Byrd, John C.

Abstract BACKGROUND: We have previously studied and reported that the combination of pentostatin (P, 2 mg/m2), cyclophosphamide (C 600 mg/m2) and rituximab (R 375 mg/m2) in previously untreated CLL is highly effective with an overall response (OR) rate of over 90% and a complete response rate (CR) of 41% (Blood109:405–411, 2007). We also found that this regimen can be effective even in older patients (>70 y), those with elevated beta-2 microglobulin levels, and patients with mildly reduced creatinine clearances (Cancer. 109:2291–2298, 2007). To determine whether similar benefit could be achieved without inclusion of an alkylating agent, we conducted a follow-up trial testing pentostatin and rituximab without cyclophosphamide and employing a higher pentostatin dose (4 mg/m2). METHODS: Eligible pts had documentation of active CLL by standard NCI-WG criteria, and were previously untreated. Treatment schema consisted of 6 cycles of pentostatin (4 mg/m2) and rituximab given every 21 days. Pentostatin was given on the first day of each cycle following infusion of rituximab. Rituximab was given at 100mg/m2 IV at day 1, then 375 mg/m2 IV on days 3 and 5 of the first treatment cycle. During cycles 2 to 6, Rituximab was given at 375 mg/m2 as a single IV infusion day 1 of week 4, 7, 10, 13 and 16. All patients were staged two months after completion of the 6 cycles of PR using the NCI-WG criteria. PATIENT CHARACTERISTICS: Overall, 33 patients were enrolled at Mayo Clinic and Ohio State University between July 2005 and February 2008. All 33 were eligible: 82% male, median age 65 (range: 45–81), with 9 (27%) being 70 years or older. 76% had a baseline ECOG PS of 0, and the rest were ECOG PS 1. Overall, 36% of patients had intermediate Rai risk (stage I–II) and 63% high Rai risk (stage 3–4) disease. Prognostic testing revealed that 36% were CD38+, 50% were Zap-70 +, and 39% had an unmutated IgVh status. Chromosome analysis by FISH found that 99% had detectable FISH panel defect, including 61%, 27% and 3% of patients with 1, 2, or 3 FISH detects, respectively. RESULTS: 28 of 33 patients (85%) completed therapy. While on treatment, 6 pts (18%) had a dose held or modified with 4 of these delays due to hematologic AE. For adverse events deemed at least possibly related to treatment, 4 (12%) pts experienced grade 3+ hematologic toxicity and 5 (15%) experienced grade 3+ non-hematologic toxicity. Out of all 33 enrolled patients, the overall response rate was 79% with 10 CR, 6 nPR, and 10 PR. At the time of this analysis, 29/33 patients are still alive with a median follow-up time of 14 months on surviving patients. To date 17/33 (52%) of patients have progressed with an estimated median time to progression of 12 months (95% CI: 8.5–21 months). 13/26 responders have progressed. Median duration of response is 12.5 months ((95% CI: 11–21 months). Finally, since eligibility were nearly identical and enrollment accrued at the same two academic centers, we compared the patient characteristics, response rates, and PFS of the 33 patients treated with PR to the 64 patients previously treated on our PCR trial. Patients in the two studies were generally similar with respect to demographic and prognostic characteristics, although patients in the PR trial had higher WBC and were less likely to be IgVH unmutated (Table). Although the differences in ORR and CR rate were not significantly different, the PFS appeared to be inferior in patients treated with PR as compared to PCR (12 months vs. 31 months; p=0.003). CONCLUSION: Although the PR regimen achieves a high OR response rate, the PFS appears inferior to PCR therapy. These findings suggest that increasing the purine nucleoside analogue dose does not eliminate the need to include cyclophosphamide in chemoimmunotherapy for patients with CLL. PCR Trial N=64 PR Trial N=33 P value Age, median(range) 63 years (38–80) 65 years (45–81) 0.34 ≥70 years(%) 28% 27% Male 77% 82% 0.61 Rai stage 0 5% 0 0.46 Rai stage I–II 42% 36% Rai stage III–IV 53% 64% White Cell Count, median(range) 79 × 109/L (11–519) 127 × 109/L (8–430) 0.04 <50 × 109/L 36% 28% 50–149 × 109/L 44% 25% >150 × 109/L 20% 47% Serum B2-microglobulin, median(range) 3.97 (1.8–13.5) 3.80 (2.0–8.2) 0.81 >2 × Upper Limit Normal(%) 57% 58% CD38 Positive 34% 36% 1.00 ZAP-70 Positive 36% 50% 0.26 IgVH Unmutated 71% 39% 0.004 FISH Normal, 11% 9% 13q- 35% 42% +12 21% 24% 6q- 2% 0 11q- 22% 18% 17p- 6% 3% other 3% 3% Overall Response Rate 91% 79% 0.12 Complete Response Rate 41% 30% 0.38 Median PFS 31 months 12 months 0.003

Abstract We previously demonstrated that apoptosis-inhibiting proteins (e.g., Bcl-2, Mcl-1, and XIAP) are important in predicting response to chemotherapy and antibody therapy for CLL. Additionally, genetic abnormalities and epigenetic control of gene expression have become increasingly important in understanding the prognosis and potential therapeutic opportunities in this disease. Greater than 80% of patients with CLL have genetic abnormalities detectable by FISH. Important subsets include those with a less favorable prognosis (deletions of 17p, 11q), an intermediate prognosis (trisomy 12), or a favorable outcome (deletion of 13q). PTPRO, encoding a receptor-type tyrosine phosphatase, is a specific tumor suppressor gene located in chromosomal region 12p12.3. PTPRO has been highly characterized as inhibiting anchorage-independent tumor growth in human lung carcinomas. This gene has been found to be silenced by promoter methylation, and can be re-expressed by hypomethylating agents in human lung tumor models. Thus, we examined the methylation profile of the promoter region of PTPRO in primary leukemic cells from patients with CLL, and in B cells obtained from normal volunteers. Analysis of the PTPRO CpG island by methylation-specific PCR and combined bisulfite restriction analysis (COBRA) demonstrated that this region was methylated in 91% of samples from 46 patients, whereas it was unmethylated in control B cells from normal volunteers. Specific quantitative determinations of important proteins related to apoptosis in the leukemic cells from 20 of these patients showed that, in the group with methylation of the PTPRO promoter, there was a significant elevation of Bcl-2 (p=0.079), Mcl-1 (p=0.004), XIAP (p=0.048), Bcl-2/BAX ratio (p=0.0024), and Mcl-1/BAX ratio (p=0.0017). There was no apparent relationship with FISH category or immunoglobulin VH mutational status. Thus, PTPRO promoter methylation analysis may identify a subset of CLL patients less likely to respond to therapy, and provide a potential reversible target for new therapeutic approaches to this disease. Finally, analysis of methylation of CLL samples by restriction landmark genome scanning also revealed methylation of genes for three additional protein tyrosine phosphatases: PTPRN2, PTPRZ2, and PTPN11. Increases in methylation of two of these (PTPRN2 and PTPRZ2) in CLL samples relative to control B cells was confirmed by COBRA. These results provide additional potential molecular targets for CLL therapy.

Abstract We performed immunophenotypic and IgVH mutation analyses on the chronic lymphocytic leukemia (CLL) cells of 307 patients for expression of CD38, ZAP-70, and IgVH mutation status to determine the relative strength of each in predicting the need for early treatment. For this we assessed the time from diagnosis to initial therapy for progressive and/or symptomatic disease. Patients confirmed to have remained untreated were censored for this endpoint. Expression of CD38 was defined as the proportion of CD38-stained CLL cells that had fluorescence intensity above a defined threshold. Similarly, cases were defined as ZAP-70+ when 20% or more of the ZAP-70-stained CLL cells had fluorescence intensity above a defined threshold, as described (NEJM351:893, 2004). CLL cells were classified as expressing mutated IgVH genes if they expressed IgVH with <98% homology with a known germline IgVH gene. The median percentage of cells expressing CD38 in our cohort was 11.5% (range 0.1% to 99.5%). In patients with multiple samples, the CD38 expression level appeared constant over time, with a median S.D. of 0.8%. We defined patient samples that had CD38 expression levels at or below the median as having low expression. There was a significant association between low expression of CD38 and lack of ZAP-70 or expression of a mutated IgVH (p < 0.0001 for each assessment by the Fisher exact test). Median level of CD38 among cases expressing mutated IgVH was 4.0%, compared to 27.4% among cases expressing unmutated IgVH (p < 0.0001 by the Wilcoxon rank sum test). Median level of CD38 among ZAP-70 negative cases was 5.3%, compared to 27.2% among ZAP-70+ cases (p < 0.0001 by the Wilcoxon rank sum test). Patients with low-level expression of CD38 had a median time to initiation of therapy of 8.2 years; whereas, patients with high-level expression of CD38 had a median time to initiation of therapy of 4.0 years (p<0.0001). Although high-level expression of CD38 was associated with a shorter time from diagnosis to initiation of therapy (p < 0.0001 in a Cox proportional hazards regression), the inclusion of IgVH mutation status (p < 0.0001) or expression of ZAP-70 (p < 0.0001) in these models reduced the contribution of CD38 status to a p-value of 0.051 (ZAP-70 model) or 0.052 (mutation status model). Although ZAP-70 expression and mutation status each contributed significantly to the hazard of initiating therapy in a Cox proportional hazards model, high-level expression of CD38 did not add significantly to the model that already addresses ZAP-70 and mutational status, p=0.425. As such, although knowledge of the CD38 expression level can be used as a predictor of the time from diagnosis to the initiation of therapy it provides only marginal value if either the IgVH mutational status or expression of ZAP-70 is known and no value if knowledge of both ZAP-70 expression and IgVH mutational status is available.

Abstract Background: MDX-060 is a fully human anti-CD30 IgG1k monoclonal antibody that induces Fc-receptor mediated killing of CD30 expressing cell lines and inhibits the growth of CD30-expressing tumor cells in a murine xenograft model. We previously reported data from the first 31 patients in a Phase I/II open-label, dose escalation study of MDX-060 in patients with relapsed or refractory CD30+ lymphomas. The purpose of the study is to determine the safety and tolerability profile of the drug, and to preliminarily evaluate efficacy. We now update the findings of this study, including reporting the results of a new higher dose cohort. Methods: In the Phase I portion of the study, MDX-060 was administered intravenously at dose levels of 0.1, 1, 5, or 10 mg/kg weekly for 4 weeks to cohorts of 3–6 patients. In the Phase II portion of the study additional patients were administered MDX-060 intravenously at 10 (n=10) or 15 (n=17) mg/kg weekly for 4 weeks. Responses were assessed at month 2. Results: To date, MDX-060 has been administered to 48 patients; 40 with Hodgkin’s disease (HD), 6 with anaplastic large cell lymphoma (ALCL) and 2 with other CD30 positive lymphomas. MDX-060 has been well tolerated, and a maximum tolerated dose has not been identified. There were 2 drug-related serious adverse events, 1 patient in the 1 mg/kg dosing cohort had Grade 3 elevated liver transaminase, and 1 patient in the 15 mg/kg cohort had Grade 3 pneumonia/Grade 4 ARDS. Objective clinical responses have been observed in 5 patients. Two patients had complete responses; 1 patient with ALCL in the 1 mg/kg cohort lasting 100 days, and 1 patient with HD in the 15 mg/kg cohort lasting 117 days. Retreatment of the ALCL patient with MDX-060 resulted in a second complete response. Three patients had partial responses; 1 patient with HD in the 5 mg/kg cohort lasting 92 days, and 1 patient with HD in the 15 mg/kg cohort, which is continuing at 176 days. In addition, 1 patient with ALCL in the 15 mg/kg cohort had a partial response. Stable disease was observed in 17 patients following MDX-060 administration with a median duration of 132 days (range:15–387 days). Baseline soluble CD30 levels in the plasma were evaluated, but did not appear to correlate with clinical outcome. Conclusions: Preliminary evidence indicates MDX-060 to be well tolerated in heavily pre-treated patients with CD30+ lymphomas, with minimal toxicity, and with clinical activity observed at 1 mg/kg, 5 mg/kg and 15 mg/kg. Additional patients are being accrued at 1 mg/kg and 5 mg/kg to more accurately assess efficacy at these dose levels.

Abstract Introduction: Aggressive chemo-immunotherapy followed by peripheral blood stem cell autografting (ASCT) in CALGB (Alliance) 59909 achieved a median progression-free survival (PFS) in MCL of 5 years (Damon et al JCO, 2009), but late recurrences occurred. Using the CALGB 59909 treatment backbone, we evaluated tolerability and efficacy of adding post-transplant bortezomib consolidation (BC) or maintenance (BM)in a randomized phase II trial. Interim results of CALGB (Alliance) 50403 were previously presented (ASH 2015) demonstrating 5 yr PFS 70% and 69% for the BM and BC arms respectively. We now report final 8 year follow up results from this trial and compare outcomes to CALGB 59909, which differed from CALGB 50403 only by the absence of post-transplant bortezomib. Methods: The primary endpoint was PFS measured from randomization for each treatment arm. Induction therapy was with 2-3 cycles of augmented R-CHOP (2000 mg/m2 cyclophosphamide) and methotrexate (300 mg/m2) followed by high-dose cytarabine/etoposide/rituximab(R)/filgrastim (EAR) stem cell mobilization and cyclophosphamide/carmustine/etoposide (CBV) ASCT. After 2 doses of post-transplant R, patients were randomized to BC (1.3 mg/ m2 IV days 1, 4, 8, 11 of a 3-week cycle for 4 cycles) or BM (1.6 mg/m2 IV weekly x4 every 8 weeks for 18 months) beginning at approximately day 90. Minimal residual disease (MRD) was analyzed using patient-specific PCR probes for the bcl-1 / IgH junction or the IgH CDR3 region. Results: 151 patients were enrolled from 10/2006 - 6/2010 at 14 sites; 147 who received treatment are included in this analysis. Median age was 59 years (29-69); stage II (2.7%), III (11.6%), IV (85.0%); MIPI low (52.4%), intermediate (30.6%), high (17.0%); blastoid histology (12.9%); bone marrow involvement (81.0%); Ki67 was evaluated in 86 and was >30% in 17.4% of patients. 118 (80%) underwent ASCT and 102 (68%) were randomized. Most withdrawals (45) were for progression (10) or adverse events (AEs) (19) including 4 treatment-related deaths. Fifty-two patients were randomized to BM and 50 to BC. Following randomization, 34 (65%) completed BM and 33 (66%) completed BC. Withdrawal for AEs occurred in 14 (28%) of BC and 7 (13%) of BM patients (p = 0.09), most for cytopenias or peripheral neuropathy. With a median follow-up 7.8 years from randomization (8.3 from registration), the median PFS from randomization for the BM arm was not reached and was 8.9 years (95% CI 7.2 to not reached) for the BC arm. Both arms had median PFS significantly greater than the null hypothesis setting median PFS to 4 years (p < 0.001; 1-sided test of exponential parameter)The 8-year PFS estimates in the BM and BC arms were 77% (95% CI 66-90%) and 58% (95% CI 44-76%), respectively. Among all 147 patients treated on CALGB 50403, 8-year PFS from time of registration was 43.6% (35.6-53.3%). PFS from registration was not significantly extended in CALGB 50403 compared with CALGB 59909 (log rank p=0.24), but using a landmark analysis from time of transplant, PFS was significantly extended in CALGB 50403 (log rank p=0.005)(fig 1). Baseline patient characteristics in the two studies were not significantly different. In CALGB 50403, 8-year PFS from registration by MIPI was 52.0% (95% CI 41.1-66.0%) in MIPI low risk, 37.5% (95% CI 25.3-55.4%) in intermediate risk, and 28.2% (95% CI 13.7-57.9%) in high-risk. Bone marrow MRD results were collected for 42 patients post-induction therapy; 8-year PFS estimates were 80.2% (95% CI 62.2-100%) (n=17) and 43.2% (95% CI 27.3-68.2%) (n=25) for MRD-negative and MRD-positive patients, respectively (p=0.009). Conclusions: Induction chemotherapy followed by ASCT and either BC or BM was efficacious and tolerable, although BC was associated with more withdrawals for toxicity. PFS was not significantly different between BC and BM. The comparison between studies 50403 and 59909 with long-term follow up continues to suggest a PFS benefit from the addition of BC or BM among patients undergoing transplant. This did not translate into a PFS benefit from time of study enrollment possibly due to the higher pre-transplant dropout rate in 50403. MRD-negativity following induction chemo-immunotherapy is highly associated with improved PFS and the role of ASCT in post-induction MRD-negative patients is currently under investigation in a randomized clinical trial. Support: U10CA180821, U10CA180882, U24CA196171; ClinicalTrials.gov Identifier: NCT00310037 Disclosures Kaplan: Bayer Pharmaceuticals: Consultancy. Maurer:Celgene: Research Funding; Morphosys: Research Funding; Nanostring: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Bartlett:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Blum:Acerta: Consultancy; Astra-Zeneca: Consultancy. LaCasce:Seattle Genetics: Consultancy, Honoraria; Humanigen: Consultancy, Honoraria; Research to Practice: Speakers Bureau; Bristol-Myers Squibb: Other: Data safety and monitoring board. Leonard:AstraZeneca: Consultancy; Juno: Consultancy; BMS: Consultancy; ADC Therapeutics: Consultancy; Gilead: Consultancy; Biotest: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; MEI Pharma: Consultancy; Celgene: Consultancy; United Therapeutics: Consultancy; Sutro: Consultancy; Genentech/Roche: Consultancy; Pfizer: Consultancy; Bayer: Consultancy. Cheson:AbbVie, Roche/Genentech, Pharmacyclics, Acerta, TG Therapeutics: Consultancy.

The critical importance of specific proteins including Bcl-2, Mcl-1, XIAP, Bax, Caspase-3, Traf-1, and ZAP70 in predicting disease progression, prognosis, and response to therapy in CLL have been extensively studied through in vitro experiments. We now report molecular data obtained from a large prospective clinical trial involving 278 previously untreated patients with CLL. This ECOG-led intergroup trial involved participation from CALGB and SWOG, and currently provides quantitative information regarding protein levels on 217 cases assessed immediately prior to treatment. NCI criteria for therapy in CLL were met before patients were randomized to receive either Fludarabine or Fludarabine plus Cytoxan. On-study analyses included: interphase cytogenetics by Fluorescence In Situ Hybridization (FISH), p53 mutational analysis, immunoglobulin variable heavy chain (IgVH) gene rearrangement, and quantitative analysis of the aforementioned specific proteins. Peripheral blood mononuclear cells were obtained from each patient at registration before starting therapy. Lysates were prepared from the patient cells and separated by SDS-PAGE. Proteins were detected by immunoblot analysis using the multiple antigen detection method previously developed by our group. Detection was performed by an enhanced chemiluminescence method and quantified by densitometry. Expression levels were calculated relative to expression in the Jurkat T cell line (for ZAP70) or the RS11846 lymphoblastoid cell line (for apoptosis-related proteins). Using a two-sided Wilcoxon rank sum test, molecular data were compared with FISH and IgVH mutational status. Patients with either p53 mutations or del(17p) had significantly lower levels of XIAP (p=0.022). Patients with del(11q) had significantly higher Bcl-2/Bax ratios (p=0.052). Patients with trisomy 12 had lower levels of Bcl-2 (p=0.066); higher Mcl-1 (p=0.026); higher Bax (p=0.09); and higher Caspase-3 (p=0.019) levels. Interestingly, these specimens also had lower ratios of Bcl-2/Bax (p=0.0016). Patients with del(13q) had significantly higher levels of Bcl-2 (p=0.0059) lower levels of Caspase-3 (p=0.047), and higher Bcl-2/Bax ratios (p=0.0569). IgVH mutated patients had significantly lower levels of Caspase-3 using either a 98% cut-off (p=0.0008) or the 97% cut-off (p<0.0001). Finally, patients with higher ZAP70 protein expression had significantly higher levels of Caspase-3 (p=0.0099) and Traf-1 (p=0.016). In 163 patients evaluable for both response and the molecular studies, either elevation of Bcl-2 protein (p= 0.01) or an abnormality of p53 (either del 17p or a p53 mutation) (p=0.08) correlated significantly with attainment of a complete remission. Potential opportunities to predict response to therapy in CLL will hopefully identify those patients likely to benefit from therapy. Based upon our previous work, these molecular parameters can be modulated both in vitro and in vivo. Therefore, understanding the relationship of these important proteins to therapeutic response can facilitate strategic plans to circumvent drug resistance to chemotherapy and immunotherapy in CLL.

Several prognostic factors including un-mutated VH mutational status, select interphase cytogenetic abnormalities [del(11q22.3), del(17p13.1)] and p53 mutations have been associated with shorter interval time from diagnosis to symptomatic disease requiring treatment, shortened progression-free survival (PFS) and overall survival (OS). Limited prospective data exists relative to the relevance of these biologic markers on PFS following treatment with modern therapies for CLL. E2997 is a randomized phase III trial of 278 previously untreated, symptomatic CLL patients who received fludarabine and cyclophosphamide (FC) versus fludarabine (F). We analyzed the response rates and PFS of the patients for whom interphase cytogenetics, VH mutational status, and p53 mutational status studies have been completed. FC therapy had a higher complete response (CR) (23% versus 5%, p<0.002), overall response [OR] (73% versus 50%, p<0.002), and median PFS (33.5 months versus 15 months, p<.0001) as compared to F. The CR and OR was not different based upon interphase cytogenetics or VH mutational status for the entire group of patients or when analyzed by arm of treatment. However, the OR for pts with del(17) or p53 mutations was lower in both the FC arm (69%, versus 76% in patients with normal p53) and F arm (27%, versus 54% in normal p53). Using the Dohner hierarchical classification [del(17p)>del(11q)>del(6q)>tri12>normal> del(13q14)], the relationship of interphase cytogenetics, VH mutational status and p53 mutational status with PFS is summarized below: Median PFS on FC (months) Median PFS on F (months) NR = median not reached; p53 mut+ = mutation present del(17p) 11.9 8.2 del(11q) 30.6 12.8 del(6q) 26.9 26.7 tri 12 NR 20.3 normal NR 11.2 del(13q) NR 21.2 del(17p)/p53 mut+ 11.9 8.9 no del(17p)/p53 mut+ NR 17.8 VH<98% NR 21.2 VH>98% 21.4 13.4 Using the interphase cytogenetic groups, there was a significant difference in PFS for both the FC (p=0.04) and F (p=0.01) treatment arms. Similarly, the presence of a p53 mutation or deletion [del(17p)] predicted for shorter PFS for both FC (p=0.005) and F (p=0.02) therapies. PFS did not statistically differ among VH mutational groups for either arm. We next investigated a Cox proportional hazards analysis to assess the impact of multiple molecular features and treatment arm on PFS. Treatment with F (hazard ratio 3.14, p<0.0001), presence of del(17p) or p53 mutation (hazard ratio 3.13, p=0.0001), and presence of del(11q) (hazard ratio 2.067, p=0.019) were highly significant in the model. Finally, VH unmutated status and del(11q) were highly associated. In conclusion, our data demonstrate that high risk genomic features including p53 mutations, del(17p), and del(11q) are highly predictive of shortened PFS. Our data provide support for a risk-stratified therapy approach for the treatment of CLL.

Abstract Deletions or mutations of the tumor suppressor p53 have been associated with more aggressive disease in many types of cancer including CLL, and are more commonly found in relapsed or refractory patients. We studied symptomatic patients (pts) enrolled on ECOG 2997, a randomized phase III trial of fludarabine monotherapy (F) versus fludarabine plus cyclophosphamide (CF) for previously untreated CLL. A total of 235 pretreatment samples were analyzed for p53 mutations in exons 5–9 using denaturing gradient gel electrophoresis (DGGE), followed by confirmation of mutations using automated sequencing. Cytogenetic analysis was also performed on 243 patient samples by Fluorescence In Situ Hybridization (FISH) using a probe to detect deletions in 17p13.1, the chromosomal location of the p53 gene. In the analysis by DGGE, a total of 25 pts (10.6%) were found to have p53 mutations. In 22 cases there was a single mutation, and in three cases there were two mutations. Mutations in exon 6 were the most common, and in each of the cases with two mutations, one of these was in exon 6. Results by exon are as follows: Exon 5 (1 pt, 0.4%), Exon 6 (15 pts, 6.4%), Exon 7 (5 pts, 2.1%), Exon 8 (3 pts, 1.3%), Exon 9 (4 pts, 1.7%). The interphase cytogenetic analysis included 24 of the 25 pts with a p53 mutation by DGGE. Of the 19 (7.8%) pts with del(17p13.1) as the leading cytogenetic anomaly, seven were cases that also had mutations in p53 by DGGE. The remaining 17 pts with mutations in p53 by DGGE had as leading cytogenetic anomaly: del(11q) (2 pts); trisomy 12 (3 pts), normal cytogenetics (2 pts), and del(13q) (10 pts). Results from both the DGGE and FISH analyses were combined to identify a group of 37 pts with either del(17p) or p53 mutation. This constituted 16% of the 230 pts in which either FISH or p53 mutational analysis was performed. Immunocytochemistry staining for p53 was performed on all pts, and no correlation was found between over-expression of p53 protein with mutation and/or deletion of this gene. The pts were then assessed by treatment arm for response and progression-free survival (PFS), relative to patients without p53 abnormality either by DGGE or FISH analysis. In the FC group: 13 pts had either a p53 mutation or del(17p), nine (69%) of whom achieved a CR or a PR, versus 56/74 (76%) of those with normal p53 (p=0.73). In the F group: 15 pts had either a p53 mutation or del(17p), of whom four (27%) achieved a CR or a PR, versus 38/70 (54%) of those with normal p53 (p=0.09). Pts with a detectable abnormality in p53 had a significantly reduced PFS, regardless of whether they received fludarabine alone (p=0.02) or fludarabine plus cyclophosphamide (p=0.005). This study suggests that routine immunocytochemistry staining should not be substituted for p53 mutational studies. While some short responses were observed in patients with p53 mutation and/or deletion receiving fludarabine and/or fludarabine cyclophosphamide, the time to progression was significantly shorter, emphasizing the prognostic significance of p53 abnormalities for this disease.

Abstract MDS with chromosome 5q deletion [del(5q)] is uniquely sensitive to clonal suppression by lenalidomide (LEN), yielding high rates of cytogenetic and erythroid response. Although haploinsufficiency for a candidate tumor suppressor gene within the common deleted segment (CDS) has been implicated in the pathogenesis of del(5q) MDS, no specific gene products account for the karyotype-dependent LEN sensitivity. Using primary AML cells, we showed that LEN is selectively cytotoxic to del(5q) cells, inducing cell cycle arrest at the G2 > G1 junctures and apoptosis in a concentration-dependent fashion. Now we show that the CDS-encoded Cdc25C and PP2A phosphatases (PPTs) serve as molecular targets for LEN. Cdc25C and PP2A are dual specificity PPTs that regulate cell cycle. The genes encoding the catalytic subunit alpha (PP2ACa), the dominant catalytic isoform, and the regulatory subunit B (beta isoform PR 52) of the PP2A holoenzyme reside within the del(5q) CDS. Real-time RT-PCR showed that Cdc25C and PP2ACa gene expression is significantly reduced in primary del(5q) AML and MDS BM-MNC compared to non-del5q and normal controls. A Cdc25C FISH probe confirmed presence of only a single allele in del(5q) specimens [n=5] vs. normal karyotype [n=5] with no overlap with the EGR1 locus. We found that LEN inhibited rHu-Cdc25C activity with IC90 inhibitory concentrations ≤10nM. Similarly, PP2A PPT activity was inhibited by LEN in enzyme immunoprecipitates from Ramos cells at sub-micromolar concentrations. In U937 cells, we confirmed intracellular Cdc25C and PP2A inhibition, showing concentration-dependent retention of the inhibitory p-Tyr15 -Cdc2 substrates for Cdc25C with corresponding phosphorylation of the PP2A substrate Ser216 Cdc25C, findings reproduced by treatment with the PP2A selective antagonist, fostreicin. Cdc25C immunoprecipitation showed that phosphorylation of Ser216 Cdc25C increased binding to the 14-3-3 protein with consequent cytoplasmic sequestration confirmed by fluorescence microscopy. Phosphorylation of histone H2A.X Ser139 was unchanged after LEN treatment, indicating that phosphorylation of the inhibitory Cdc25C serine does not arise from upstream DNA damage activated kinases. Importantly, siRNA suppression of Cdc25C gene expression in U937 cells promoted LEN-specific apoptosis in 45% of cells, which increased to 68% after fostreicin inhibition of PP2A. These data indicate that haplodeficiency for the Cdc25C and PP2ACa genes promotes selective sensitivity of del(5q) cells to LEN-induced apoptosis. Haplodeficiency of critical drug-able targets may represent a novel strategy for tumor selective therapy.

Abstract Abstract 2406 The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. Disclosures: Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.