ALBERT

Description: We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10−16). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.

Description: Background and Significance : Chronic lymphocytic leukemia (CLL) is the most heritable hematologic malignancy; however, no common CLL predisposition genes are known. Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by small accumulations of B lymphocytes in the peripheral blood. MBL has a CLL-like immunophenotype, may progress to overt CLL, and is over represented in CLL families. Therefore, MBL observed in the context of familial CLL may be a marker of inherited risk for development of CLL. Detailed characterization of family-associated MBL may also provide mechanistic insights into the pathogenesis of familial CLL. Our strategy was to detail the biologic characteristics of CLL in family-associated MBL. Methods : Persons with MBL were identified by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. Flow cytometry was used to determine the surface immunophenotype including CD38 and intracellular ZAP-70. We defined MBL as populations of CD19+, CD5+, CD20lo, CD23+ B cells that comprised at least 2% of the CD19+ peripheral B cell compartment and did not exceed 5.0 × 109 MBL cells/L. RNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences. MBL cells were sorted in bulk for FISH for loci associated with clinical CLL. Results : Twenty-two out of 190 (12%) unaffected family members were found to have MBL. We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 32%; range 2%–97%). Nonetheless, the absolute size of the MBL clone was small, 15 of 17 individuals had 〈 200 × 106 MBL cells/L. CD38 expression (defined as CD38 surface expression in ≥30% of MBL cells) was observed in 8 of 18 subjects tested. ZAP-70 (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 4 of 12 participants. Among 12 subjects tested, 5 MBL expressed both surface IgD and IgM, 3 expressed IgD only, 2 expressed IgM only, and 2 did not express IgD or IgM. Analysis of immunoglobulin heavy and light chains has been completed in 8 individuals. Both immunoglobulin heavy chain variable (IgVH) region mutated (n = 12) and unmutated (n = 4) sequences were observed. Four of 8 individuals had 2 or more unrelated MBL clones (range 2–5), including one individual with both unmutated and mutated clones. Among the 16 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IgVH genes were 3–07 (3 MBL clones), 3–15 (3), and 4–34 (3). No VH1 family gene rearrangements were observed. In one individual, a VH3–07 MBL clone showed intraclonal diversification suggestive of antigen driven immunoglobulin sequence changes. Twenty productive light chain rearrangements were identified among the 16 MBL clones, with 11 Vλ and 9 Vκ genes used. We observed 6 productive rearrangements of Vλ1–51. MBL cells were bulk sorted for FISH from 9 subjects. Mono or biallelic deletion of 13q14.3 was observed in 5 subjects, the other 4 were normal. Conclusions : Our findings confirm that MBL is commonly observed among the unaffected family members from CLL kindreds. We found that some MBL clones express ZAP-70, CD38 or have unmutated IgVH genes and thus are similar to clinical CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. Small MBL clones are commonly oligoclonal. Importantly, although the immunoglobulin heavy and light chain genes rearranged in these MBL clones are all commonly used in CLL, the absence of VH1 and abundance of Vλ1–51 rearrangements do suggests differences in BCR usage between CLL and MBL. Further investigation of family associated MBL may clarify the genetics and immunobiology of familial CLL.

Description: The incidence of chronic lymphocytic leukemia (CLL) is significantly lower in African Americans than whites, but overall survival is inferior. The biologic basis for these observations remains unexplored. We hypothesized that germline genetic predispositions differ between African Americans and whites with CLL and yield inferior clinical outcomes among African Americans. We examined a discovery cohort of 42 African American CLL patients ascertained at Duke University and found that the risk allele frequency of most single nucleotide polymorphisms known to confer risk of development for CLL is significantly lower among African Americans than whites. We then confirmed our results in a distinct cohort of 68 African American patients ascertained by the CLL Research Consortium. These results provide the first evidence supporting differential genetic risk for CLL between African Americans compared with whites. A fuller understanding of differential genetic risk may improve prognostication and therapeutic decision making for all CLL patients.

Description: Abstract 1636 Background: CD37 is a tetraspanin protein expressed on the surface of normal and transformed B-cells across a wide range of maturational stages. TRU-016 is a novel humanized anti-CD37 protein therapeutic that has demonstrated significantly greater direct killing of CLL cells than rituximab and greater Fc mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab in pre-clinical models. Preclinical in vitro and in vivo models of NHL have demonstrated significant activity of TRU-016 against multiple cell lines. A recent phase 1 study in 57 relapsed and/or refractory CLL patients treated with TRU-016 identified the maximum tested dose (20 mg/kg) as safe and tolerable. We now report an extension of that study in patients with NHL. Methods: Patients with relapsed/refractory follicular NHL (FL), mantle cell (MCL), or Waldenström's (WM), adequate organ function, ECOG ≤2, and absolute neutrophil count 〉500/μL were eligible. Patients received 20 mg/kg of TRU-016 administered IV once a week for 8 doses followed by 4 monthly doses. Responses were determined using standard disease specific criteria. Results: 16 patients (8 FL; 4 MCL; and 4 WM) received TRU-016 in the expanded cohort with 4 patients in follow up at the time of abstract submission. Patient characteristics: median age 63 yrs (range, 41–81), median prior regimens 4 (1–7), refractory to last regimen 56% (9/16), refractory to anti-CD20 therapy 53% (8/15), bulky nodes ≥5 cm 57% (8/14), prior autologous stem cell transplant 13% (2/16). The most frequent adverse events were neutropenia 44% (7/16), fatigue 38% (6/16) and diarrhea, nausea and thrombocytopenia, at 25% (4/16) each. The most frequent Grade 3/4 adverse events were neutropenia 38% (6/16) and thrombocytopenia 13% (2/16); there were no Grade 3/4 infections. There were 2 serious adverse events reported by 2 patients, both were considered unrelated to TRU-016 by the investigators (a death due to an anaphylactic reaction to CT contrast and Grade 2 hypotension in another patient). Lymphocyte reduction of ≥50% was observed in 25% (4/16) of patients. Lymph node reduction of ≥50% by CT scan measurements was seen in 18% (2/11). One FL NHL patient had a PR, 6 had SD and 1 had PD. Two MCL patients had SD and 2 had PD. One WM patient had a Minor Response, 2 had SD and 1 had PD. Conclusions: TRU-016 treatment has a favorable safety profile. Clinical activity was observed in this small cohort of highly refractory and heavily pretreated heterogeneous group of B-cell NHL patients including a reduction in lymphocyte count, reduction in lymph node size by CT scans and one PR. The single-agent clinical activity of TRU-016, and the synergistic or additive effect of TRU-016 with multiple agents in pre-clinical models warrants further clinical investigation in NHL. A combination trial of TRU-016 with bendamustine and rituximab has been initiated in relapsed indolent B-cell NHL patients. Updated results from ongoing patient follow-up will be presented at the meeting. Disclosures: Stromatt: Emergent Product Development Seattle, LLC, Seattle, WA: Employment. Gopal:Abbott: Research Funding.

Description: Abstract 3430 Poster Board III-318 Background and Significance Chronic lymphocytic leukemia (CLL) is a malignant B cell lymphoproliferative disorder characterized by the clonal expansion and accumulation of CD5+ B cells. CLL is quite heterogeneous in clinical behavior, where some patients remain asymptomatic and require no therapy for many years, whereas other patients follow an aggressive clinical course with early need for therapy and short overall survival. Those patients with somatic deletions and / or mutations of p53, a central mediator of cell cycle control, apoptosis, and a potent tumor suppressor, follow the most aggressive clinical courses of all patients with CLL. Furthermore, these patients tend to be refractory to conventional CLL chemotherapy regimens, and therefore novel therapeutic approaches are needed. Cenersen (Eleos Inc.) is a 20-mer antisense molecule and binds to a coding region of exon 10 in p53 mRNA and has been shown in vitro to increase apoptosis in lymphoma cell lines. We hypothesize that the addition of cenersen to chemo-immunotherapy will enhance leukemic cell apoptosis and thus increase chemotherapy sensitivity in vivo for patients with aggressive CLL. Here we report early clinical responses to cenersen in combination with FCR chemo-immunotherapy at a planned interim analysis for efficacy and safety after enrollment of 17 patients. Methods We have undertaken a clinical trial at Duke University Medical Center of cenersen in combination with chemo-immunotherapy for patients with CLL or SLL (ClinicalTrials.gov identifier: NCT00636155). Patients with relapsed CLL or previously untreated patients with a deletion or mutation of p53 requiring therapy were enrolled. Cenersen was administered at 2.4 mg/kg/day as a continuous infusion over 24 hours starting on day 1 and ending on day 4. Fludarabine was administered daily at 25 mg/m2 on days 2 through 4, cyclophosphamide was administered daily at 250 mg/m2 on days 2 through 4, and rituximab was administered at 375 mg/m2 on day 2. Cycles were repeated every 28 days for a maximum of 6 cycles. The primary outcome measure was the overall response rate (complete and partial responses by iwCLL response criteria), and secondary outcome measures included progression free survival, event free survival and overall survival. All patients received growth factor support as well as prophylaxis with acyclovir and trimethoprim / sulfamethoxazole. Interphase cytogenetics and sequencing analysis for somatic mutations of p53 was performed in all patients. Results To date, 17 patients have been enrolled to a protocol-defined interim analysis of efficacy and safety. Of the enrolled patients, 1 was previously untreated, and the remaining 16 were relapsed (median number of prior therapies 4, range 1 - 8). Among previously treated patients, the mean response duration of the most recent prior regimen was 2 months. 15 of 16 had previously received fludarabine and 14 of 16 patients had previously received rituximab. 10 of 16 patients tested showed 17p deletions by FISH. Among the 14 patients tested to date, 8 showed somatic mutations in p53 by sequence analysis, of these, 6 showed both deletion of 17p as well as somatic mutation of p53. Of the 17 patients enrolled, 14 are evaluable for response: 1 patient has not yet received 3 cycles of therapy and 2 patients voluntarily discontinued trial participation. To date, 5 patients show a partial response by iwCLL criteria, including 2 patients who have concluded all therapy and show a partial response with incomplete hematologic recovery (PRi) with no detectable CLL B cells by flow cytometry analysis of bone marrow aspirate. One of these patients has subsequently undergone allogeneic stem cell transplantation. The remainder of the patients had stable disease (SD), though 3 were removed from study prior to the completion of 3 cycles for inadequate response as determined by the treating physician. There have been 4 episodes of febrile neutropenia, and there have been no toxicities specifically attributed to cenersen. Conclusions We have observed encouraging clinical responses to the combination of cenersen with FCR chemo-immunotherapy among a group of extensively pretreated patients with high risk genetic features. There does not appear to be any excess toxicity associated with the investigational agent. The results to date suggest that there may be therapeutic benefit to p53-targeted therapies in high risk CLL. This study will be continued to full accrual. Disclosures Lanasa: Genentech: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Rituximab, in combination with fludarabine and cyclophosphamide, for the treatment of CLL. Cook:Eleos: Employment. Rizzieri:Eleos: Research Funding.

Description: Abstract 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts 〉1.9 × 109 / L, progress to CLL, “low count” MBL (

Description: Abstract 2883 CLL-like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotypic features and cytogenetic abnormalities with CLL and is generally perceived as its premalignant state. The World Health Organization has set a consensus cut-off of 5×109/L circulating B cells to discriminate between what constitutes ‘disease’ and what not. However, the clonal size within MBL is extremely variable. High-count (HC), clinical MBL is associated with absolute lymphocytosis and progresses to CLL requiring treatment at a rate of 1–2% per year, whereas low-count (LC) MBL is found in the general population through high-sensitivity techniques and carries a risk of progression that is limited if any. Given the high frequency of CLL-like MBL in the general population, it is important to understand the underlying mechanisms and also identify biological markers endowing malignant potential that may distinguish between the different forms. To this end, we performed a detailed immunogenetic profiling of 334 CLL-like MBL cases (78 LC and 256 HC) for a total of 355 productive VDJ rearrangements (including double rearrangements), 91 from LC MBL and 264 from HC MBL. We also compared the immunoglobulin (IG) gene repertoires of MBL to 544 CLL Rai Stage 0 (CLL-0) that were part of an IG sequence dataset of 7424 CLL cases previously analyzed by our group. LC and HC MBL had distinct IG gene repertoires, with over-representation of the IGHV1–69 and IGHV4–34 genes in HC and the IGHV4–59/61 genes in LC MBL, respectively (p

Description: Introduction: Lenalidomide is an immunomodulatory small molecule with efficacy in CLL, though monotherapy responses are typically partial and delayed. Plerixafor, a small molecule inhibitor of the CXCR4 receptor responsible for homing CLL to the microenvironment, has been safely utilized in CLL. The combination of plerixafor with lenalidomide offered a novel non-cytotoxic alternative to improve anti-tumor activity through targeting of the essential pro-survival microenvironment. Figure 1 Figure 1. Methods: The primary objective of this single institution, open label, phase I clinical trial (NCT01373229) was to evaluate safety of daily lenalidomide in combination with escalating dose levels of plerixafor. Secondary objectives examined objective response rates, disease symptom improvement, and laboratory correlatives of the drugs’ synergy or resistance. The 3 stage study design is outlined in Figure 1. All adverse events (AEs) on combined therapy were recorded according to CTCAEv4, with dose limiting toxicity (DLT) assessment period defined as the first 28 days of combination therapy. Blood was collected throughout study conduct for analyses including an extended CLL phenotyping by flow cytometry with analysis of CLL subpopulations by CXCR4 expression levels. Phospho-protein analysis was performed by phospho-flow cytometry (phosflow). Figure 2 Figure 2. Results: Fifteen subjects (4 female) with a median age of 62 years were enrolled, and all have completed treatment on the trial. Subjects received a median 4 (range 1-8) prior chemotherapy regimens (100% with previous alkylator and rituximab, 93% with prior fludarabine) and had poor prognostic features (73% with del 17p, 93% with unmutated IGHV). Ten subjects initiated plerixafor, and 7 completed at least 1 combination cycle. Two dose limiting toxicities (neutropenia, thrombocytopenia) occurred in plerixafor dose 2 (0.32 mg/kg), thus MTD was exceeded at this level. A total of 6 subjects completed dose cohort 1 confirming 0.24 mg/kg as the MTD. Most common grade 3/4 toxicities (〉20%) on combination therapy included neutropenia (60%), thrombocytopenia (60%), and anemia (40%). Six patients reached interval response assessment and each had stable disease (SD). All response-evaluable patients derived improvement in nodal/splenic mass and disease symptoms. Of 4 subjects who completed the planned combination therapy, 2 continued on amended lenalidomide monotherapy with 1 subject obtaining a PR. Phenotyping demonstrated CD40, CD95, CD80 and CD86 significantly increased within the CXCR4 high population 4 hours post first plerixafor dose. For the five subjects completing greater than 4 months of combination treatment with end on study (EOS) samples, CD52 declined significantly; and although total WBC decreased over treatment course, the proportion of the high CXCR4 population increased with time (Figure 2). Phosflow demonstrated a significant increase of p-Erk (T202/Y204) by EOS. Conclusions: The MTD was oral lenalidomide 10mg daily with plerixafor SQ 0.24mg/kg thrice weekly for 3 weeks of a 28 day cycle. The majority of grade 3/4 toxicities (and DLTs) were cytopenias (anemia, neutropenia, thrombocytopenia). These toxicities may have been augmented from the combination treatment, or may be a reflection of study subjects with preexisting disease or therapy related marrow suppression. Other emerging lenalidomide clinical results suggest that lower doses may produce less myelosuppression while maintaining or improving clinical benefits by decreasing treatment disruptions that precipitate disease symptoms or tumor flare. Overall, the SD and single PR among heavily pretreated subjects with very high risk features are encouraging given the duration these subjects remained on trial without progression. Correlates demonstrated a decrease in CD52, a marker that has been previously associated to poorer CLL outcomes, and suggests a possible therapeutic effect given these subjects did not have disease progression while on treatment. Increases of CD40, CD95, CD80 and CD86 on lenalidomide monotherapy within 4 hours of plerixafor addition suggest immunomodulatory actions in circulating CLL cells (CXCR4 high expressing). Increased p-Erk by the end of trial may be an emerging resistance marker, and exploration of this effect in select patients could translate into other combination strategies that target this mechanism. Disclosures Brander: Celgene: Mentor received research funding Other. Off Label Use: This is a phase I clinical investigational study of lenalidomide and plerixafor combination in previously treated CLL/SLL.. Rizzieri:Celgene: Consultancy, Speakers Bureau; Sanofi: Consultancy. Lanasa:MedImmune, LLC: Employment; Genentech/Roche: Consultancy; Celgene: Research Funding.