), double-refractory (DR-group), or Others‘-group for outcome-analysis. Median age was 69 years 67% had Rai-stage III/IV with median 3 prior therapies. Overall response-rate was 20%; significantly lower in BFR (8%, p = 0.01) and DR (20%, p = 0.01) than in ‘Others‘ (31%). Time-to-treatment-failure (months) was significantly longer in ‘Others‘ (9.2) than in BFR/DR (5.3/4.4) (p < 0.01) and significantly longer (p < 0.05) in antibody-treated patients (9.1) compared to other regimens (5.2). Early-death occurred in 5%, ≥ grade III-infections in 20%. Median overall-survival (OS) was 18 months; 29 in BFR vs 13 in DR (p = 0.054). Male sex was the only prognostic factor on OS (p = 0.01, HR 2.2, multivariate-Cox-regression). Our results, without external referrals, facilitate interpretation of non-randomised trials/novel drugs in advanced-stage-CLL.

A Phase II Multi-Center Trial Of Pentostatin Plus Cyclophosphamide With Ofatumumab (PCO) In Older Previously Untreated Chronic Lymphocytic Leukemia (CLL) Patients

▪ IntroductionThe seminal phase 3 trial conducted by the German CLL Study Group demonstrated that the addition of the anti-CD20 monoclonal antibody [mAb] rituximab to the FC platform (FCR) improved response rates, progression free survival and also overall survival. However, FCR showed considerable hematologic toxicity, particularly among patients over age 70. Pentostatin demonstrated similar response frequency to other purine analogues in CLL. Furthermore, its relative lack of myelotoxicity has allowed to use it with improved tolerability particularly when administered in combination with myelotoxic agents such as cyclophosphamide. Ofatumumab is a fully human anti-CD20 mAb with clinical activity as a single agent in patients with fludarabine-refractory CLL. Ofatumumab appears to have greater single agent clinical activity than rituximab in patients with previously treated CLL and also has activity in rituximab-refractory patients. Given the reported efficacy of chemo immunotherapy [CIT] in CLL and the activity and toxicity profile of pentostatin combinations, we designed a trial of pentostatin, cyclophosphamide, and Ofatumumab for previously untreated older patients with CLL. MethodsPatients with CLL who required therapy (2008 NCI-WG guidelines) aged ≥ 65 years and ECOG PS of 0-2 were enrolled to receive Pentostatin 2 mg/sqm and Cyclophosphamide 600 mg/sqm both as intravenous infusions at day 1 of each 21 day cycle and Ofatumumab administered as intravenous infusions (Cycle 1: 300 mg day 1 and 1000 mg day 2, subsequent cycles: 1000 mg at day 1). Ofatumumab premedication included acetaminophen, antihistamine and glucocorticoid. Treatment duration was of 6 cycles. The primary endpoint was overall response rate (ORR) including detection of minimal residual disease (MRD) and secondary endpoints included, progression-free survival (PFS) overall survival (OS) and safety. Patients49 patients from 12 centres from the italians regions of Lombardy and Piedmont were included. Baseline demographics were: Median age 72.8 years with 64% aged over 70, among them 32 were males (65%). Disease characteristics in 32 patients evaluable at this point were: 76% Binet stage B and 24% C; 45% of patients had unmutated IGVH, 7 % showed 17p deletions. ResultsORR was 93.7% with 41% CR(11)/CR(2) with incomplete marrow recovery [CRi]. All six intended courses of treatment were administered to 30 (94%), and 90% of these patients received full-dose treatment. The reason for discontinuing treatment before completing six courses was myelosuppression occurring in 2 patients. The primary reason for dose reduction was again myelosuppression. Grade ≥3 AEs that occurred from start of treatment until 60 days after the last dose were experienced by 62% of patients receiving PCO with the most common being neutropenia [Total number of patients with at least one Grade 3 or 4 event: 19 patients] while anemia and thrombocytopenia were detected only as grade 1 to 2 in 41% and 25% of cases respectively. Of the grade 1 to 2 toxicities, fever occurred in 2 patients (6%), hypotension occurred in 2 patients (6%), nausea and vomiting occurred in 3 patients (9%), skin rash of grade 1 occurred in 2 patients. Grade 3 infusion-related AEs were reported in 12% of patients. There were no grade 4 toxicities associated with any Ofatumumab infusion. Grade 3 infection was reported in 1 patient (3%) being a pneumonia. No deaths during treatment occurred in these 32 subjects. ConclusionOfatumumab added to Pentostatin and Cyclophosphamide demonstrated clinically important results and is well tolerated in patients with previously untreated CLL. In this preliminary report the efficacy of this ofatumumab-based CIT compares favorably to the historical rituximab-based CIT using the same chemotherapeutic agents with a more manageable side effect profile in this older population. Further data in a higher number of enrolled patients and including MRD detection will be presented at the Meeting. Disclosures:Montillo:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rambaldi:Novartis: Honoraria; Sanofi: Honoraria; Italfarmaco: Honoraria.

Rituximab and High-Dose Dexamethasone (R-dex) Is Effective In The Treatment Of Relapsed/Refractory Chronic Lymphocytic Leukemia

▪ IntroductionHigh-dose methylprednisolone (HDMP) in combination with rituximab is active in the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) but serious infections are frequent. Recently published data suggest that high-dose dexamethasone might be equally effective to HDMP despite lower cumulative dose. AimsTo assess efficacy and safety of high-dose dexamethasone combined with rituximab (R-dex) in relapsed/refractory CLL. Patients and MethodsA total of 60 pts (pts) with relapsed/refractory CLL treated at a single tertiary center between September 2008 and October 2012 were included in this retrospective analysis. Basic characteristics are summarized in Table 1. The schedule of R-dex consisted of rituximab 500 mg/m2 i.v. day 1 (375 mg/m2 in cycle 1) and dexamethasone 40 mg orally on days 1-4 and 10-13. Treatment was repeated every 3 weeks for a maximum of 8 cycles. All pts received antimicrobial prophylaxis with sulfamethoxazole/trimethoprim and aciclovir.Table 1Basic characteristics.Total number of patients60Age (median, range)69 (54-83)Males73%Rai stage III/IV65%Bulky lymphadenopathy (≥ 5cm)58%Previous treatment lines (median, range)2 (1-7)Unmutated IgVH82%Del 11q42%Del 17p19%Fludarabine-refractory50%Bulky fludarabine-refractory33% ResultsMedian number of administered R-dex cycles was 6 (range, 1-8). The overall response (ORR)/complete remissions (CR) were achieved in 75/3%. At the median follow-up of 9 months, median progression-free survival was 8 months and median overall survival 24 months. Significant predictors of short PFS in univariate analysis were bulky lymphadenopathy (p=0.023) and refractoriness to fludarabine (p=0.02). Interestingly, activity of R-dex in bulky fludarabine-refractory CLL was similar to ofatumumab (ORR 62 %, median PFS, 4 months, median OS, 12 months) (Wierda et al., 2010). R-dex was successfully used as a debulking regimen before allogeneic stem cell transplantation in 8 patients. Serious (CTCAE grade III/IV) infections occurred in 29% of patients; 19% pts developed steroid diabetes requiring temporary short-acting insulin. ConclusionsOur data show that R-Dex is an active and feasible treatment for patients with relapsed/refractory CLL; however, major infections remain relatively frequent despite combined antimicrobial prophylaxis. In addition, durable responses are infrequent. Therefore, further optimization of this therapeutic approach is warranted. Updated results will be presented. Disclosures:No relevant conflicts of interest to declare.

Bendamustine plus alemtuzumab is safe and feasible treatment for fludarabine refractory chronic lymphocytic leukaemia (CLL)

Bendamustine plus alemtuzumab is safe and feasible treatment for fludarabine refractory chronic lymphocytic leukaemia (<scp>CLL</scp>)

NOTCH1, SF3B1, and TP53 mutations in fludarabine-refractory CLL patients treated with alemtuzumab: results from the CLL2H trial of the GCLLSG

AbstractWe studied the incidences, associations, and prognostic roles of NOTCH1 and SF3B1 mutations (NOTCH1mut, SF3B1mut) as compared with TP53mut in fludarabine-refractory chronic lymphocytic leukemia (CLL) patients treated with alemtuzumab in the CLL2H trial. We found NOTCH1mut, SF3B1mut, and TP53mut in 13.4%, 17.5%, and 37.4% of patients, respectively. NOTCH1mut and SF3B1mut were mutually exclusive, whereas TP53mut were evenly distributed within both subgroups. Apart from correlation of SF3B1mut with 11q deletion (P = .029), there were no other significant associations of the mutations with any baseline characteristics or response rates. However, NOTCH1mut cases had a significantly longer progression-free survival (PFS) compared with wild-type cases (15.47 vs 6.74 months; P = .025), although there was no significant difference with overall survival (OS). SF3B1mut had no significant impact on PFS and OS. In multivariable analyses, NOTCH1mut was identified as an independent favorable marker for PFS. This clinical trial is registered at www.clinicaltrials.gov as #NCT00274976.

Ofatumumab in Rituximab-Refractory Autoimmune Hemolytic Anemia Associated With Chronic Lymphocytic Leukemia: A Case Report and Review of Literature

Clinical Practice Points Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in the Western world and usually affects individuals older than the age of 65. The main clinical manifestations are lymphocytosis, lymphadenopathy, hepatosplenomegaly, anemia, thrombocytopenia, hypogammaglobinemia, and autoimmune disorders including autoimmune hemolytic anemia (AIHA). Chronic lymphocytic leukemia cells aberrantly coexpress CD5 (a T-cell marker) andCD19 andCD23 (B-cell markers) and they have low-level expression of CD20. Despite advancements in molecular characterization and treatment strategies, CLL remains incurable and most patients do relapse despite chemotherapy. Currently the fludarabine, cyclophosphomide, and rituximab regimen seems to be the best regimen in terms of prolonging survival and maintaining diseasefree progression in untreated and relapsed cases. In relapsed cases, rituximab (a chimeric monoclonal antibody against CD20) monotherapy has been used because of its low adverse effect profile and has been reported to show significantly long response duration in patients after fludarabine induction. Rituximab has been used to treat autoimmune complications of CLL, such as AIHA. Recently, ofatumumab, a fully human monoclonal antibody, that targets a unique epitope on the CD20 molecule that differs from the one targeted by rituximab, has been approved in treatment of fludarabine-refractory CLL. Here we report a case of a 70-year-old woman with the diagnosis of CLL who developed AIHA that was refractory to steroids and rituximab, yet had complete resolution of the hemolysis after ofatumumab treatment. To our knowledge, this is the first case report of ofatumumab for the treatment of AIHA.

Abstract 597: The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B.

Abstract Chronic Lymphocytic Leukaemia (CLL) is the most common hematological malignancy in the western world. Fludarabine, a nucleoside analogue, is commonly used in combinational treatments for CLL. However, patients commonly develop resistance to fludarabine based therapies. Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), showed synergistic apoptotic responses when combined with fludarabine in human leukemic cells and primary CLL cells. The mechanism for this synergistic apoptotic response is unknown. We found that fludarabine and VPA treatment increased activates caspases-8, -9, -3, and caspase-2 and a decreased in expression of anti-apoptotic proteins Mcl-1 and XIAP. Treatment with fludarabine alone or in combination with VPA led to the loss of lysosome integrity suggesting a leakage of the lysosomal content into the cytosol in response to these drugs. Chemical inhibition of a specific lysosomal protease, cathepsin B, using CA074-Me was sufficient to stabilize Mcl-1 and XIAP while reducing caspase activation and apoptosis. Addition of purified cathepsin B to leukemic cell lysates led to the reduction in protein levels of Mcl-1, XIAP and pro-caspase-2, thus suggesting that the re-localization of cathepsin B into the cytosol is sufficient to degrade these proteins. VPA treatment increased cathepsin B levels in both leukemic cell lines and primary CLL cells. VPA also increased cathepsin B activity. In addition, six relapsed CLL patients who had received at least one prior therapy with fludarabine were treated with VPA alone or in combination with fludarabine. No responses were seen after 28 days using VPA alone but in five patients who continued on VPA with fludarabine, three patients showed reduced lymphocyte count after receiving at most five cycles of the combination therapy. When the CLL cells from VPA-treated CLL patients were examined, VPA administration induced increased levels of histone-3 acetylation and cathepsin B expression in vivo. Thus, VPA and fludarabine synergistic apoptotic response is mediated by cathepsin B activation leading to a decrease in anti-apoptotic proteins in CLL cells and likely contributes to the clinical response observed in relapsed, fludarabine-refractory CLL patients. Citation Format: Ju Yoon Yoon, David Szwajcer, Ganchimeg Ishdorj, Pat Benjaminson, Rajat Kumar, James B. Johnston, Spencer B. Gibson. The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2013-597

BIRC3 but Not BIRC2 mRNA Expression in Chronic Lymphocytic Leukemia Correlates with Disease Progression and Mediates Decreased Fludarabine Sensitivity in Vitro

AbstractAbstract 563 IntroductionThe molecular characterization of chronic lymphocytic leukemia (CLL) with whole genome sequencing has recently begun to foster a novel understanding of the disease, identifying pathways of importance in CLL biology. One of the target genes that was identified by this approach was BIRC3 (Rossi et. al. Blood 2012). BIRC3 (cIAP2) and its close homolog BIRC2 (cIAP1), are members of the IAP (inhibitior of apoptosis) protein family, that unlike other IAPs, do not directly interfere with apoptosis but are key components of the canonical and non-canonical NFKB signalling pathways. As such they play an important role in B-cell differentiation, survival and proliferation. In the murine system BIRC2 and BIRC3 were shown to be redundant (Gardam et. al. Blood 2011). Their combined loss rendered murine B-cells independent of some survival factors and led to uncontrolled B-cell accumulation. In CLL, Rossi et. al. reported recurrent mutations in BIRC3 that were rare at diagnosis (<5%), but associated with increased non-canonical NFKB signalling, worse prognosis and fludarabine refractory disease, independent of p53. We aimed to determine if BIRC3 mutations might be a flag for an importance of the pathway in a larger context of CLL biology and wanted to know whether BIRC3 expression levels are important for clinical outcome. Materials and MethodsPeripheral blood MCs of 63 fully characterized CLL patients were collected. mRNA levels of BIRC2 and BIRC3 were analysed using Real Time PCR and normalized to 18S as housekeeping control. Cutoffs to detect different time to treatment intervals were determined by ROC analyses. In vitro culture of CLL cells was performed under standard culture conditions in presence or absence of accessory cells (NIH 3T3 fibroblasts +/− CD40L transgene) in order to abrogate spontaneous apoptosis or achieve CLL cell activation for 3 days. siRNA transfections were performed with non-targeting siRNA (NTC), BIRC2 or BIRC3 siRNA using lipofection as recommended by the manufacturer (Qiagen). ResultsBIRC3 and BIRC2 mRNA levels were compared with established risk factors and clinical course. We detected significantly downregulated BIRC3 expression levels (P < 0.001) in CLL with unmutated (N = 24) as compared to CLL with a mutated B-cell receptor status (N = 33) and in CLL of advanced RAI stages (N = 19, P < 0.01). Low expression of BIRC3 mRNA was furthermore significantly correlated to decreased time to first treatment (P < 0.001, Fig 1A), which highlights the prognostic impact of BIRC3 mRNA levels independent of potential BIRC3 mutations. Since our test population was previously untreated and cut-offs for prediction were close to the median, this effect was presumably independent of the occurrence of BIRC3 mutations, that should be rare in such a population. To clarify this, mutation analysis is currently ongoing. Despite the proposed functional redundancy, the mRNA levels of the homologue BIRC2 did not correlate with prognostic factors or time to treatment (Fig 1B) giving first hints, that in the context of CLL BIRC3 may be more important as compared to its homolog, which is also reflected in the dominance of BIRC3 mRNA levels as compared to those of BIRC2 (N = 63, P < 0.0001). To further address the question of functional redundancy we performed siRNA mediated downregulation of either BIRC2 or BIRC3. We found that BIRC3 but not BIRC2 siRNA mediated downregulation significantly inhibited fludarabine induced cell death of CLL cells in vitro (N = 6, Fig 1C). Discussion and ConclusionHere we demonstrate for the first time that in addition to the reported effect of mutations in BIRC3, mRNA levels were related to negative prognostic factors and predictive for shorter time to treatment. We further provided evidence that BIRC3 and BIRC2 potentially have non-redundant roles indicated by their different behavior, opposite clinical parameters and different outcomes in response to siRNA mediated downregulation in vitro. Importantly and consistent with a potential selection for mutations, downregulation of BIRC3, but not BIRC2, increased in vitro resistance of CLL cells against fludarabine, the backbone of CLL therapy. Considering the accumulation of BIRC3 mutations in fludarabine refractory CLL (Rossi et. al. Blood 2012), BIRC3 might serve as a novel biomarker, that together with aberrations in p53, could characterize CLL patients with suboptimal therapy success and inferior outcome. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NOTCH1, SF3B1 and TP53 Mutations in Fludarabine-Refractory CLL Patients Treated with Alemtuzumab: Results From the CLL2H Trial of the Gcllsg

AbstractAbstract ▪710▪This icon denotes a clinically relevant abstractGenetic alterations play a major role in stratifying prognostic risk groups in chronic lymphocytic leukemia (CLL). Recently, next generation sequencing strategies revealed novel recurrent mutations in genes such as NOTCH1 and SF3B1 in CLL. Correlations have been reported for SF3B1 mutations (SF3B1mut) with deletion 11q and for NOTCH1 mutations (NOTCH1mut) with trisomy 12. Both mutations have been associated with unmutated IGHV, refractory disease and inferior outcome in heterogeneous cohorts outside clinical trials.We assessed the incidence and prognostic impact of NOTCH1, SF3B1 and TP53 mutations in the CLL2H trial of the GCLLSG, a multicenter phase II trial of subcutaneous alemtuzumab (3 × 30mg/week, max. 12 weeks, n=103, NCT00274976) for fludarabine-refractory CLL patients. Genetic studies were performed in the central reference laboratory of the GCLLSG on 97/103 (94%) trial patients based on availability of DNA. NOTCH1 was analyzed by Sanger sequencing of a PCR fragment from the PEST domain (exon 34, chr9:139,390,619–139,391,290), while SF3B1 (exons 13–16) and TP53 (exons 2–11) were analyzed by DHPLC (WAVE® 3500HT, Transgenomic Inc.) followed by sequencing of aberrant fragments.NOTCH1mut were found in 13/97 (13.4%), SF3B1mut in 17/97 (17.5%), and TP53mut in 37/99 (37.4%) cases. Two different types of NOTCH1mut (c.7541_7542delCT, n=11; c.7444delC, n=2) and 13 different point mutations of SF3B1 were identified. All SF3B1mut were heterozygous missense mutations resulting into 11 different amino acid changes in exons 14, 15 and 16. Of note, in none of the 97 cases concurrent NOTCH1mut and SF3B1mut was found. IGHV was unmutated in 71/90 (79%) cases overall, in 100% of NOTCH1mut (p=.062) and in 60.0% of SF3B1mut (p=.078). Moreover, SF3B1mut was associated with 11q deletion (p=.042), but we observed no significant correlation of NOTCH1mut with trisomy 12 (p=.321).TP53mut was found together with NOTCH1mut in 3/12 (25%, p=.349) and with SF3B1mut in 7/17 (41%, p=1.000) cases.The overall response rates (ORR) among the 97 cases was 34% (3% CR and 31% PR). After a median follow-up time of 33.9 months, the median progression-free survival (PFS) was 7.7 months, and the median overall survival (OS) was 18.3 months. The ORR was not significantly different when comparing the groups with or without mutations (50.0% vs. 34.2% for NOTCH1, p=.341 and 41.2% vs. 35.1 % for SF3B1, p=.781, respectively). PFS in NOTCH1mut cases was significantly superior as compared to wild type cases (median 15.47 months vs. 6.74 months, p=.025, see Figure) but there was no significant difference in OS (median not reached vs. 18.3 months, p=.181). For SF3B1mut, we found no significant difference between mutated and wild type cases in PFS (median 4.76 vs. 7.72, p=.974) and OS (median 29.0 vs. 17.1, p=.243). Multivariate analysis of PFS and OS was performed including age, ECOG performance status, WBC, thymidine kinase, β2-microglobulin, IGHV status, 11q deletion, 17p deletion, NOTCH1mut, SF3B1mut and TP53mut as potential factors. Regarding PFS, ECOG performance status > 1 (HR 2.080, p=.057), increased thymidine kinase (cont.) (HR 1.479, p=.007) and elevated β2-microglobulin (cont.) (HR 1.188, p=.0003) emerged as independent unfavorable prognostic factors, whereas NOTCH1mut (HR 0.375, p=.049) was a favorable marker. Regarding OS, none of the gene mutations showed significant prognostic impact, whereas age at increments of 10 years (HR 1.035, p=.024), ECOG performance status > 1 (HR 2.575, p=.017) and increased thymidine kinase (HR 1.568, p=.002) were identified as independent unfavorable factors.In summary, our analysis performed in a multicenter trial of fludarabine-refractory CLL patients showed NOTCH1mut in 13.4%, SF3B1mut in 17.5% and TP53mut in 37.4% cases, with NOTCH1mut and SF3B1mut being mutually exclusive. As compared to non-refractory CLL cohorts, the incidence of NOTCH1mut and SF3B1mut do not appear to be increased which is in contrast to TP53mut. NOTCH1mut was found as a favorable prognostic marker for PFS, but none of the mutations impacted OS in this trial. [Display omitted] Disclosures:Hallek:Roche: Consultancy, Honoraria, Research Funding. Stilgenbauer:Genzyme: Consultancy, Honoraria, Research Funding.

Therapeutic advancement of chronic lymphocytic leukemia.

Despite the combinations of chemotherapy with monoclonal antibodies have further improved response rates, chronic lymphocytic leukemia (CLL) remains an incurable disease with an extremely variable course. This article reviews the ongoing clinical advances in the treatment of CLL in both previously untreated and relapsed disease and focuses on the benefit of different therapeutic strategies, the most effective therapy combinations and the potential activity of novel agents. Novel agents and combination therapies have been investigated by several studies in both the upfront and relapsed setting, particularly for patients with 17p deletion, TP53 mutation and fludarabine-refractory CLL. While these agents and combination therapies have improved initial response rates, ongoing studies are continued to determine and improve the efficacy and safety. Despite advancements in the treatment of CLL have led to high response rates, allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option and reduced-intensity conditioning (RIC) allo-HSCT must be strongly considered whenever feasible. As such, ongoing studies of these agents and other novel approaches in clinical development are needed to expand and improve treatment options for CLL patients.

Ofatumumab retreatment and maintenance in patients with fludarabine-refractory CLL.

6584 Background: In Study 406, the anti-CD20 monoclonal antibody ofatumumab (ofa), given as monotherapy over 6 months, showed 47% overall response rate (ORR) in patients (pts) with chronic lymphocytic leukemia (CLL) refractory to fludarabine and alemtuzumab (FA-ref), or to fludarabine with bulky (&gt;5cm) lymphadenopathy (BF-ref). The effects of ofa retreatment (retx) and maintenance (mt) are unknown. Methods: Pts who responded to ofa and then progressed or had stable disease (SD) in Study 406, were offered retx in Study 416 (NCT00802737; GSK/Genmab) with ofa 1 x 300 mg + 7 x 2000 mg weekly followed by mt with ofa 2000 mg monthly for up to 2 years (if SD or better). Primary endpoint was ORR (1996 NCI-WG). Safety and time to event outcomes were also assessed. Results: Of 29 pts enrolled, 7 had SD and 22 had partial response (PR) and progressed in Study 406. Pts were defined per Study 406: 17 FA-ref, 11 BF-ref, 1 “other”. Pretreatment characteristics were similar between groups. Pts received a median of 12 doses (range 3-32); 86% had 8 doses, 3% received all 32 doses. 72% of pts had infusion-related adverse events (AEs), 41% at 1st infusion, mostly grade 1-2. The most common grade &gt;3 AE occurring up to 60 days after last tx was infection (38%); the most common was pneumonia (17%). 3 pts (10%) had fatal infections, all bronchopneumonia. Clinical efficacy is shown in Table. Comparative analysis to Study 406 is ongoing. Conclusions: The response to ofatumumab (ofa) as induction retreatment and progression-free survival in this limited number of pts was similar to 1st treatment (Study 406). Ofa maintenance had some clinical benefit for pts with advanced CLL. Ofa was well-tolerated with no unexpected toxicities. [Table: see text]

Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia

AbstractThe genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

The Hsp90 inhibitor SNX-7081 synergizes with and restores sensitivity to fludarabine in chronic lymphocytic leukemia cells with lesions in the TP53 pathway: a potential treatment strategy for fludarabine refractory disease

Drug resistance in chronic lymphocytic leukemia (CLL) associated with lesions in the ATM/TP53 pathway represents a major challenge in clinical management. Evidence suggests that heat shock protein-90 (Hsp90) inhibitors may represent a therapeutic option in combination with more conventional therapies. We explored the effects of combining the Hsp90 inhibitor, SNX-7081, with fludarabine in vitro against CLL cells and hematological cell lines. In seven cell lines and 23 patient samples synergy between SNX-7081 and fludarabine (2-FaraA) was apparent in the three TP53 mutated cell lines and at significantly lower concentrations in TP53 or ATM dysfunctional patient cells. In 11/13 2-FaraA-resistant patient samples, SNX-7081 reduced the 50% inhibitory concentration to within a clinically achievable range. Synergy between SNX-7081 and 2-FaraA was evident in both the cell lines and patient samples as a significant decrease in cell viability. Our data suggest that combining SNX-7081 and fludarabine may be effective in the treatment of fludarabine-refractory CLL.