ALBERT

Description: We examined a large cohort (N=2,457) of chronic lymphocytic leukemia (CLL) patients evaluated by the CLL Research Consortium (CRC) and found 63 (2.6%) used IGHV3-21. Comparing the Ig heavy chain third complementarity determining region (HCDR3) of the IGHV3-21 cases: 25/63 cases (39.7%) had a conserved amino acid motif (motif 1: DANGMDV) in the otherwise highly variable Ig HCDR3, as described by Tobin et al. Blood 2003. All but one of these Ig heavy chains (IgH) were paired with a lambda light chain encoded by IGLV3-21. In addition, we found that 3/63 cases (4.8%) had a previously unrecognized conserved HCDR3 amino acid motif (motif 2: DPSFYSSSWTLFDY). In contrast, these IgH invariably were paired with kappa immunoglobulin light chains (IgL) encoded by IGKV3-20. Similarly to that noted for CLL cases that use IgH encoded by unmutated IGHV1-69 (Widhopf et al. Blood Epub First Edition 2007), the pairing of IgH encoded by IGHV3-21 with IgL appears governed by the HCDR3. The non-stochastic pairing of IgH with IgL argues strongly that antigen plays a role in selecting the Ig expressed in CLL. To examine for the antigen(s) recognized by the most common Ig encoded by IGHV3-21, we isolated IgH and IgL genes expressed by IGHV3-21/IGLV3-21 CLL cases and generated recombinant antibodies, which we examined for binding to antigen(s) present on microarray of self or environmental antigens. We found that Ig encoded by IGHV3-21/IGLV3-21 had apparent specific binding for protein L, a multi-domain cell-wall protein isolated from Peptostreptococcus magnus, a Gram-positive commensal bacteria that comprise a large portion of the human bacterial gut flora. Prior studies identified that protein L is a superantigen capable of binding human Ig kappa light chains encoded by IGKV genes of the I, III, and IV subgroups, but not human Ig lambda light chains. The specific binding of IGHV3-21/IGLV3-21 to protein L suggested that protein L might play a role in the development of CLL cells that express such Ig. To test this hypothesis, we examined the capacity of various recombinant antibodies to bind protein L by ELISA. We found that lambda IgL encoded by IGLV3-21 could bind to protein L with similar activity, independent of whether this lambda IgL paired with the native IgH, IgH encoded by IGHV3-21 lacking the DANGMDV HCDR3 motif, or even irrelevant IgH encoded by IGHV4-39 that are not found paired with IGLV3-21 in the Ig expressed in CLL. Moreover, Ig formed by pairing IgH encoded by IGHV3-21 that has the DANGMDV HCDR3 motif with an IgL encoded by an IGLV that was irrelevant to IGLV3-21 did not bind protein L. These results reveal a previously unrecognized capacity of human IgL encoded by IGLV3-21 to bind the protein L superantigen of Peptostreptococcus magnus, a bacteria commonly found in the human gastrointestinal tract. However, because the binding of IGLV3-21 does not depend upon the non-stochaistic pairing of IgH and IgL observed in CLL, we reason that the capacity of IGLV3-21 to bind protein L cannot account for the selected Ig repertoire expressed in CLL, suggesting that it actually does not play a role in CLL leukemogenesis. This finding suggests that caution should be exercised when defining an antigen that is found capable of binding the restricted Ig expressed in CLL as the driving factor responsible for leukemogenesis.

Description: Introduction: Fludarabine treatment has been shown to be beneficial for patients with Chronic Lymphocytic Leukemia (CLL), and fludarabine-based combinations may even further improve outcomes in patients with CLL. However, most CLL patients eventually become fludarabine refractory, a state which is associated with a relatively short survival. Treatment of fludarabine-refractory patients is challenging, with a median survival of about 10 months. Recently, 2 phase II clinical trials (Chanan-Khan et al. JCO 2006 and Ferrajoli et al. ASH 2006) demonstrated the clinical efficacy of lenalidomide, an immunomodulatory agent, in relapsed/refractory CLL patients. We conducted a subset analysis to examine the efficacy of lenalidomide in patients who are fludarabine refractory. Methods: All patients enrolled on the 2 phase II single agent lenalidomide clinical trials were evaluated and patients with fludarabine-refractory disease (progressed while on or within 6 months of fludarabine-based therapy) were assessed for clinical efficacy of lenalidomide. Lenalidomide was given orally either at 10 mg daily for 28 days followed by 5 mg increments every 28 days to a maximum dose of 25 mg or given at 25 mg on days 1–21 of each 28-day cycle. Response was assessed using the NCI-WG 1996 criteria. Results: A total of 80 patients were collectively enrolled in these clinical studies. Among these, 29 were identified to have fludarabine-refractory disease. Important clinical characteristics of these patients are reported in Table 1. The overall response rate in fludarabine-refractory patients was 34.5% (10/29). Complete remission was observed in 2 (6.8%) patients. Conclusion: Lenalidomide is a novel agent with immunomodulating properties demonstrating clinical efficacy in relapsed or refractory CLL patients. Interestingly, clinical responses to single agent lenalidomide were noted despite refractoriness to fludarabine (a subset of CLL patients with poor survival and limited therapeutic options). This observation of the clinical benefit of lenalidomide independent of responsiveness to prior fludarabine is encouraging and warrants further evaluation. Table 1 Ferrajoli et al. Chanan-Khan et al. Fludarabine-refractory (N=12) Fludarabine-refractory (N=17) ORR, overall response rate; PFS, progression-free survival; OS, overall survival. Median age, years (range) 62 (51–82) 68 (53–75) Sex, female/male 4/8 4/13 Median no. prior therapies (range) 4 (3–15) 4 (1–10) Median beta microglobulin (range) 5 (3–10) 5 (2–10) Advance Rai Stage (III/IV), n (%) 7 (58.3) 13 (76.4) ORR, n (%) 3 (25.0) 7 (41.2) Median PFS, months 12 14.9 Median OS, months All alive (range, 7–19) 23

Description: Abstract 2381 Poster Board II-358 The natural history of patients (pts) who fail or relapse after chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) has not been established. Three hundred pts received FCR as initial therapy for progressive or advanced chronic lymphocytic leukemia (CLL) (Tam CS; Blood 112(4):975-980, 2008). Fifteen (5%) pts failed to respond, 72% achieved a CR and 22% a PR. Treatment failure occurred in 18 pts because of the development of AML, MDS or Richter's transformation and there were 15 deaths in remission (infection (7), cancer (6), or cardiac events (2)). Fourteen relapsed pts have not received therapy and are considered to be “watch and wait.” One hundred and twelve pts have received therapy. A large variety of treatment programs were administered at time of relapse during the ten years of the study. The most commonly used were FCR-like regimens (33) with or without lumiliximab or bevacizumab, FCR + alemtuzumab (CFAR) 9 pts, rituximab-based regimens (28) +/- GMCSF or steroids, Campath-based regimens (16) +/- rituximab, and a variety of other phase I and miscellaneous salvage treatments. 79 pts received salvage therapy at M. D. Anderson Cancer Center (MDACC) and the 33 others in their local community. 17 patients (16%) achieved a CR and 46 a PR (4%). CR rates were 15% for FCR, 56% for CFAR, 4% for rituximab regimens, 31% for alemtuzumab regimens and 4% for other regimens. While higher CR rates were noted in alemtuzumab regimens, no difference in time-to-treatment failure or survival was noted. The median overall survival was 33 months with a 40% five-year survival rate. A number of characteristics shown in Table 1 associated with complete remission and overall response rate. Outcome of 1st Salvage – FCR Relapsed/Refractory (112 pts) Characteristic Value Patients %CR %OR Med Surv (Mths) Age / years

Description: Abstract 2347 Poster Board II-324 The human T cell leukemia/lymphoma 1 (TCL1) oncogene was initially identified as a target of chromosomal translocations and inversions at the 14q32.1 chromosome breakpoint region in T-cell prolymphocytic leukemia (T-PLL). Increased TCL1 expression is seen in follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia (CLL). Transgenic mice over-expressing TCL1 under control of the mu immunoglobulin gene enhancer develop a CD5+ B cell lymphoproliferative disorder that mimics human CLL, indicating that TCL1 plays a central and/or causal role in the pathogenesis of CLL. However, chromosome aberrations that constitutively activate TCL1 have not (yet) been identified in the vast majority of CLL patients, and therefore the oncogenic mechanism(s) of TCL1 activation in CLL remain unclear. There is growing evidence that external signals from the microenvironment control and regulate the survival and proliferation of CLL cells. Marrow stromal cells (MSC) are highly effective in protecting CLL cells from spontaneous and drug-induced apoptosis, and are used as a model system to study the marrow microenvironment. In order to explore the molecular cross talk between CLL cells and MSC, we co-cultured CLL cells with different MSC and analyzed gene expression changes induced by co-cultures with MSC, an approach similar to our recent study with nurselike cells (Blood 113:3050-8, 2009). For this, RNA was extracted from 19-purified CLL cells from 10 different patients (baseline expression, day 0). Also, the same patients' samples were co-cultured on stroma cells (KUSA-H1, NK-Tert) for 2 and 7 days. At these time points, RNA again was isolated after CD19-purification. Then, gene expression was determined using HG U133 plus 2.0 oligonucleotide arrays from Affymetrix. Gene expression changes were analyzed in individual patients' samples, comparing baseline samples' gene expression to samples after 2 and 7 of co-culture on MSC. We observed relatively homogeneous gene expression changes in CLL cells after co-culture with MSC. We found that TCL1 was among the top 5 genes that were most highly up-regulated by MSC, based on at least 3-fold up-regulation in at least 6 of the paired samples. We also found an up-regulation of TCL1 at the protein level when assessed by immunoblotting and flow cytometry in CLL samples after co-culture with MSC. These findings indicate that MSC can induce and regulate TCL1 expression in CLL, suggesting that the microenvironment plays an even greater role in the pathogenesis of this disease than previously recognized. Disclosures: No relevant conflicts of interest to declare.

Description: The majority of patients with acute myelogenous leukemia (AML) who achieve complete remission (CR) with initial chemotherapy will eventually relapse. Most relapses occur in the first three years and rare patients relapse after being in CR for more than 5 years. We have reviewed retrospectively 2347 patients with AML treated at M D Anderson Cancer Center from January 1980 to July 2008. Of the total cohort, 1366 patients achieved CR and among these 942 patients relapsed. We identified 11 patients (1.16%) who relapsed after being in CR 〉5 years; these patients are the focus of this analysis. There were 4 females and 7 males. Their median age was 66 years (range 37–79), and their median presentation white blood cell (WBC) count was 2.3 x109/L (range, 1.1–92.3). The FAB classification was M2 in 5 patients, M1, M4, M5, M6 in 1 patient each, and unknown in 2 patients. Initial cytogenetics were diploid in 6, del(7q) in one, miscellaneous in 1, and unavailable in 3 patients. Initial therapy was with combination of idarubicin (Ida) and cytarabine (Ara-C) in 4 patients (1 with additional fludarabine), amsacrine based in 3, daunorubicin (Dauno) single agent in 2 and other agents in 2 patients. All patients except one achieved CR after the first induction course; one patient needed 2 cycles of induction to achieve CR. None underwent an allogeneic stem cell transplant in first CR. The median duration of CR was 81 months (range, 60–137). At the time of relapse, median WBC count was 4.4 x109/L (range 1.7–48.8). Karyotype at relapse was diploid in 2, del(5)del(7) in 1, del(6)del(7) in 1, trisomy 8 in 1, hyperdiploid in 2, add(2q) and add(6q) in 1 each and unavailable in 2 patients. The karyotype at relapse was different from the initial finding in 8 of 8 patients with available data at both time points. Treatment for relapse included Ida (or Dauno) with Ara-C in 8 patients (1 with additional fludarabine), and other agents in 3 patients. The response to treatment was CR in 4, partial remission in 2, resistant in 4 and unknown in 1. No patient underwent an allogeneic stem cell transplant in second CR. The median duration of the second CR was 2 months (range 0–37). The median survival after relapse was 6.4 months (range 1–39). Median survival from initial diagnosis of AML was 107 months (range 68–143). We conclude that late relapses in AML (〉5 years after CR) are infrequent (1.16% of all relapses) and response to their subsequent therapy is poor; best responses occur with a regimen similar to the initial induction regimen. The karyotype at relapse is frequently different raising the question of a second AML versus relapse with the original clone.

Description: Abstract 1261 Poster Board I-283 Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle except G0 and is widely used as a marker of cellular proliferation in human tumors. We recently demonstrated that levels of plasma circulating Ki-67 (cKi-67) are significantly higher in patients with newly diagnosed acute lymphoblastic leukemia (ALL) than in healthy control subjects, and that elevated levels of cKi-67 are associated with a shorter survival in ALL patients. Here we examined the associations of cKi-67 levels with laboratory and clinical variables in patients with chronic lymphocytic leukemia (CLL). The study included 194 patients with CLL and 96 healthy control subjects. The cKi-67 levels in plasma were determined using electro-chemiluminescence-based immunoassay using the Mesoscale Discovery platform. Since usually Ki-67 is used as an index of tumor cell proliferation, we took into account the lymphocyte count of the CLL patients in peripheral blood and normalized the levels of the cKi67 to the absolute number of lymphocytes in the peripheral blood establishing plasma cKI-67 index (cKi-67 level ng/1000 circulating lymphocytes/μL plasma). Median (range) levels of absolute cKi-67 were significantly higher in patients with CLL than in control subjects (914.65 [102.0-4975.12] ng/mL vs 353 [35.76-2830.65] ng/mL; P

Description: Abstract 206 Lenalidomide 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: 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.

Description: Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL–MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL–MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell–cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC–CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.