ALBERT

Description: Abstract 1786 The Wnt/beta-catenin pathway is highly active in chronic lymphocytic leukemia (CLL) and confers an anti-apoptotic effect in vitro and possibly also in vivo. As such, inhibition of this pathway represents a potential therapeutic target. Here we report preclinical studies using agelastatin A (AgA), a naturally occurring alkaloid extracted from the marine sponges Agelas dendromorpha and Cymbastella sp. We tested AgA using SW480 cells transfected with a beta-catenin dependent reporter gene expression system. Using this assay, we found that AgA is a potent Wnt signaling inhibitor with IC50 of 12nM. AgA also inhibits the expression of reporter genes in HEK293 cells cotransfected with either Wnt1 or mutated (dominant-active) beta-catenin, suggesting that its mechanism of action is independent of the beta-catenin degradation complex. Moreover, real time-PCR results show that Lef1, a classic Wnt/beta-catenin target gene highly expressed in CLL, is down-regulated in primary CLL cells incubated with AgA at nanomolar concentrations. AgA, but not structurally related compounds agelastatin C and agelastatin D, selectively induces apoptosis in CLL B-cells (mean IC50 = 56nM) compared with peripheral blood mononuclear cells from healthy volunteers (mean IC50 = 250nM). Interestingly, AgA induces apoptosis at nanomolar concentrations even in samples from CLL patients who had no clinical response to fludarabine, samples with 17p deletion or TP53 gene mutations, and samples in which p53 deficiency was determined by in vitro chemoresistance and absence of inducible p21, CD95, and DR5 after irradiation (Figure 1). In addition, AgA induces apoptosis on CLL cells that are maintained in co-culture conditions with stromal cells, which typically increases leukemia cell viability. This suggests that AgA is capable of disrupting pro-survival signals derived from the microenvironment, including Wnt mediated activation. In conclusion, AgA has potent activity against CLL cells in vitro. Our studies show that AgA inhibits Wnt/beta-catenin signaling at nanomolar concentrations, induces apoptosis in CLL cells independently of p53, and is able to disrupt pro-survival signaling derived from stromal cell support. AgA warrants further studies and clinical development for the treatment of CLL and potentially other malignancies associated with Wnt/beta-catenin signaling. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2544 The clinical presentation of chronic lymphocytic leukemia (CLL) is variable and includes at least two distinct clinical subtypes: indolent or progressive disease. Several prognostic markers such as the mutational status of the IgVH genes or ZAP-70 expression identify patients that will have more aggressive clinical courses. We have previously reported that ZAP-70 expression in CLL requires the support and stability provided by the Hsp90 activated complex (Hsp90-AC) and that Hsp90 inhibitors induce apoptosis in CLL cells preferentially in patients with high–risk/rapid progressive disease. Hsp90 requires the conformation of a multimeric complex with other co-chaperones (Hop, p23) to become active and function as a chaperone for mutated, over-expressed, misfolded or constitutively activated proteins. However, to measure Hsp90-AC activity is challenging and requires protein assays that are semi-quantitative and not always can provide a functional read out. To address this problem we designed a test to measure Hsp90-AC by taking advantage of the higher avidity of this complex to bind Hsp90 inhibitors such as 17-AAG. We hypothesize that Hsp90-AC activity is an independent prognostic marker of disease progression in CLL and to test this we measured the ability of 17-AAG to inhibit Hsp90-AC and promote apoptosis in vitro in a cohort of previously untreated CLL patients. This evaluation was performed in samples collected at the time of diagnosis and the results were correlated with the time from diagnosis to first treatment (t-Tx1) as well as the presence of other known prognostic markers. Ninety-five previously untreated CLL patients were included in this cohort. In vitro sensitivity/apoptosis to Hsp90-AC inhibition using 17-AAG (1μg/ml) was measured by flow cytometry using DiOC6 and PI after 48 hours of incubation. Only samples with more than 60% viability were included and we selected a cut-point of ≥ 53% induction of apoptosis to determine if a sample was sensitive or not to Hsp90-AC inhibition (This cut-point was selected using a ROC analysis for optimal diagnostic performance). We found that 37 patients (39%) were sensitive to 17-AAG. These patient had a median time from diagnosis to first treatment (t-Tx1) of 7 years compared to 4.2 years in patients that were resistant to 17-AAG (p=0.04). In addition, 37 patients (39%) had unmutated IgVH genes with a median t-Tx1 of 8.7 years compared to 4.2 years in patients with mutated IgVH genes (p=0.02) and 41 patients (43%) had high levels of ZAP-70 expression (〉20% by flow cytometry) and this group of patients had a median t-Tx1 of 7 years vs. 4.2 years in ZAP-70 negative patients (p=0.03). We evaluated the association between the degree of Hsp90-AC inhibition (17-AGG sensitivity) and IgVH mutational status and found a strong correlation between these two variables with shorter t-Tx1 in patients that were IgVH unmutated and sensitive to Hsp90 inhibitor (p=0.0003). We found a similar strong correlation between ZAP-70 expression and sensitivity to Hsp90-AC inhibitor (p=0.003). In our patient cohort, we saw that IgVH, ZAP-70 and Hsp90 inhibitor sensitivity are independent prognostic markers in CLL (Cox regression, p=0.002). We found that the sensibility and specificity of ZAP-70 test for high-risk CLL (sensitivity 81.1% [95% IC 67–95%] and specificity 77.6%[95% IC 65–90%]) was increased when the Hsp90-AC inhibitor sensitivity test was added (sensitivity 87% [95% IC 71–100%] and specificity 91%[95% IC 79–100%]). In conclusion, in vitro inhibition of Hsp90-AC in CLL using 17-AAG is an independent prognostic maker and strong predictor of the need for treatment. Hsp90-AC inhibition showed a high correlation with IgVH mutational status and ZAP-70 expression. Additionally, patients sensitive to 17-AAG whose leukemic cells also highly express ZAP-70 appear to have the worst prognosis with the shortest t-Tx1. Our data suggest that in vitro sensitivity to 17-AAG at the time of diagnosis serves as a quantitative surrogate marker for Hsp90-AC activity and correlates with disease progression in CLL. This is the first time that such correlation has been established in a cohort of cancer patients and shows the relevance of the Hsp90 chaperone system in cancer biology and disease progression. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4474 Chronic lymphocytic leukemia (CLL) remains incurable with standard therapies and a large number of patients become refractory to treatment or develop toxicities that prevent further treatment and contribute to their mortality. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is the only known curative treatment for CLL. However, management and prevention of toxicities as well as patient / donor selection remain as the most significant challenges that limit the success of this treatment. In this observational study we evaluated the efficacy and tolerability of Allo-HSCT in relapse / refractory patients with CLL and correlated the clinical outcome with prognostic markers as well as the donor type. All patients were enrolled at the University of California, San Diego - Moores Cancer Center between November 1998 and July 2009. Eighteen patients with progressive relapse / refractory CLL were included (9 males [8 Caucasian, 1 Hispanic], 9 females [8 Caucasian, 1 Hispanic]), nine patients received stem cells from related donors and the other nine from unrelated donors. The median age was 50 years (age range, 26–71), median interval between diagnosis and Allo-HSCT was 83 months, and the median number of regimens prior to transplant was 5.3 (range, 2–11). Five out of eighteen patients were fludarabine-refractory and one was not evaluable (NE). All patients had a high-risk disease at the time of transplantation, with advanced stages according to Rai classification, and high prevalence of IgVH unmutated state, as well a high expression of CD38 and ZAP-70. Six patients had cytogenetic abnormalities, ten were normal and two were not evaluated. The most common pre-transplantation cytogenetic alteration was deletion 17p (33.3%), followed by deletion 13q (22.2%) and deletion 11q (22.2%). Prior to Allo-HSCT, three were in CR, ten patients were in PR and five had PD. CMV serologic reactivity was present in 67% of patients. Seventeen patients received non-myeloablative conditioning regimens and one patient was treated with a myeloablative regimen. Disease status was assessed 30 and 100 days, 1, 2 and 3 years post Allo-HSCT. 13 out of 18 patients showed response to Allo-HSCT (6 PR and 7 CR) with the remaining patients having PD. Two patients had acute GVHD and 3 patients had extensive chronic GVHD. We observed no differences in the rate of GVHD in unrelated vs. related donor recipients (p=0.1). Five patients died, three due to progression of the disease, one due to post-transplant organ failure and one for acute GVHD. Survival analysis showed a median OS of 60.1 ± 3.9 months. We observed a significant difference in terms of OS in patients who had unrelated vs. related transplants (HR=3.1 [95% CI 1.5–18.7]). We observed no difference in survival of patients discriminated by the IgVH mutational status (HR =1.8 [95% CI 0.2–17.5]) or ZAP-70 expression (HR =1.0 [95% CI 0.9–1.1]). The median progression free survival was 56 months. Five of the 18 patients were retreated with donor lymphocyte infusions or a second Allo-HSCT after relapse or lost of chimerism. Three of these patients died of PD while the other two are still alive. In conclusion, Allo-HSCT induces long-term remission and survival in high-risk relapse / refractory CLL patients including patients with multiple previous treatments and progressive disease status at the time of transplantation. The response to Allo-HSCT is independent of prognostic markers such as IgVH and ZAP-70 expression. Patients that received stem cells from unrelated donors have a better clinical outcome including OS with no evidence of increased GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2866 Despite advances in the treatment of patients with chronic lymphocytic leukemia (CLL), the disease still remains incurable and eradication of minimal residual disease (MRD) being one of the most challenging goals of treatment. Alemtuzumab (Campath-H1™) has been shown to effectivily eradicate MRD from the bone marrow and induce long-term remissions, however its use is limited to patients without bulky disease. Futhermore, combination of alemtuzumab with chemotherapy has resulted in serious adverse events. In this study, we evaluate the toxicity and efficacy of alemtuzumab as consolidation therapy for CLL patients following induction with high-dose methylprednisolone in combination with rituximab (HDMP-R). Twenty-one patients with evidence of residual disease after treatment with HDMP-R received additional treatment with alemtuzumab. This antibody was administered three times a week for a total of 8 weeks. Patients received antibiotic prophylaxis with trimethoprim-sulfamethoxazole 160/800 mg twice a day × 3 per week, fluconazole 100 mg / day and valganciclovir 900 mg / day. The median age was 60 years (range, 49–73), with Rai stage III-IV in 81% of the patients. Twelve patients (57%) had evidence of unmutated IgVH gene and thirteen (62%) had high level of ZAP-70 expression. Cytogenetic and FISH analysis showed eight patients with deleletion 13q, three patients with trisomy 12, one patient with deletion 11q, five patients with no chromosomal abnomalities and in six patients data was not available. The median number of previous treatments was 1.3 (range, 0–5) and the median time from the end of HDMP-R treatment to initiation of alemtuzumab was 5 months (range, 1–14). After HDMP-R, nine patients (43%) achieved CR and twelve (57%) were in PR; all of them had evidence of residual disease in the bone marrow by 4-color flow cytometry analysis. Eight additional patients achieved CR after consolidation with alemtuzumab for a total of 17 patients (81%) in CR at the end of the study. We found no evidence of MRD (MRDneg) in 12 of those patients (57% of the total and 71% of CR patients). Of the remaining patients, one had PR and three patients had progressive disease for an overall response rate of 86%. The median progression-free survival (PFS) was 63 months (range, 6–84) for all patients. The median PFS in CR MRDneg patients has not been reached at a median follow-up of 46 months (range, 18–84), with 8/12 patients that have not progressed after a time at risk of 3.8 years. CR MRDpos patients have a median PFS of 48 months (range, 6–48). The treatment was well tolerated and there were no deaths attributed to therapy. Adverse events were classified following the NCI common terminology criteria for adverse events (CTCAE) Version 4.0. Two patients (9.5%) developed infections. The first event occurred during the administration of alemtuzumab and required hospitalization of the patient for management of pneumonia galactomannan positive suspicious for invasive aspergillosis (Grade 3), the second event was in a patient with aspegillus sp. infection of the skin that occurred four months after completion of alemtuzumab (Grade 2). Both patients recovered completely. We observed no CMV or other opportunistic infections. Three patients (14%) developed cytopenias; two patients with (Grade 4) thrombocytopenia and three patients with (Grade 4) neutropenia. In conclusion, alemtuzumab consolidation for residual disease after treatment with HDMP-R was well tolerated and effective in patients with CLL. We observed a near two-fold increase in the number of patients that achieved CR and the majority of these (71%) had no evidence of MRD. Moreover, patients with CR MRDneg have an exceptionally long PFS. The low rate of infection and lack of treatment related mortality compares very favorably with previous studies using alemtuzumab consolidation after chemotherapy treatment in which toxicities including treatment related death were found to be prohibitive. These encouraging results provide the rationale for additional studies using this combination therapy. Disclosures: James: Celgene: Research Funding.