Unmutated IgVH Research Articles

ZAP-70 Expression Assessed by Immunohistochemistry Correlates with Time to First Treatment in Patients with Chronic Lymphocytic Leukemia.

Abstract 4686 BackgroundZAP-70 expression is a prognostic indicator in chronic lymphocytic leukemia (CLL). Initial studies measured intracellular ZAP-70 expression by flow cytometry and defined a cut-off of 20% as predictive of time from diagnosis to initial therapy and overall survival. However, due to technical challenges, standardization of methods for evaluation of ZAP-70 expression has been difficult. This has resulted in a limitation of applicability for a potentially useful clinical test. Immunohistochemistry (IHC) is easy to perform and interpret, and therefore may be a more reliable and accurate means for assessing ZAP-70 expression. We investigated the potential prognostic implications of ZAP-70 expression assessed by IHC. MethodsWe searched our CLL database for all patients seen at Weill Cornell Medical College between 2007 and 2009 with an identifiable date of diagnosis, date of first therapy, IgVH mutation status, and ZAP-70 status. All patients gave informed consent to participate in the research database. Paraffin-embedded cell blocks were prepared from formalin-fixed peripheral blood mononuclear cells and evaluated by IHC for PAX5, CD3, and ZAP-70. The entire cell clot was reviewed. If more than 20% of cells faintly expressed ZAP-70, the ZAP-70 status was considered to be positive. Patients had IgVH mutations status determined by a commercial laboratory with <98% sequence homology used as a cut-off. Time from diagnosis to first treatment (TT) was estimated by the Kaplan-Meier method; comparisons were performed by log-rank test. Pearson's pairwise correlation coefficient was used to evaluate the relationship between ZAP-70 and IgVH. ResultsOne-hundred thirty-seven patients with date of diagnosis and date of first treatment were identified in the database. Sixty-six patients have been treated to date. ZAP-70 status was available for 106 patients, 62 were positive, 44 were negative. In a subset of 20 patients, two blinded pathologists reviewed IHC and found perfect agreement on ZAP-70 status. IgVH mutation status was available for 86 patients, 34 were unmutated, and 52 were mutated. Eighty-two patients had both ZAP-70 status and IgVH mutation status available. There was good correlation between ZAP-70 and IgVH status (r=0.36, p=0.0008). Seventy-five percent of those with unmutated IgVH were ZAP-70 positive. Sixty-one percent of patients with mutated IgVH were ZAP-70 negative. However, only 57% of ZAP-70 positive patients were IgVH unmutated. The predicted median TT was 54 months for the whole cohort (range 0-376+ months, 95% CI 45-96 months). The median TT was 27 months (range 0-112 months) for ZAP-70-positive patients compared to 96 months (range 0-376+ months) for ZAP-70-negative patients (p=0.002). The median TT was 32 months (range 0-112 months) for IgVH unmutated patients compared to 88 months (range 0-376+ months) for IgVH mutated patients (p=0.01). ConclusionsZAP-70 as measured by IHC was feasible in a large cohort of patients and ZAP-70 status correlated with time to first treatment. These data suggest that ZAP-70 assessment by IHC may be a reliable prognostic tool in CLL and should be evaluated in larger, multicenter clinical studies. Disclosures:No relevant conflicts of interest to declare.

Treatment with Fludarabine and Rituximab Produces Extended Overall Survival (OS) and Progression-Free Survival (PFS) in Chronic Lymphocytic Leukemia (CLL) without Increased Risk of Second Malignancy: Long-Term Follow up of CALGB Study 9712.

Abstract 539 Introduction:The addition of rituximab to fludarabine-based regimens in CLL has been shown to produce high response rates with extended remissions. The long-term follow-up of these regimens with respect to progression, survival, and development of secondary malignancies has been limited. Patients and Methods:We report the long-term follow up of the chemoimmunotherapy trial CALGB 9712 (Blood 2003;101:6-14). This trial randomized 104 untreated, symptomatic patients to receive either 6 monthly cycles of fludarabine plus rituximab (FR) followed 2 months later by 4 weekly doses of rituximab (concurrent arm) or 6 monthly cycles of single agent fludarabine followed by rituximab consolidation using 4 weekly doses (sequential arm). With a median follow up of 92 months (range: 60-107), we analyzed the updated CALGB database and flow sheets submitted by treating physicians. Results:The overall response rate (ORR) was 84% (95% CI: 77%-91%), with a 90% ORR in the concurrent group (95% CI: 82%-98%) and a 77% ORR in the sequential group (95% CI: 66%-89%). Complete response (CR) was seen in 38% of patients (95% CI: 30%-45%), and partial response (PR) in 46% (95% CI: 38%-54%). The median OS was 85 months (95% CI: 71-95), with 71% of patients alive at 5 years (95% CI: 61%-79%). The median PFS was 37 months (95% CI: 27-45), with 27% progression-free at 5 years (95% CI: 19%-36%). With long-term follow up, the estimated median OS and PFS for the concurrent group were 84 months (95% CI: 57-100) and 32 months (95% CI: 23-55), respectively; the median OS and PFS for the sequential group were 91 months (95% CI: 71-110) and 40 months (95% CI: 23-50), respectively. Patients with del(17p13.1)/del(11q22.3)(18 patients) and unmutated IgVH(43 patients) continue to have an inferior OS (P=0.01 and P=0.04, respectively) and PFS (P=0.03 and P=0.04, respectively) compared to those without these abnormalities. We next assessed the frequency of therapy-related myeloid neoplasms (t-MN) and other cancers occurring after this chemoimmunotherapy regimen. No patient has developed MDS or AML prior to relapse. One patient (1%) developed t-MDS following relapse and receipt of FCR 41 months after completing trial therapy; t-MDS was diagnosed 9 months later. Richter's transformation was noted in three (3%) of the CALGB 9712 patients with large cell (n=2) or Hodgkin lymphoma (n=1). Second malignancies have included localized basal cell or squamous cell skin cancer in 12 (12%) patients whereas 11 (11%) have developed other epithelial malignancies including 4 GI, 3 lung, 3 melanomas, and 1 prostate cancer. Conclusions:Long-term follow up of patients enrolled on CALGB 9712 demonstrates extended OS and PFS with fludarabine plus rituximab, given either concurrently or sequentially, with an estimated 17%(95% CI: 9%-27%) of responders still in remission 8 years later. Looking at other published data, patients treated with FR administered concurrently or sequentially do not appear to have an increased risk of t-MN or second cancers. These long-term data reaffirm that FR is one of several acceptable frontline treatments for symptomatic patients with CLL. Disclosures:Morrison:Genentech: Speakers Bureau.

Combination Therapy with Lenalidomide and Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL).

Abstract 206Lenalidomide is an immunomodulatory agent that has clinical activity in CLL. In patients (pts) with relapsed/refractory CLL treatment with single agent lenalidomide induces an overall response rate of 32–47% when used as monotherapy (Chanan-Khan A.A. et al. 2006; Ferrajoli A. et al. 2008). Rituximab has modest activity as monotherapy, but significally synergizes with chemotherapy agents when administered to pts with CLL. The addition of rituximab to lenalidomide resulted in clinical responses in a small number of pts with CLL that had progressed while on lenalidomide monotherapy (Chanan-Khan A.A. et al. 2006). Because lenalidomide stimulates NK cell proliferation (Wu et al. 2008) we hypothesized that lenalidomide will enhance the activity of rituximab.We, therefore, designed a phase II study to evaluate the combination of lenalidomide and rituximab in pts with relapsed CLL.Pts with CLL and active disease were eligible if they had received prior treatment with purine analog-based therapy. Standard inclusion criteria were required in terms of organ function and performance status, and pts with any ANC or platelet count were eligible.All pts received rituximab (375 mg/m2) intravenously on days 1, 8, 15 and 22 of cycle 1, and then once every 4 weeks during cycles 3–12. Lenalidomide was given orally at the dose of 10 mg/day starting on day 9 of cycle 1 and continued daily for 12 cycles. Each cycle consisted of 28 days of treatment. During the first two weeks of therapy, allopurinol at the dose of 300 mg daily was prescribed as prophylaxis for tumor lysis.Sixty pts were accrued between October 2008 and July 2009. Thirty-seven pts have received treatment for at least 6 cycles and are evaluable for response and toxicity. The median age is 59 yrs (range 44–83), 15 pts (41%) have Rai stage III-IV disease and the median beta-2M level was 3.6 mg/dL (1.5–9). The median number of prior treatments was 2 (1–9), 9 pts (24%) were refractory to fludarabine and all pts had received prior rituximab. Twenty-six pts (70%) had unmutated IgVH, 9 pts (24%) had chromosome 17p deletion and 10 pts (37%) had 11q deletion by FISH analysis.After 6 cycles of treatment, 25 pts achieved a response [6 nodular PR (16%), 19 PR (51%)] for an OR of 68% (according to 1996 NCI-WG criteria). Six pts (16%) attained stable disease or clinical improvement and are continuing on treatment, and 6 pts (16%) failed to respond, including one death that occurred on day 34 owing to infectious complications. Responses according to pts characteristics are summarized in the table: [Display omitted] Most common grade 3–4 treatment related adverse events observed were: neutropenia (16 pts, 43%), fatigue (6 pts,16%) and thrombocytopenia (4 pts, 11%). One pt (3%) developed grade 3 tumor lysis syndrome and 1 pt (3%) had grade 3 joint pain. Infectious complications occurred in 9 pts (24%): neutropenic fever (6 pts), pneumonia (2 pts) and urosepsis (1 pt). Lenalidomide-associated tumor flare reaction was limited to grade 1 (8 pts, 22%) and grade 2 (1 pt, 3%).We examined the effect of therapy with lenalidomide and rituximab on the distribution of circulating B, T, and NK cell subsets. When compared to the baseline, there were significant decreases in the percentage of CD19+CD20+ B cells along with significant increases in the percentages of CD4+ T, CD8+ T, CD4+CD25hiCD127− regulatory T, and CD3−CD16+CD56+ NK cells after 3 cycles of therapy (paired sample t test).In conclusion, our results suggest that the combination of lenalidomide and rituximab is superior to single agent lenalidomide, despite all our pts having received prior rituximab. Additionally, there was no increase in toxicity and lenalidomide-associate tumor flare reaction was less frequent and less severe with this combination compared to single agent lenalidomide. Disclosures:Ferrajoli:Celgene: Research Funding; Genentech: Research Funding. Off Label Use: The use of Lenalidomide for the treatment of CLL is considered investigational.. O'Brien:Celgene: Consultancy; Genentech: Research Funding. Wierda:Genentech: Honoraria; Celgene: Speakers Bureau. Keating:Celgene: Data Monitoring Committee, Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

Health Related Quality of Life (HRQOL) in Patients Receiving Chemoimmunotherapy with Fludarabine (F), Cyclophosphamide (C), and Rituximab (R) (FCR) or Fludarabine and Cyclophosphamide (FC) for First Line Therapy with Advanced Chronic Lymphocytic Leukemia (CLL).

Abstract 3438Poster Board III-326 INTRODUCTIONChemoimmunotherapies like the FCR combination have been shown to increase complete remission rates and progression-free survival in patients with CLL in comparison to chemotherapies without biologicals (Hallek et al., ASH 2008). Until now little is known on HRQOL outcome of CLL patients receiving chemoimmunotherapy. Therefore we assessed the HRQOL in patients with advanced CLL, who were randomized between FC and FCR treatment within an international randomized trial of the German CLL Study Group (GCLLSG). METHODS817 pts with good physical fitness as defined by a cumulative illness rating scale (CIRS) score of up to 6 and a creatinine clearance (cr cl) ≥ 70 ml/min were enrolled between July 2003 and March 2006. Pts were randomly assigned to receive 6 courses of either FC (N=409; F 25mg/m2 i.v. d1–3 and C 250 mg/m2 i.v. d1–3; q 28 days) or FC plus R (N=408; 375 mg/m2 i.v. d 0 at first cycle and 500 mg/m2 d1 all subsequent cycles; q 28 days). The EORTC C30 questionnaires were sent to all patients included in Germany or Austria at baseline, after 3, 6, 12 months (mo) and then in yearly follow-up (FU). In all other countries questionnaires were handed out to the patients personally on the same time points during their visits in the study center. The analysis of the questionnaires was performed according to the EORTC recommendations (Aaronson et al., 1993). The questionnaire contained a global health scale, five functional scales (physical, role, cognitive, emotional and social), three symptom scales (fatigue, pain, nausea and vomiting) as well as six single items (dyspnea, appetite loss, sleep disturbances, financial impact, constipation and diarrhea). Mean score values of the EORTC scales ranged between 0 and 100. High scores in the functional scales represent good HRQOL, low scores in the symptom scales a low symptom burden. RESULTSHRQOL was evaluated in 763 (93%) of the included patients who completed at least one questionnaire (376 (49%) FC and 387 (51%) FCR treated patients). The compliance rate was significantly higher in those countries, where the questionnaire was handed out personally (96% in other countries versus 92% in Germany and Austria; P=0.013). Pts answering the baseline questionnaire and at least one further questionnaire (444; 58%) were compared to those how did not (319, 42%): pts with only one or a missing baseline questionnaire had a significantly higher CIRS score (1,7 vs 1,4; P=0.007) and more frequently leukocytopenias (24% CTC grade 3 and 4 leukocytopenias vs 13%; P< 0.001). Age, distribution of Binet stages, gender, poor prognostic factors (del(11q) or del(17p), unmutated IgVH) and treatment arms were similar distributed between both groups. There were also no differences in the rate of other toxicities or response rates. A total of 482 questionnaires were available initially, 406 at interim staging, 454 at final staging, 496 after 12 mo FU, 414 after 24 mo FU and 198 after 36 mo FU.A comparison of the two treatment arms at interim or final staging after 3 and 6 months respectively showed no significant difference between both arms with regards to the global health status, functional scales and symptom scales. Dyspnoe was scored significantly higher during FC treatment in comparison to FCR (23 versus 18; P = 0.023). At 12, 24 and 36 months of FU no significant difference between FC and FCR in all functional scales, symptom scales, single item and global health status was found. Both treatment arms showed slight improvement (defined as difference of 5-10 points) of global health status at 12 months FU in comparison to baseline (FC: 62 at baseline vs 68 at FU 12; FCR: 62 vs 70). CONCLUSIONSAlthough the FCR regimen is associated with a higher rate of cytopenias in patients' perception this increased hematological toxicity does not result in a difference in HRQOL between both treatment arms. After a median observation time of 38 mo the better efficacy of the FCR regimen with regards on response rates and progression-free survival does not yet result in an improved HRQOL. For the final evaluation of HRQOL outcome after chemoimmunotherapy a longer is FU is needed. DisclosuresEichhorst:Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Hospira: Honoraria. Fischer:Roche: Travel Grand; Munipharma: Travel Grand. Fink:Roche: Travel Grand. Fingerle-Rowson:OrthoBiotech: Employment; Roche: Honoraria. Westermann:Roche: Travel Grand. Wendtner:Roche: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; BayerSchering: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Hallek:Roche: Research Funding, Speakers Bureau; Mundipharma: Research Funding, Speakers Bureau.

ZAP-70 Disrupts Dipeptidyl Peptidase 2 (DPP2)-Regulated Quiescence in Chronic Lymphocytic Leukemia (CLL).

Abstract 1251Poster Board I-273CLL is a common hematologic malignancy with heterogeneous outcomes. Overexpression of ZAP-70 and unmutated B-cell receptor (BCR) heavy chain gene (IgVH) confer an adverse prognosis. While it is accepted that ZAP-70 augments BCR signaling in CLL B-cells, it remains unclear how ZAP-70 contributes to disease propagation. Peripheral blood CLL B-cells accumulate in G0. We have previously shown that CLL B-cells express DPP2, a serine protease involved in maintenance of quiescence of resting but not activated lymphocytes. We identified two subsets of CLL: sensitive CLL (S-CLL), where CLL B-cells undergo apoptosis upon inhibition of DPP2, and resistant CLL (R-CLL), where inhibition of DPP2 does not cause cell death. Here we sought to validate our preliminary observation and establish the role of ZAP-70 and BCR signaling in resistance to apoptosis in CLL.The patient cohort included 152 subjects with B-CLL from the Hematology clinics at Tufts Medical Center, Dana-Farber Cancer Institute (both in Boston, MA), and the Lahey Clinic (Burlington, MA). IgVH mutational status, ZAP-70 expression and history of treatment were analyzed. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique. Cells were treated with ValboroPro (VbP, Point Therapeutics), a non-specific inhibitor of DPPs, or AX8819 (ActivX), a DPP2-specific inhibitor. To interfere with ZAP-70, cells were treated with 17-Allylaminodemethoxygeldanamycin (17-AAG, Calbiochem), an inhibitor of hsp90, a protein involved in stabilization of ZAP-70. For apoptosis analysis cells were stained with propidium iodide and Annexin V at 16 h of incubation and assayed by flow cytometry. A CBA assay was used to measure tyrosine-phosphorylated p72Syk and ZAP-70. Expression of p27 and p130 proteins was assessed by western blot analysis.Of 152 CLL patients 99 were males (65.1%). Median age was 63 years. Median follow up was 6 years. When the study samples were obtained, 107 patients (70.4%) were untreated. In apoptosis assays, 97 (63.8%) samples were categorized as S-CLL and 55 (36.2%) as R-CLL. The small molecule inhibitor data correlated with DPP2 anti-sense experiments. Patients with R-CLL were more likely to receive treatment of their disease and had a shorter treatment-free interval from disease diagnosis compared with S-CLL (HR=4.79, 95% CI, 4.7 to 15.2; p<0.0001).In the R-CLL subgroup, B-CLL cells also exhibited unmutated IgVH and expressed high level of ZAP-70 (p<0.001). R-CLL B-cells exhibited higher level of p72Syk phosphorylation compared with S-CLL (p<0.05), suggesting that those cells are partially activated. Meanwhile, S-CLL B-cells had high p27 and p130 protein level, characteristic of a quiescent state. ZAP-70 was phosphorylated at a similar level between the two subsets, consistent with earlier observations that ZAP-70 does not depend on its phosphorylation to enhance BCR signaling. Upon inhibition of DPP2, S-CLL B-cells became activated as evidenced by dramatic phosphorylation of p72Syk. Concomitantly, p27 and p130 protein levels decreased indicating inappropriate cell cycle entry. Co-inhibition of DPP2 and hsp90 in R-CLL B-cells increased apoptosis by a mean of 8.1%, indicating that their survival is dependent on ZAP-70.CLL can be categorized into two prognostic groups based on the susceptibility of B-cells to DPP2 inhibition-induced apoptosis. Resistance to apoptosis correlates with unmutated IgVH and high ZAP-70 and is associated with an unfavorable disease course. The distinction between the two subsets of CLL stems from the aberration in the quiescence program. While S-CLL B-cells rest in true G0, R-CLL B-cells are partially activated due to ZAP-70 co-stimulatory signal and escape apoptosis. Destabilization of ZAP-70 reverses the resistant phenotype. DPP2 inhibition alone or with concomitant inhibition of ZAP-70 warrants investigation as a therapeutic modality in CLL. DisclosuresNo relevant conflicts of interest to declare.

PHA767491, a Dual Cdc7/CDK9 Inhibitor, with Potential to Target Both Proliferation and Survival in CLL.

Abstract 2366Poster Board II-343CLL is characterized by a combination of a block in apoptosis allied to the steady expansion of a small proliferative compartment, which is more pronounced in marrow and nodal tissue than the peripheral blood. Current treatments for CLL are not curative and factors associated with resistance to standard therapy include IgVH mutations and abnormalities of p53. It is thought that persistence of cells within the proliferative nodal compartment contributes to relapse.PHA-767491 was originally identified as a potent inhibitor of the S phase promoting kinase Cell division cycle 7 (Cdc7), but has also been shown to have inhibitory activity against the cyclin dependent kinase 9 (CDK9). PHA-767491 has been shown to selectively induce apoptosis in cancer cells in a p53 independent fashion (Montagnoli et al Nat Chem Biol. 2008 Jun;4(6):357-65). An orally bioavailable form of PHA767491, NMS-354 is now in phase I clinical trials in solid tumors. Since transcriptional inhibition resulting from CDK9 inhibition could result in depletion of anti-apoptotic proteins with short half-life, such as Mcl-1 and XIAP, both critical for CLL survival, we hypothesized that PHA-767491 and NMS-354 may have considerable activity in CLL, targeting both survival and proliferation pathways and inducing cell death independently of p53 abnormalities. These studies detail the mechanism of action of PHA-767491 on primary CLL cells.Primary cell samples were isolated from a total of 20 patients with CLL with various stages of disease (8 Binet A, 6 Stage B, and 4 Stage C) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12), IgVH mutation, CD38 expression and B2M levels.PHA-767491 was equally effective at inducing apoptosis in CLL cells with both favourable and unfavourable prognostic features, including 17p del, 11q del and unmutated IgVH. The concentration of PHA-767491 required to induce cell death in 50% of the CLL cells (IC50) approximated to between 500 nM and 1 mM at 12 hours of treatment. Following treatment with 1 mM significant apoptosis was seen in all samples by 6 hours, and was maximal by 12 hours. Induction of apoptosis (assessed by TMRE, Annexin-V, caspase 3 activation and PARP cleavage) was noted as early as 4 hours. The mechanism of action of PHA-767491 was investigated by western blotting for a variety of apoptotic proteins following incubation with the compound. Rapid downregulation of Mcl-1 was noted within 4 hours of treatment and this was followed later (6-10 hours) by downregulation of XIAP. Levels of Bcl2 and Bcl-XL were unchanged.We next conducted cell cycle analysis, which showed that the CLL cells were in G0/G1 indicating that the likely mechanism of action in peripheral blood cells ex-vivo is likely to be due to Mcl-1 downregulation rather than Cdc7 inhibition. Subsequent immunohistochemical staining of lymph node biopsies from patients in this study showed evidence of Cdc7 activity in CLL cells (Cdc7, Mcm2 phosphorylation), providing evidence of a proliferative compartment potentially targetable by Cdc7 inhibition. Experiments to explore this aspect are ongoing.In summary these data suggest that PHA-767491 and related dual Cdc7/CDK9 inhibitors may have a role in treatment of high risk CLL and that further investigation is warranted. Disclosures:Montagnoli:Nerviano Medical Sciences: Employment.

Relationship Between Expression of TLRs and Disease Activity in CLL.

Abstract 4379 BackgroundToll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well known that chronic lymphocytic leukemia (CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course. AimsTo evaluate the correlation between the gene expression of TLRs in 41 patients with CLL (mean age±SD 66±18 years, range 42-90, 15 female and 26 male), the clinical course of the disease and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations). MethodsThe gene expression of TLR4, TLR9, and TLR10 was studied. Total RNA was extracted from B cells of patients and controls (pool of 10 healthy donors), cDNA was synthesized, and real-time PCR was performed. For each reaction 50 ng of cDNA were mixed with 1.25μl of TaqMan primer/probe set and 10.25μl of TaqMan Universal Master Mix. The TLR4, TLR9, and TLR10 expression was normalized according to GAPDH as an internal control gene and it was expressed as percentage of control. ResultsTLR4 gene expression was significantly lower in CLL patients compared to controls (18±3%, mean±SE) while TLR9, and TLR10 gene expression was higher (3457±500% and 2897±440%, respectively). Moreover, considering “progressive” CLL patients (n=20), TLR4 gene expression was significantly lower compared with “indolent” ones (n=21) (10.2±2.2% vs 24.9±4.9%, p=0.01). Patients with unmutated IgVH expression and unfavorable cytogenetic alterations (mostly 11q-) showed even more reduced TLR4 gene expression, but the small numbers did not allow statistical evaluation. TLR4 gene expression was lower in patients with infective diathesis, although not significantly (11.5±2.9% vs 20.0±3.9).The 6 “progressive” CLL patients with autoimmune diseases (hemolytic anemia, Evan's syndrome and phemphigus) showed significantly higher TLR4 gene expression compared with “progressive” patients without autoimmune phenomena (17.7±4.4% vs 6.9±2.1%, p=0.02), possible expression of activated immune system. ConclusionThe reduced TLR4 gene expression in CLL patients with “progressive” disease, unmutated IgVH status, and unfavourable cytogenetic alterations is consistent with a reduced ability to a proper immune response to Gram-negative bacterial lipopolysaccarides and consequent increased susceptibility to infections. Disclosures:No relevant conflicts of interest to declare.

Rituximab Maintenance Following Chemoimmunotherapy Improves Outcome in B-Cell Chronic Lymphocytic Leukemia.

Abstract 2364Poster Board II-341The treatment goal of B-cell chronic lymphocytic leukemia (B-CLL) has already shifted from symptom palliation to the attainment of maximal disease control combining purine analogs with monoclonal antibodies. This immunochemotherapeutic approach resulted both in more complete responses (CR) and longer response duration, often remaining only a minimal residual disease (MRD) detectable by flow cytometry. We treated in first line 120 B-CLL symptomatic patients (pts), median age 62 years, with six monthly courses of intravenous or oral fludarabine at conventional doses and then, after a median time of 31 days, with four weekly doses (375 mg/sqm) of rituximab (rtx). Fourteen pts had a low Rai stage, 103 an intermediate stage and 3 a high stage. We defined as high risk pts having at least two of these markers: unmutated IgVH, CD38>30%, ZAP-70>20%, intermediate unfavorable cytogenetics (trisomy 12 or del11q or del17p). Based on NCI criteria, 92/120 (77%) pts achieved a CR, 24/120 (20%) a partial remission (PR) and 4/120 (3%) no response or progression. Ten pts underwent grade 3 (WHO) infective lung toxicity, 1 patient acute fatal B hepatitis and 2 pts progressed towards Richter's syndrome. Hematologic toxicity included mainly neutropenia (grade 3 and/or 4 in 56 pts) and thrombocytopenia (grade 3 and/or 4 in 8 pts). Fifty-four pts either in CR with B-CLL bone marrow cells >1% (MRD+, n=16 pts) or in CR MRD negative, but with B-CLL peripheral cells going up >1000/microl within 1 year after induction (n=22 pts) or in PR (n=16 pts), underwent consolidation and maintenance therapy with four monthly cycles of rtx at 375 mg/sqm followed by twelve monthly low doses of rtx (150 mg/sqm). The median follow-up duration was 50 months. All treated pts experienced a long progression-free survival from the end of induction treatment (40% at 9 years). On the other hand, global overall survival (OS) was 54% at 10 years. Nevertheless, CLL pts undergoing consolidation and maintenance therapy (n=54) showed a longer response duration vs MRD+ not consolidated pts (n=16; 75% vs 9% at 4 years; P<0.00001, Figure). Noteworthy, persistently MRD negative (>1 year) pts (n=43) showed a very long response duration (79% at 6 years, Figure). Moreover, OS was shorter in MRD+ not consolidated pts (0% vs 79% at 15 years; P=0.0007). Noteworthy, within the high risk subset (n=48), consolidated pts (n=17) showed a longer response duration (56% vs 0% at 2.5 years, P=0.003) vs MRD+ not consolidated pts (n=11). Therefore, rituximab consolidation and maintenance immunotherapy improve response duration in B-CLL, thus potentially increasing OS, also within the high risk subset. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Differential Effects of Microenvironmental Elements On Tumor Cells Survival in Chronic Lymphocytic Leukemia Patient Subsets with Good or Poor Prognosis.

Abstract 2333Poster Board II-310Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH): patients with unmutated (UM) IgVH have a worse prognosis than patients with mutated (M) IgVH. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival.We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Leukemic cells purified by negative selection from the PB of UM CLL patients showed significantly higher rates of spontaneous apoptosis after long-term in vitro culture as compared to CLL cells isolated from M patients. Both M and UM CLL cells showed high basal level of Bcl-2 expression and NF-kB activity soon after purification. In vitro spontaneous apoptosis of purified UM CLL cells was associated with a progressive downregulation of the intracellular expression of Bcl-2 and with a complete loss of the active nuclear form of NF-kB. On the contrary, the higher long term viability of M CLL cells was paralleled by a maintained Bcl-2 and NF-kB expression. IL-4 and CD40L, used alone or in combination, as well as murine and human BMSC were capable of rescuing UM tumor cells from apoptosis. The pro-survival effect of these stimuli was exerted through the upregulation of Bcl-2 and was totally independent from the recovery of NF-kB nuclear translocation. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (PBMC) as compared to purified leukemic cells. This higher cell viability was associated with a retained expression of Bcl-2 and of the nuclear form of NF-kB, thus suggesting the presence of a pro-survival element in the peripheral blood of these patients. The extremely low numbers of NLCs generated from PMBC of UM patients ruled out a role for these cells in supporting the survival of unpurified leukemic cells. Conversely, a pro-survival effect on UM CLL cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM cells were cultured in the presence of autologous peripheral blood T cells (PBT). The prosurvival effect of circulating T cells was particularly evident at high T:B ratio, did not require a cell-cell contact and was mediated by the upregulation of Bcl-2 and the activation of NF-kB in leukemic cells.These data indicate that the survival of UM tumor cells is highly dependent on the action of multiple microenviromental stimuli. Conversely, M cells are intrinsically more resistant to apoptosis and minimally influenced by the local microenviroment. The higher dependency of UM CLL cells from extrinsic signals might be exploited to develop new therapies targeting the tumor microenvironment and to improve the outcome of more aggressive CLL. Disclosures:No relevant conflicts of interest to declare.

The PI3K Inhibitor ON 01910.Na Inhibits Critical Survival Pathways and Induces Apoptosis in CLL Cells through Induction of NOXA and BIM.

Abstract 412Chronic Lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a clinically heterogeneous disease characterized by the accumulation of mature B lymphocytes. Disease progression is driven by cell survival and proliferation signals provided by the tumor microenvironment through different receptors including the B-cell receptor, TNF, chemokine and cytokine receptors, and through other cell-cell interactions. ON 01910.Na (Onconova, Therapeutics) belongs to the family of styryl benzyl sulfones, a novel family of non-ATP competitive compounds that are currently under development as potential anticancer treatments. These compounds have shown activity in a variety of xenograft mouse models. Interestingly, it has recently been described that ON 01910.Na exerts potent antitumor activity against Mantle Cell Lymphoma (MCL) cells by inhibition of PI-3K/Akt/mTOR pathway and downregulation of Cyclin D1 translation. Here, we evaluated the cytotoxic effect of ON 01910.Na after 48 hours of in vitro exposure on PBMCs from CLL and normal donors by flow cytometry using AnnexinV-PE. We co-stained with CD3-APC and CD19-FITC to assess the effect on B and T cells separately. ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested (n=24), without affecting T-cell viability. Notably, ON 01910.Na did not affect B and T cells from normal donors at concentrations that induced apoptosis of CLL cells. Thirteen CLL samples were highly sensitive towards ON 01910.Na (mean B-cell IC50 0.71 μM) a concentration readily achieved in phase I clinical trials, eight samples were sensitive (mean B-cell IC50 1.38 μM) and three showed moderate sensitivity (mean B-cell IC50 4.26 μM). In contrast, the IC50 of T-cells was >8 μM in all samples. No significant differences were found in ON 01910.Na cytotoxicity against CLL expressing mutated (n=9, mean B-cell IC50 1.45 μM) or unmutated (n=15, mean B-cell IC50 1.34μM) IgVH sequences. ON 01910.Na was also effective against tumor cells having 17p (n=4, mean B-cell IC50 1.45 μM) or 11q deletions (n=3, mean B-cell IC50 0.96 μM). ON 01910.Na activated the mitochondrial apoptotic pathway, as shown by flow cytometry using conformation specific antibodies to detect activation of Bax and Bak, leading to mitochondrial depolarization (measured by mitotracker) and caspase-3 activation. ON 01910.Na also induced the upregulation of the proapoptotic BH3-only proteins Noxa and Bim and decreased expression of Mcl-1 at 4 hours, well before the onset of apoptosis. Inhibition of Noxa expression by retroviral transfection reduced the sensitivity to drug by 60%. ON 01910.Na induced reactive oxygen species (ROS) that may contribute to Noxa induction. Consistently ROS blockade using N-acetyl-cysteine reduced ON 01910.Na cytotoxic activity. To further delineate the biological processes underlying ON 01910.Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro for 4 and 10 hours with ON 01910.Na. GEP revealed the induction of Noxa and ATF3, consistent with an oxidative stress response. Noteworthy, Gene Set Enrichment Analysis (GSEA) of microarray data showed an induction of an AP-1 gene signature, (False Discovery Rate (FDR) = 0.10), validated by the nuclear accumulation of c-jun at 4 hours and a FOXO gene signature (FDR=0.13) that correlated with the upregulation of the proapoptotic BH3-only protein Bim. Strikingly, ON 01910.Na repressed B-cell receptor (FDR=0.023), NF-kB (FDR=0.084) and PI3K gene signatures. Consistent with inhibition of the BCR/PI3K/AKT axis, ON 01910.Na inhibited AKT phosphorylation by 65% after in vitro BCR activation of CLL cells. Our results identify ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. These data support the development of ON 01910.Na in CLL and a clinical trial has been initiated at our institution. Disclosures:No relevant conflicts of interest to declare.

Bendamustine Combined with Rituximab (BR) in First-Line Therapy of Advanced CLL: A Multicenter Phase II Trial of the German CLL Study Group (GCLLSG).

Abstract 205 Introduction:Bendamustine has shown considerable activity in monotherapy for lymphoid malignancies including chronic lymphocytic leukemia (CLL). In vitro studies have demonstrated synergistic pro-apoptotic effects of bendamustine and the CD20 antibody rituximab (BR) in primary CLL cells. Encouraging results have also been obtained using the BR combination treatment in previously treated CLL. This multicenter phase II trial (CLL2M) is the first study assessing the efficacy and toxicity of bendamustine in combination with rituximab in previously untreated CLL patients (pts). Patients and Methods:Between March 2007 and September 2008 117 pts with untreated CLL requiring therapy were enrolled into the protocol. Bendamustine was given at a dose of 90 mg/m2 on day 1 and 2, combined with 375 mg/m2 rituximab for the first cycle and 500 mg/m2 for subsequent cycles. BR treatment was administered every 28 days for up to 6 courses. Blood samples were taken for analysis by fluorescence in situ hybridization (FISH), the IgVH mutational status and expression of ZAP70/CD38. Minimal residual disease (MRD) was evaluated in peripheral blood and bone marrow by 4-color flow cytometry. Results:Data on the entire study population of 117 pts (median age 64 years) with a total of 583 treatment cycles are available. As of June 2009 the median observation time was 15.4 months (mo). 11.1% of the pts presented with stage Binet A, 41.0% with Binet B and 47.9% with Binet C disease. A mean number of 5.0 courses were delivered. 114 pts were evaluable for toxicity, 110 for response and 113 for progression free survival (PFS). The most frequent adverse events based on 583 treatment cycles were myelosuppression and infections: grade 3/4 anemia occurred in 4.9%, grade 3/4 leukopenia in 14.6%, grade 3/4 neutropenia and thrombocytopenia in 6.5% and 6.1% of all given courses, respectively. 29 episodes of CTC grade >3 infections were documented (5.1% of all courses). Treatment related mortality occurred in 2.6% of the pts: one liver failure after attempt of suicide with antihistamines, one fatal pneumonia and one sepsis in neutropenia. The overall response rate was 90.9% with 32.7% (36 pts) clinical complete remissions (CR). A nodular partial remission (nPR) was achieved in 2.7% (3 pts) and a partial response (PR) in 55.5% of the pts (61 pts), respectively. 9.1% of the pts (10 pts) had stable disease (SD) whereas none of the pts was progressive (PD). After 18 mo 75.8% of the pts were still in remission, median PFS has not been reached. An MRD level below 10E-4 was observed after completion of therapy in 29 of 50 evaluable pts in peripheral blood, while 7 of 25 pts achieved MRD negativity in bone marrow. Differences in response were observed among the genetic subgroups: 19 of 21 pts with 11q- achieved a remission with 10 PR and 9 CR (ORR: 90.5%). Accordingly, 17 of 19 patients with +12 responded (14 PR, 3 CR, ORR: 89.5%). In the high-risk group with 17p-, 3 of 7 pts showed a partial response (ORR: 42.9%). 56 of 63 pts (ORR: 88.9%) with unmutated IgVH status responded to BR. Conclusion:Bendamustine plus rituximab (BR) is effective and safe in the first-line treatment of CLL. Major side effects (myelosuppression and infections) were infrequent. Based on these encouraging phase II data the German CLL Study group is presently investigating the efficacy of BR in comparison to fludarabine-based immunochemotherapy (FCR) in the first-line treatment of CLL within a randomized phase III trial (CLL10 protocol). Disclosures:Fischer:Roche: travel expenses. Cramer:Roche: travel grants. Stilgenbauer:Bayer: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Fink:Roche: . Boettcher:Roche: Research Funding. Ritgen:Roche: Research Funding. Kneba:Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Döhner:Roche: Research Funding. Eichhorst:Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Hospira: Honoraria. Hallek:Roche: Consultancy, Honoraria, Research Funding. Wendtner:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Schering: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Immunochemotherapy with Fludarabine (F), Cyclophosphamide (C), and Rituximab (R) (FCR) Versus Fludarabine (F), Cyclophosphamide (C) and MabCampath (Cam) (FCCam) in Previously Untreated Patients (pts) with Advanced B-Chronic Lymphocytic Leukemia (B-CLL) : Experience On Safety and Efficacy within a Randomised Multicenter Phase III Trial of the french Cooperative Group On CLL and WM (FCGCLL/MW) and the “Groupe Ouest-Est d'Etudes Des Leucémies

Abstract 538▪▪This icon denotes an abstract that is clinically relevant. Introduction:Recent data suggest that FCR immunochemotherapy improves response rates and Progression-Free Survival (PFS) of previously untreated CLL pts. The monoclonal antibody alemtuzumab, a humanized anti-CD52 antibody (Campath), has shown activity alone and in combination in CLL pts. In order to validate the place of Campath in combination with the synergic association FC, the FCGCLL/MW and the GOELAMS conducted a multicenter French and Belgian phase III trial, CLL2007FMP, to evaluate the efficacy and toxicity of FCCam versus FCR in previously untreated patients with advanced CLL. PFS was the primary-end-point of this trial. Methods and Patients:A cohort of 178 fit medically pts (cumulative illness rating scale (CIRS) score of up to 6), less than 65 years old, without 17p deletion, were enrolled between November 2007 and January 2009. Pts were randomly assigned to receive 6 oral courses of FC (F 40mg/m2 d1-3 and C 250 mg/m2 d1–3; q 28 days) in combination with either R (n=83; 375 mg/m2 i.v. d 0 at first cycle and 500 mg/m2 d1 all subsequent cycles; q 28 days) or Cam (n=82; 30 mg s/cut d1-3; q 28 days). Patients were stratified according to IgVH mutational status and 11q deletion. Anti-infective prophylaxis included trimethoprim-sulfamethoxazole and valaciclovir during immunochemotherapy and until the CD4-positive lymphocyte count reached 0,2 109/l. In the FCCAm arm, CMV monitoring was monthly performed by either PCR or antigenemia. The use of GSCF was recommended. The trial's recruitment was stopped in January 2009 after 165 pts had been randomized (83 and 82 respectively in the FCR and FCCam arms) because of an excess of mortality in the FCCam arm, while 13 patients were enrolled but not randomized because of this decision. Results:We reported here a preliminary analysis including baseline characteristics and response rates in the first 100 included pts but safety analysis of the whole cohort ; among the first 100 pts, 81% were Binet B, 19 % Binet C ; median age was 56.8 years (range 52.8 to 60.6). Percentages of pts with 11q deletion, unmutated IgVH status, and elevated β2m, were 18.5%, 48.4%, and 77.6%, respectively. A number of 76.5% (FCR arm) and 71.4% (FCCam arm) of pts received 6 cycles. Reasons for discontinuation were mainly related to persistent grade 3-4 neutropenia. Safety analysis data were availabel for 165 pts. Number of patients reported with Common Toxicity Criteria (CTC) grade 3-4 adverse events were observed in 87.8% (FCCam arm) versus 90.2% (FCR arm) (p = 0.76). Grade 3-4 neutropenia was the most frequently reported adverse event (79.6% with FCCam and 74.6% with FCR arm). Interestingly, percentage of observed grade 4 neutropenia was stable during FCR treatment (17.6% for cycle 1 and 17.9% for cycle 6) but increased during FCCam treatment (28.4% for cycle 1 and 45.5% for cycle 6). A total of 63 Serious Adverse events (SAE) were declared (19 with 18 pts in FCR arm, and 44 with 35 pts in FCCam arm) consisting mostly of the cases in febrile neutropenia (13 with FCR arm, 27 with FCCam arm); 7 patients died, all in the FCCam arm : 3 of B diffuse large B-cell lymphoma (one of them EBV positive), 1 of mucormycosis, 1 of septic shock due to P.aeruginosae and 2 of heart failure during neutropenia. The Overall Response Rate ( ORR) in the first 100 patients was 96% in the FCR arm compared to 85% in the FCCam arm (p=0.086). The Complete Response rate was 78% (FCR arm) versus 58% (FCCam arm) (p=0.072). PFS and OS are not yet evaluable. Conclusion:the FCCam regimen for the treatment of advanced CLL appeared to be associated with an unfavourable safety profile representing a significant limitation of its use in this indication. Other combinations with Alemtuzumab may be studied. Disclosures:No relevant conflicts of interest to declare.

Dic(17;18)(p11.2;p11.2) Is a Recurring Abnormality in Chronic Lymphocytic Leukemia Associated with Aggressive Disease.

Abstract 1233Poster Board I-255 IntroductionCytogenetic analysis has become a routine part of the evaluation of patients with chronic lymphocytic leukemia (CLL) because specific chromosomal aberrations have been shown to assist in predicting disease progression, treatment efficacy, and overall survival. One such abnormality is del(17p13.1), which has been found to be associated with a need for early therapy, poor response to conventional treatment, and shortened survival. Identification of recurring abnormalities in this region represents great interest to identify genes relevant to CLL progression and poor prognosis. Patients and MethodsWe performed an extensive review of 1213 patients undergoing metaphase cytogenetics study at our institution and identified 16 (1.3%) with a recurrent unbalanced translocation between the p arms of chromosomes 17 and 18 that results in a dicentric chromosome with loss of much of 17p and 18p. The clinical features of these patients were characterized. ResultsA total of 16 patients were identified to have dic(17;18)(p11.2;p11.2) representing a 1.3% occurrence rate. The median age at diagnosis of these patients was 57 years (range 37-68). The dic(17;18)(p11.2;p11.2) was associated with a complex (3 or more unrelated cytogenetic abnormalities) karyotype in 12 patients (75%) at the time that the abnormality was first identified, and eventually associated with a complex karyotype in 94% of patients. This abnormality was associated with trisomy 12 in 7 patients (44%) and with del(13q) in 5 patients (31%) with no overlap between these two abnormalities. IgVH mutational analysis was un-mutated in 88% of cases. Except for one patient who was diagnosed with CLL incidentally during a workup for metastatic tonsillar cancer, all patients identified with dic(17;18)(p11.2;p11.2) met criteria for disease treatment, with a median time from diagnosis to first treatment of 15 months. Of the 12 patients who received fludarabine-based therapy, 7 (58%) were refractory. Three patients have received stem cell transplant for recurrent/refractory disease, and 4 are currently undergoing chemotherapy. With a mean follow-up of 54 months, 4 patients have died, 21, 42, 49, and 92 months after diagnosis. ConclusionsOur study combined with small series reported by others demonstrate that dic(17;18)(p11.2;p11.2) is a novel recurrent cytogenetic abnormality in CLL. Here we demonstrate the clinical significance of this recurring abnormality that is associated with young age at diagnosis, accelerated disease progression, decreased response to fludarabine, and shortened overall survival. The dic(17;18)(p11.2;p11.2) is frequently accompanied by other negative prognostic markers including a complex karyotype and unmutated IgVH status. Additional studies to further characterize the prevalence of this abnormality and genes that are disrupted by the translocation are warranted. DisclosuresNo relevant conflicts of interest to declare.

First-Line Treatment with Fludarabine (F), Cyclophosphamide (C), and Rituximab (R) (FCR) Improves Overall Survival (OS) in Previously Untreated Patients (pts) with Advanced Chronic Lymphocytic Leukemia (CLL): Results of a Randomized Phase III Trial On Behalf of An International Group of Investigators and the German CLL Study Group.

Abstract 535 Introduction: In 2008, first results of a multicenter, international randomized phase III trial (CLL8) were presented, showing superiority of FCR chemoimmunotherapy for response rates and progression-free survival (PFS) when compared to FC chemotherapy. We now report updated results with a longer median observation time of 37.7 months (mo). Methods and Patients: 817 treatment-naive patients (pts) with good physical fitness and CD20-positive CLL randomly (1:1) received treatment with 6 courses of either FCR or FC therapy. Both treatment arms were well balanced with regard to sex, age, stage, genomic aberrations and IGVH gene status. The median age was 61 years (range 30 to 81), 25.7% pts were female in both arms, 64.1% were Binet B, 31% Binet C and 4.9% Binet A. The median cumulative illness rating scale (CIRS) score was 1 (range 0-8). The overall incidences of trisomy 12 and abnormalities of 13q, 11q23, and 17p13 detected by FISH were 12%, 57%, 25%, and 8%, respectively. A mean number of 5.2 treatment courses were delivered in the FCR arm vs 4.8 courses in the FC arm (p=0.006). Dose reductions by more than 10% in at least one treatment course were performed in 47% (FCR) and 27% (FC) of pts (p Results: As of June 2009, the median observation time was 37.7 (mo). 761 pts (FCR 388; FC 371) were evaluable for response, 790 pts (FCR 401; FC 389) for PFS and all for OS. FCR induced a higher overall response rate than FC (95.1 vs 88.4%) and more complete remissions (44.1 vs 21.8%; p As previously reported more hematologic adverse events, particularly neutropenia, were observed with FCR treatment, but this did not result in an increased infection rate. More deaths have occurred in the FC arm (86/396, 21.7%) than in the FCR arm (65/404, 16.1%). In the majority of cases, the underlying cause of death was progressive disease (FC 48/86, FCR 33/65), secondary malignancies (FC 13/86, FCR 5/65) or unrelated causes of death such as myocardial infarction (FC 15/86, FCR 17/65). Treatment related mortality occurred in 8 (2.0%) of pts in each arm. Of these, 7 FC-treated pts and 5 FCR-treated pts died from infections related to treatment. In 7 pts (3 FC vs. 4 FCR), treatment was discontinued before the third treatment course due to fatal toxicity. Multivariate analysis was performed to evaluate factors predicting outcome. Age, sex, FCR-treatment, response, number of cycles (0-3), 17p-deletion, increased serum levels of thymidine kinase and β2-microglobulin and unmutated IGVH genes were independent prognostic factors predicting OS or PFS. Conclusion: Treatment with FCR chemoimmunotherapy is more effective than FC chemotherapy. The partial failure to demonstrate a benefit for FCR in Binet stage C patients may be related to an insufficient treatment intensity in these patients with higher tumor load. To our knowledge, this is the first time that a randomized trial is able to demonstrate that a specific first-line treatment for CLL results in an improved overall survival. The results corroborate the recommendation to use FCR as standard therapy in physically fit pts with CLL requiring therapy. Disclosures: Hallek: Roche: Consultancy, Honoraria, Research Funding. Fingerle-Rowson: Roche: Honoraria, travel grants. Fink: Roche: Travel grants. Mayer: BMS: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding. Hensel: Roche: Honoraria, Travel Grants; Bayer: Honoraria. Hopfinger: Roche: Membership on an entity9s Board of Directors or advisory committees. Hess: Roche: Honoraria. Bergmann: Roche: Honoraria for monitoring and CRFs. Catalano: Roche: Honoraria, Research Funding, Travel grants. Seymour: Bayer Schering: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Travel grants; Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Travel Grants. Berrebi: Roche: travel grants. Jaeger: Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Travel grants. Trneny: Roche: Honoraria, Research Funding. Westermann: Roche: Travel Grants. Wendtner: Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Eichhorst: Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Hospira: Honoraria. Staib: Roche: Research Funding. Boettcher: Roche: Honoraria, Research Funding, Travel grants. Ritgen: Roche: Research Funding. Mendila: Roche: Employment. Kneba: Roche: Honoraria, Research Funding. Doehner: Roche: Research Funding. Stilgenbauer: Roche: Consultancy, Honoraria, Research Funding, travel grants. Fischer: Mundipharma: Research Funding; Roche: travel grants.

Towards a Comprehensive Prognostic Score in CLL Based On a Combination of Genetic Parameters.

Abstract 1234Poster Board I-256CLL is a heterogeneous disease with a variable clinical course. In this study the prognostic power of chromosome banding analysis (CBA), interphase FISH and IgVH status was evaluated. In total 399 untreated cases were analyzed. First, we could confirm the prognostic significance of established parameters such as age (≥65 yrs), white blood cell count (≥20.000/μl), IgVH status, TP53 deletion and 11q deletion in our cohort. In addition, a negative prognostic impact of translocations involving the IgH locus, especially t(14;18)(q32;q21) and of the complexity of the karyotype measured by the number of clonal chromosome aberrations in CBA was observed. Furthermore it became obvious that some parameters discriminated better for overall survival and other for time to treatment. While the impact of the IgVH status on overall survival was low within the first 5 years after diagnosis (mutated 88.5% surviving vs unmutated 82.0% surviving, log rank test p=0.022), an unmutated IgVH status was strongly correlated with a shorter median time to treatment (18.3 months unmutated vs 110.7 months mutated, log rank test p<0.0001). On the other hand the impact of TP53 deletion was more pronounced on overall survival as compared to time to treatment. Age was associated with a shorter overall survival but was not significantly associated with time to treatment. Based on these results we propose a score for overall survival (OS) based on: age ≥65 yrs, WBC ≥20.000/μl, unmutated IgVH status, TP53 deletion, t(IgH), and the number of chromosome aberrations observed in CBA. Three respective risk groups showed considerable differences in OS (94.5% vs 64.3% vs 41.1% surviving at 5 yrs, p<0.0001). In contrast, time to treatment (TTT) was predicted best by unmutated IgVH status, ATM deletion, t(IgH) and number of chromosome aberrations. Four subgroups could be separated with median TTT of 110.7 months, 39.8 months, 19.5 months, and 3.8 months, respectively (p<0.0001). In conclusion, our data show that in combination with established prognostic markers such as an unmutated IgVH status, TP53/17p deletions or 11q deletions also the newly defined complexity of the karyotype measured by the number of chromosome aberrations has an important impact both on overall survival and also on time to treatment in CLL. These newly combined parameters translate into a more distinct separation of prognostic subgroups within the first years after diagnosis as compared to other prognostic systems using FISH data only or based on FISH data in combination with IgVH status. Prospective studies should evaluate the power for early stage CLL patients. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Expanded Proliferation Centers (PCs) Identify a Histological Subtype of Chronic Lymphocytic Leukemia (“Accelerated” CLL) with Adverse Clinical Outcome.

Abstract 2335Poster Board II-312The proliferation compartment in lymphoid tissues involved by CLL is essentially represented by large-cells that form proliferation centers (PCs) whose biological and clinical significance is a matter of investigation. For this, tissue biopsies consecutively obtained from 100 patients diagnosed with CLL were analyzed. Histological review was independently performed by three investigators (EC, AM and DM) in a BX51 Olympus microscope. Each biopsy was analyzed for the presence and size of proliferation centers (PCs), delineated by p27 negative immunostains, and for the proliferation rate inside the PCs assessed by means of mitosis count and Ki-67 immunostain. Histological characteristics were correlated with the main clinical characteristics and survival from the time of biopsy. To define the best cut-offs for histological variables in predicting survival, the Maximally Selected Rank Statistics was applied. CLL/SLL diagnosis was established in 78 cases, whereas DLBCL transformation was observed in 22 cases. Upon variables selection by the Maximally Selected Rank Statistics, CLL cases with enlarged and confluent PCs (broader than 20x field) (p=0.001), >2.4 mitosis per PCs (p=0.019), or >30% Ki-67 per PCs (p=0.024) were considered as having “accelerated” CLL (n= 23). Positive p53 immunostaining was observed in 24% “accelerated” CLLs compared to 5% CLLs without adverse features in the biopsy (p=0.02). The presence of p53 positivity was also frequently observed in DLBCL transformed cases (67%). “Accelerated” CLL cases were detected early in the course of the disease (median time from diagnosis: 16 months; 39% of cases at diagnosis), as opposite to DLBCL transformed cases (median time form diagnosis: 45 months; 14% at diagnosis). Tissue biopsy was performed in 73% of cases to rule out a transformation into an aggressive lymphoma. “Accelerated” CLL/SLL displayed high beta-2-microglobulin levels, elevated ZAP-70 expression and unmutated IgVH status more frequently than CLL/SLL without adverse features in the biopsy (p< 0.05). Median survival of “accelerated” CLLs from the time of biopsy was 34 months, whereas it was of 75 months (HR 2.2, 95%CI HR 1.21-3.86; p=0.008) for cases without adverse features in the tissue biopsy. Patients diagnosed with DLBCL transformation (n=22) had a median survival of 4 months. Notably, only one patient with an “accelerated” CLL pattern transformed into DLBCL after a median follow-up of 5 years. In conclusion, in lymph node biopsies from CLL/SLL patients, enlarged and confluent PCs and/or an increased proliferation activity in the PCs without DLBCL criteria identifies a new histological subtype of CLL/SLL (“accelerated CLL) with adverse biological features and inferior survival. Whether these patients require specific therapeutic approaches warrants investigation. Disclosures:No relevant conflicts of interest to declare.

Influence of ZAP-70 Expression On Migration of Chronic Lymphocytic Leukemia (CLL) and Lymphoma Cells.

Abstract 2345Poster Board II-322ZAP-70 (ξ-associated protein) is a tyrosine kinase (PTK) of the Syk/ZAP family normally expressed in T and NK cells. Increased ZAP-70 expression in CLL correlates with unmutated IgVH genes, a short time of progression, and a short survival. Mechanisms by which increased ZAP-70 protein expression can influence clinical outcome are not fully understood. After B-cell receptor (BCR) stimulation, activated ZAP-70 can cooperate with Syk, the tyrosine kinase of the BCR signal pathway normally expressed in B-cells, thus inducing activation of the CLL cells. Stimulation of the BCR leads to the triggering of Syk protein kinase to the cytoplasmatic tails of the receptor initiating subsequent activation of critical effector enzymes such as PI3K and PLCγ2. The PI3K/Akt pathway is related to survival and protection from BCR-induced apoptosi. Against this background, we analyzed the functional consequences of abnormal expression of ZAP-70 protein in B-lymphoma cell lines that normally does not express ZAP-70, particularly analyzing the impact of increased ZAP-70 expression oncell metabolism and cell migration. Mec-1 (CLL), Raji and Ramos (Burkitt) cell lines were stably transfected with the ZAP-70 expression vector pEGFP-ZAP-70 as well as the control pEGFP. BCR stimulation was induced in the stable Ramos cell line by IgM at several time points, and phosphorylation of downstream proteins was analyzed by Western Blot. Proliferation, cell cycle, calcium flux, adhesion molecules and cell migration were analyzed in all stable cell lines after ZAP-70 phosphorylation. After IgM stimulation, in Ramos cell line phosphorylation of ZAP-70 was observed at residue Y319 and, simultaneously, phospho-Syk expression was reduced. In addition, Erk protein was strongly activated in ZAP-70 transfected cell line compared to the control cell line, this activation lasting more than 24 hours. Notably, calcium flux detected by flow cytometry was higher in ZAP-70 transfected Ramos. Migration experiments showed that after IgM stimulation, transfected ZAP-70 Ramos migrated significantly more rapidly than cells transfected with the vector alone. Of note, after IgM stimulation, ZAP-70 transfected Ramos cells expressed a higher number of adhesion molecules (CXCR7 and others) on surface membrane than non-transfected cells. This effect on migration stimulation was also observed with the Raji cell line transfected with ZAP-70. In conclusion, addition of ZAP-70 expression to a heterologous B-cell system enhances BCR downstream signalling, calcium mobilization, and migration. Ongoing studies are determining the influence on the gene expression profile dependent of ZAP-70 expression. Altogheter, these data suggest that additional ZAP-70 expression in CLL and other lymphoproliferative disorders enhances tumoral activity, this explaining in part the poor clinical prognosis of patients with increased expression of ZAP-70. Disclosures:No relevant conflicts of interest to declare.

Phase I Study of RO5072759 (GA101) in Relapsed/Refractory Chronic Lymphocytic Leukemia.

Abstract 884 Background:RO5072759 (GA101) is the first humanized and glycoengineered type II monoclonal anti-CD20 antibody to enter clinical trials. In vitro, GA101 exhibits increased antibody-dependent cytotoxicity (ADCC) and strongly enhanced direct cell death compared to rituximab. Methods:A Phase I study was initiated to determine the safety, tolerability, dose-limiting toxicity (DLT), and pharmacokinetics of GA101 given as a single agent to patients with CD20+ B-CLL for whom no therapy of higher priority was available. A flat, intravenous dose ranging from 400 mg to 2000 mg was given in a safety-driven, dose escalating, 3 × 3 design on days 1, 8 and 22 and subsequently every 3 weeks for a total of 9 infusions. Here, we present the phase I data on 13 B-CLL patients, 33% [9/13 evaluable] with high risk cytogenetics (17p or 11q) and 70% [7/10 evaluable] displaying unmutated IgVH status, who received a median of 3 [1-8] prior regimens, including fludarabine (13/13) and rituximab-containing therapy, (8/13). Baseline median hematology parameters included hemoglobin 12.6 g/dL [9.4 -14.9], WBC 51.8 × 109/L [10-124], platelets 191 × 109/L [48-404], lymphocytes 47.4 × 109/L [7.0-119.3]. Results:GA101 was well tolerated with no DLTs and no dose reductions. The most common adverse events were Grade 1 or 2 infusion related reactions essentially limited to the first infusion. GA101-related Grade 3/4 hematological toxicities included transient neutropenia (n=9), febrile neutropenia (n=1) and transient thrombocytopenia (n=1). Neutropenia recovered spontaneously or with G-CSF. SAEs were reported in 3 patients (febrile neutropenia, thrombocytopenia, bronchitis, gingivitis, neutropenia and tumor lysis syndrome). Ten patients had infections (17 episodes with only three being Grade 3). No significant changes to baseline immunoglobulin levels were observed. Measurement of plasma cytokines during and immediately after the 1st infusion showed a transient increase in IL6 and IL8 with smaller increases in IL10, IFN-γ and TNFa. Activation of complement was not observed (C3a, C4a, C5a). Concurrent to cytokine increase was a decrease in T-cell subsets and NK cell counts (peripheral blood) after the first infusion. At the end of treatment, CD3 and CD8 counts had recovered while median CD4 and CD16/56 counts remained just below the normal range, with no clinical sequelae observed. Immunologic monitoring is ongoing.B-cell (CD19+) depletion was almost complete for all 13 patients and sustained following the first infusion. Best overall response rate according to IWCLL criteria was 62% (8/13) with 1 CRi, 7 PR and 5 SD observed across all FcγIIIRA [158F/V polymorphism] genotypes with no clear dose relationship established. Responses are ongoing with durations ranging from 3.5+ to 8+ months. End of treatment minimal residual disease (MRD) from 7/11 evaluable patients was detectable for 6 patients (median reduction of 2 log, range 2-4) and negative for one (despite a stable disease, as assessed by CT-scan). GA101 pharmacokinetics were characterized by one linear and one time-dependent saturable clearance components, consistent with target-mediated disposition, which is also observed with rituximab. Whilst the plasma concentrations demonstrate a dose-dependent increase, there was significant inter and intra-patient variability. Time-dependent clearance is consistent with a reduction in target-mediated antibody clearance with increasing duration of treatment. With the same doses of GA101, clearance is faster in B-CLL patients than NHL patients (Salles et al ASH 2008, 2009). Conclusion:These phase I results indicate that GA101 has promising activity when given as single agent to heavily pre-treated B-CLL patients and has a similar safety profile to that observed in NHL patients (Salles et al, ASH 2008, 2009) with an increased incidence of transient neutropenia in B-CLL. GA101 is currently being explored as a single agent in phase II in relapsed/refractory B-CLL and indolent/aggressive NHL and in combination with chemotherapy in a phase Ib study. Disclosures:Morschhauser:Roche Products Limited : Honoraria. Cartron:Roche Products Limited : Consultancy, Honoraria. Milpied:Roche Products Limited : Honoraria. Weisser:Roche Diagnostics : Employment. Birkett:Roche Products Limited : Employment. Salles:Roche Products Limited : Honoraria.

14q Deletions Are Recurrent Aberrations in CLL and Other Mature B-Cell Neoplasms and Show a High Coincidence with An Unmutated IgVH Status and Trisomy 12, and Are Associated with An Intermediate Overall Survival and a Shorter Time to Treatment.

Abstract 1262Poster Board I-28414q deletions are rare aberrations in malignant neoplasia but recurrent in hematologic malignancies, where they occur most frequently in CLL and other mature B-cell neoplasms. The breakpoints and the size of the 14q deletion have not been exactly characterized in a larger cohort yet. Based on chromosome banding analysis we identified 47 cases with 14q deletion out of 3054 cases (1.5%) with mature B-cell neoplasms (CLL: 1863, CLL/PL: 92, other: 1099). 32 cases with 14q deletion were classified as CLL, 5 as CLL/PL and 10 as other mature B-cell neoplasms based on immunophenotyping or cytomorphology. Therefore, the incidence of 14q deletions was 1.7% in CLL, 4.7% in CLL/PL and 0.9% in other mature B-cell neoplasms. These cases were further analyzed by a panel of FISH probes localized in 14q22.1 (BAC RP11-218E20), 14q24.1 (BACs PR11-35D12, RP11-226F19), 14q32.33 (BAC RP11-521B24) (BlueGnome, Oxford, UK) and the IgH-locus (14q32.3). The study revealed that 14q deletions are heterogeneous in size. A breakpoint cluster on the centromeric side was identified in 14q24.1 between BAC PR11-35D12 and BAC RP11-226F19 (62% of cases), whereas the most frequent breakpoint on the telomeric side was within the IgH-locus (14q32.3) between the IgH3'-flanking probe and IgHV probe (45% of cases). In 16 cases (34%) breakpoints occurred within 14q24.1 and within 14q32.3. In 19% of patients the 14q deletion was the sole abnormality whereas 30% showed one, 15% two and 36% three or more additional aberrations. Trisomy 12 (45%) was the most frequent aberration accompanying 14q deletion. In comparison to a cohort of 1579 cases with mature B-cell neoplasms without 14q deletion which were analyzed for IgVH mutation status in the same time period, patients with 14q deletions showed more often an unmutated IgVH status (39% vs. 61%, p=0.001). In the subgroup with breakpoint in 14q24.1 and 14q32.3 even 82% showed an unmutated IgVH-status. This subgroup was also associated with a CLL immunophenotype (81%). Clinical follow up data was available in 28 patients. CLL and CLL/PL with 14q deletion were compared to 390 CLL and CLL/PL cases without 14q deletion. While overall survival (OS) did not differ between both groups (OS at 3 yrs 86.4% vs. 88.7%, p=0.195), time to treatment (TTT) was significantly shorter in cases with 14q deletion (21.0 months vs. 80.1 months, p=0.015). Next, all 418 cases were assigned into 6 subgroups with known prognostic relevance: 1) 14q deletion, 2) 17p deletion, 3) 11q deletion, 4) trisomy 12, 5) 13q deletion as the sole abnormality, and 6) none of the mentioned abnormalities. OS at 3 yrs was 86.4%, 31.4%, 77.5%, 96.9%, 93.4% and 93.9%, respectively. For TTT the respective data were 21.0 months, 21.6 months, 20.9 months, not reached, 79.6 months and 147.0 months. In conclusion, 14q deletions are recurrent abnormalities in CLL and other mature B-cell neoplasms. Comparable to 13q deletions the size of 14q deletions varies. However, recurrent breakpoints in 14q24.1 and 14q32.3 were identified. Our data suggest to add FISH probes for 14q24.1 and for the IGH locus to the standard FISH panel in CLL and other mature B-cell neoplasms to pick up cases with 14q deletion, who would otherwise be assigned to “normal” FISH subgroup. In conclusion, 14q deletions are frequently accompanied by trisomy 12, an unmutated IgVH status and are associated with an intermediate overall survival and a shorter time to treatment comparable to other high risk cytogenetic aberrations (17p deletion, 11q deletion). DisclosuresReindl:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

The SET Oncogene, a Potent PP2A Inhibitor, Is Elevated in CLL and Antagonism of SET Induces Apoptosis.

Abstract 802 Background and Significance:Even though we have treatments for CLL, it remains an incurable leukemia. We need new and better treatments for this disease. The Akt kinase is usually constitutively activated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. The tumor suppressor protein phosphatase 2A is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We developed apoE-mimetic peptides that potently decrease phosphorylation of Akt and MAPKs, decrease TNF and nitric oxide synthase expression, and display anti-inflammatory activity in vitro and in vivo by antagonism of SET, a potent physiological inhibitor of PP2A. Others have reported that PP2A activity is reduced and that SET is overexpressed in cells of chronic myelocytic leukemia patients. Increased SET expression with consequent decreased PP2A activity leads to dysregulated kinase signaling. We show here that SET is also overexpressed in CLL cells, and that SET antagonist apoE-mimetic peptides kill CLL cells. Methods:Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. ApoE-mimetic peptides were prepared by chemical synthesis. Western blotting was used with anti-SET and anti-beta-actin antibodies. Apoptosis assays were performed with the annexin-V:propidium iodide staining method. Results:Samples from 17 CLL patients and 5 normal volunteers were examined by Western blotting. SET protein levels were 6.2-fold higher in CLL cells than in normal B cells. The apoE-mimetic COG compounds are peptides of 17 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein, and they act by binding to SET and preventing the inhibition of PP2A. This results in a net increase in PP2A activity in CLL cells. We have examined 11 COG peptides. Each of the 11 peptides displayed some cytotoxicity for CLL cells in vitro, irrespective of the patients' stages and other good or bad prognostic findings. 12 of 17 CLL patients were Rai stage 0 at presentation, and 5 were stage 1 or 2. They had been followed 4.2 yr (median; range 1.0 – 24.1 yr). 2 of 16 were CD38 positive, and 6 of 15 were Zap-70 positive. Of 15 analyzed, 6 had unmutated IgVH gene. 11 of 17 patients had not been treated. Peptide COG449 was the most potent, while a control peptide with an inverted apoE sequence had no activity. COG449 induced cell death in a dose-dependent fashion in all patients' samples, with a mean ED50 of 80 nM. The ED50 of COG449 for normal B cells was very high (> 10,000 nM). Annexin-V staining indicated that apoptosis was induced at concentrations in good agreement with the ED50 for cytotoxicity of the compounds tested. In vivo studies in normal mice using COG449 show no toxicity even at doses of 100 mg/kg when delivered by subcutaneous injection. Conclusions:We demonstrated SET overexpression in CLL cells and that apoE-mimetic peptides bind SET to de-inhibit PP2A. This results in apoptosis and death of CLL cells in vitro with high efficacy and potency (low nanomolar ED50s). CLL cells are killed preferentially compared to normal PBMC and B cells. Preliminary studies show that the peptide is nontoxic in normal mice. Trials in CLL patients will help determine the efficacy in vivo. Disclosures:Christensen:Cognosci Inc.: Employment. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.