Citations

Abstract Background: B-cell non-Hodgkin lymphomas (NHL) are common lymphoid malignancies in which malignant B-cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. The microenvironment that supports the growth and survival of NHL B cells consists of a complex network of immune cells and cytokines and its specific composition has been shown to have significant clinical implications. The intratumoral immune cells include effector and regulatory T (Treg) lymphocytes that are more than simple residual elements from the normal lymph node structure. We previously explored whether the extent of T cell infiltration played a role in the clinical outcome of patients with B-cell NHL and found that an increased percentage of CD4+ T cells in the diagnostic biopsy of lymphoma patients significantly correlated with an improved 5-year overall survival. We have also shown that intratumoral Treg cells significantly suppress the anti-tumor response. Based on the fact that intratumoral T-cells are important in B-cell NHL, we evaluated whether genetic variation in genes that regulate the T-cell response may be associated with lymphoma risk. Methods: We genotyped 257 single nucleotide polymorphisms (SNPs) from 50 candidate genes related to T-cell differentiation and function in a clinic-based study of 441 Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. Tagging and nsSNPs were selected from HapMap. The most prevalent homozygous genotype was used as the reference group and each SNP was modeled individually as having a log-additive effect, expressed as ordinal odds ratios (OR) per variant allele and 95% confidence intervals, in an age- and sex-adjusted logistic regression analysis. We also evaluated the consistency of the findings for the most common types of B-cell NHL - diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and CLL/SLL. For gene-level analyses, we used principal components (PC) analysis using all SNPs from a gene; those PCs that explained > 90% of the variability were then modeled in a logistic regression analysis and significance was determined using a likelihood ratio test. We also evaluated the association of haplotypes from each gene with risk of NHL using the score test implemented from HAPLO.SCORE. Results. The mean age at diagnosis was 60.1 years for cases and 58% were male; among controls, the mean age at enrollment was 61.7 years and 55% were men. In the PC gene analyses, PRF1 (perforin gene - involved in cytotoxicity; p=0.004), CD276 (B7-H3 gene - involved in co-stimulation; p=0.01), TBX21 (T-bet gene – regulates Th1 cell development; p=0.02), and IL6 (p=0.02) were significant at p<0.05; haplotype analysis showed similar results, with the addition of CARD15 (NOD2 gene – regulates Th1 responses; p=0.02). There were 2 SNPs in PRF1, and both the intronic SNP rs3758562 (OR=1.38; 1.13–1.69) and the synonymous coding SNP rs885821 (OR=0.80; 0.63–1.02) were associated with NHL risk overall and for each subtype. There were 2 SNPs in CD276, and both the intronic SNP rs7176654 (OR=1.29; 1.07–1.56) and the mrna-utr SNP rs3816661 (OR=0.82; 0.68–0.99) were associated with NHL risk overall and with the CLL/SLL and follicular NHL subtypes specifically. There were 2 SNPs in TBX21, and only the mrna-utr SNP rs7502875 (OR=0.73; 0.59–0.92) was associated with NHL risk overall and for each subtype. There were 11 SNPs in IL6, and the intronic SNP rs2069835 (OR=1.88; 1.24–2.84) and the locus-region SNP rs2069824 (OR=1.73; 1.20–2.50) were each associated with NHL risk overall and each subtype. Finally, there were 9 SNPs in CARD15, with only the nonsynonymous coding SNP rs5743291 (OR=1.43; 1.02–1.98) being associated with overall NHL risk, and risk was specific to FL. Conclusions. Genetic variation in several genes that play critical roles in T-cell maturation and function was associated with lymphoma risk. These genes appeared to be associated with the differentiation and function of effector T-cells particularly Th1 and Th17 cells. These results provide potentially important insights into the role of T-cells in lymphomagenesis.

Abstract Background: Reactive oxygen species cause DNA damage and altered immunologic responses, and have been linked to the development of NHL. Diets rich in fruits and vegetables are excellent sources of antioxidants, and vitamins C, E, polyphenols, and carotenoids, along with selected micronutrients such as zinc are thought to be responsible for most of the antioxidant activity in foods. We evaluated the hypothesis that vegetables, fruits and nutrients involved in antioxidant activity would protect against developing NHL. In a secondary analysis, we also assessed heterogeneity of this hypothesis for the most common subtypes of NHL: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma, and diffuse large B cell lymphoma (DLBCL). Methods: We evaluated dietary antioxidant intake and NHL risk in a clinic-based study of 416 newly diagnosed NHL cases and 926 frequency-matched controls enrolled at the Mayo Clinic from 2002–2007. Usual diet two years before diagnosis/enrollment was assessed using a self-administered, 128-item food frequency questionnaire. Dietary intake of antioxidants was estimated using the Food Processor SQL system, further supplemented with data from US Department of Agriculture nutrient databases. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusted for age, sex, residence, and total energy. NHL subtypes were centrally reviewed, and subtype-specific risks were estimated using polychotomous logistic regression. Results: The mean age at diagnosis was 60.8 years for cases and 57% were male; for controls, the mean age at enrollment was 60.8 years and 54% were men. NHL risk was inversely associated with intake of total vegetables (OR for highest compared to lowest quartile, 0.46; 95% CI 0.32–0.68; p-trend<0.001), and specifically green leafy (OR=0.54; 95% CI 0.39–0.76; p-trend<0.001) and cruciferous (OR=0.62; 95% CI 0.43–0.88; p-trend=0.02) vegetables, but not legumes or red/yellow/orange vegetables. There was no association with total fruit intake or intake of citrus fruits in particular. When modeled together, the association for green leafy vegetables was unchanged while the association for cruciferous vegetables attenuated (OR=0.75; 95% CI 0.50–1.13; p-trend=0.3). Higher intake of total vitamin A (OR=0.60; 95% CI 0.41–0.88; p-trend<0.001) and vitamin E (OR=0.53; 95% CI 0.34–0.82; p-trend<0.001), but not vitamin C, were associated with lower NHL risk. For vitamin A, there was no association with preformed Vitamin A, but there was an inverse association with beta-carotene (OR=0.56; 95% CI 0.39–0.81; p-trend<0.001); only weak inverse associations were observed for other carotenoids (lutein/zeaxanthin, lycopene, and cryptoxanthin). When modeled together, the inverse association for vitamin E was unchanged while the association for beta-carotene attenuated. For micronutrients, there were inverse associations for manganese (OR=0.60; 95% CI 0.40–0.90; p-trend=0.02), selenium (OR=0.47; 95% CI 0.27–0.80; p-trend=0.01), and zinc (OR=0.46; 95% CI 0.27–0.80; p-trend<0.001) but not copper; when modeled simultaneously, both manganese (p-trend=0.03) and zinc (p-trend=0.02) remained significantly and inversely associated with NHL risk. Further adjustment for education, family history of NHL, smoking, alcohol use, and body mass index did not alter these results. There was no NHL subtype heterogeneity for the associations with total vegetables, green leafy vegetables or vitamin E, while inverse associations were not observed for manganese and follicular lymphoma or zinc and CLL/SLL. Conclusion: Higher intakes of vegetables, particularly green leafy vegetables, vitamin E, manganese, and zinc were associated with lower risk of NHL. These finding extend a growing literature from case-control and cohort studies that support a protective role of foods and nutrients that function in antioxidant related pathways against development of NHL, and therefore may represent one of the few known modifiable risk factors for this cancer.

Abstract Abstract 2835 Background: Extrinsic growth and survival signals provided by nonneoplastic cells present within the tumor microenvironment promote the growth and survival of CLL cells. For example, bone marrow stromal cells, blood-derived “nurse-like” cells and follicular dendritic cells have been shown to promote the survival of CLL cells via the provision of both soluble and cell-contact dependent factors. More recently, monocytes were convincingly shown to support the survival of CLL cells during in vitro culture, and many of the factors (e.g. BLyS/BAFF, SDF-1, CD14) previously implicated in supporting the growth and survival of CLL cells are produced by monocytes. To explore the potential clinical implications of these in vitro findings, we evaluated the relationship between the absolute monocyte count (AMC) at diagnosis and disease progression in a well defined cohort of CLL patients. We hypothesized that the presence of a relative monocytosis at presentation in patients newly diagnosed with CLL may create a nurturing environment for CLL cells and be associated with more rapid disease progression. Methods: Previously untreated patients diagnosed with CLL/SLL within 12 months of presentation to the Mayo Clinic (from 1/1/2000-8/31/2010) were identified (n=1396). The AMC was obtained from routine complete blood counts (CBC) with automated or manual differentials that were performed within 3 months of diagnosis (n=947). The primary study objective was to determine whether the AMC is associated with time to first treatment (TTFT) in CLL. The secondary objective was to determine whether the AMC had independent prognostic value after adjusting for CD38/CD49d/Zap-70 expression, IGHV gene mutation status, and cytogenetic (11q-, 17p-) findings by FISH analysis. Results: The median age was 66 years and the median AMC was 550/μL (380–812/ μL, 1st-3rd quartile) at diagnosis (normal range: 300–900/μL). No significant difference in the median AMC was observed by age, gender; CD38, CD49d, or Zap-70 expression; IGHV gene mutation status or cytogenetic findings. Patients were followed for a median of 3.2 years (range: 0–10.3), with disease progression requiring the initiation of treatment observed in 268 (28%) patients. On multivariate analysis using a Cox proportional hazards model which incorporated age, gender and Rai stage, the AMC (as a continuous variable) was associated with TTFT with a hazard ratio (HR) of 1.099 (per unit change; 95% CI, 1.003–1.205; p=0.04). The optimal cutpoint for the association between AMC and TTFT was determined to be ≥1, 170/μL, a threshold that remained an independently significant predictor of TTFT after adjusting for age, gender and Rai stage with a HR of 5.1 (95% CI, 2.56–10.10, p<0.0001). The median TTFT was 1.0 year for patients with an AMC ≥1, 170/μL and 7.4 years for those with an AMC <1, 170/μL (p<0.0001). In order to evaluate whether the AMC had independent prognostic value after adjusting for other prognostic biomarkers, CD38, CD49d, Zap-70, IGHV gene mutation status and cytogenetic findings (11q- or 17p-), were individually incorporated into the multivariate analysis. In each case, the AMC remained an independent predictor of TTFT, with HRs ranging from 4.31 to 5.43 (p<0.0001 for all analyses). Conclusions: The AMC at the time of diagnosis as measured by a routine CBC is associated with accelerated disease progression in CLL/SLL independent of other prognostic biomarkers. These results suggest that the in vitro observation that nonneoplastic cells present in the tumor microenvironment promote CLL cell survival may have direct clinical implications for the prognostic evaluation of CLL patients and further substantiate the pathogenic role of monocytic cells in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2836 Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods. Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results: S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions: Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment: This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). Disclosures: Taylor: Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., >90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Abstract Background: In B-CLL, the observation of interphase cells with hemizygous D13S319 deletion at 13q14 (13q-x1) as a sole anomaly in blood is widely considered a favorable prognosis. The observation of cells with 11q-, +12 or 17p- has been associated with a relatively poor prognosis. Over the past 1.5 yrs, 16.2% (174/1,076) of patients (pts) referred for fluorescence in situ hybridization (FISH) testing for B-CLL in our clinical practice had a clone with homozygous D13S319 deletion (13q-x2), but the prognostic significance of this observation is poorly understood. Moreover, 39.3% (142/361) of pts with unfavorable FISH anomalies have 13q-x1 and/or 13q-x2 and the clinical significance of this observation is also unknown. Thus, we investigated pts with 13q- (with or without other chromosome anomalies) to establish the relative clinical significance of 13q- in B-CLL. Methods: We studied 333 pts with B-CLL sampled between 9/1999 and 6/2004 who had FISH performed on interphase nuclei from blood. The FISH probe set was designed to detect 6q-, 11q-, +12, 13q-, 17p-, and translocations involving IgH at 14q32. We classified pts into four groups: 13q-x1 only (group 1), 13q-x1 and 13q-x2 only (group 2), 13q-x2 only (group 3) and 13q-x1 and/or 13q-x2 plus other FISH anomalies (group 4). FISH groups were compared with gender, age, Rai stage, treatment status, time to treatment, CD38 and IgVH mutation. Results: Of the 333 pts, 171 (51.3%) had a 13q-: 71 were in group 1, 25 in group 2, 26 in group 3 and 49 in group 4. %CD38+ differed significantly across FISH groups; in pairwise analyses, the proportion of pts with >30% CD38+ was significantly greater for pts in group 4 vs. group 3 (p=0.0015) although no significant differences were observed for group 3 vs. group 1 or vs. group 2. Pts in group 3 were not significantly different from other FISH groups for Rai stage, IgVH mutation or gender. The median percentage of abnormal nuclei for pts with group 1 was 54.5% vs. 79.5% for pts in group 4 (p<0.0001). The median percent abnormal nuclei for pts in groups 3 and 2 was 72.5% and 68.5%, respectively. Median % abnormal nuclei for group 3 was not significantly different than the other FISH groups. Treatment status was available on 147 pts, where the proportion of pts who had treatment in each group was as follows: group 1, 15/66; group 2, 2/22; group 3, 4/21; and group 4, 9/38. Due to limited sample size and heavy censoring, any analysis on time to treatment is preliminary; however, these early analyses suggest group 4 pts have a lower median time to treatment (9 yrs) compared to groups 3 and 1 (12.3 and 13 yrs, respectively). Conclusions: This study has generated new information about the 13q- anomaly in B-CLL. First, known prognostic markers for B-CLL pts with 13q-x2 are not significantly different than for pts with 13q-x1 or 13q-x1/13q-x2. Second, 13q-x1 and/or 13q-x2 occurring with other unfavorable FISH anomalies have an unfavorable prognosis; i.e. potential benefits of 13q- are trumped when it is observed with unfavorable FISH anomalies. Thus, patients with any form of 13q- alone may have indolent disease while patients with 13q- and unfavorable FISH anomalies should be considered to be in a more aggressive phase.

Abstract The most common recognized cytogenetic change in B-CLL is a deletion involving band 13q14.3. Dewald found a heterozygous or homozygous 13q– in 92% of CLL patient samples using the FISH probe D13S319 that hybridizes to 13q14.3 (BJH 121:287, 2003). This contrasts with the usually normal conventional chromosome analysis (CCA) results partly because CLL cells divide infrequently but also because the 13q– is often visualized only by FISH (Stockero, Ca Genet Cytogenet 166:152, 2006). Mayr recently showed that CpG stimulation can reveal a chromosomally abnormal CLL clone in metaphase cells (Blood 107:742, 2006). The purpose of the present study was to compare the incidence of microdeletion and visible 13q deletion in CLL using interphase FISH and CCA after CpG culture on the same peripheral blood specimens. METHOD: We compared the chromosome 13 results by interphase FISH (fresh uncultured cells) and by a 20 metaphase CCA from 5–day CpG cultures. Our unselected CLL cohort (n=40) represented a typical and relatively high risk population: median age 61 (range 36–75), 48% CD38–positive, 62% ZAP–70 positive, and 48% IgVH unmutated. In addition 70% were previously treated for progressive disease, with all Rai stages represented. CpG RESULTS: By CCA of 40 patients, CpG stimulation revealed a clonal (multiple abnormal cells) or nonclonal (one abnormal cell) abnormal karyotype in 32 (80%) and a normal karyotype in 8 of the 40 patients (20%). Among the 32 abnormal cases, each chromosome 13 pair appeared normal in 17 and was abnormal in 15 (one was monosomy 13, 8 were 13q–, and 6 had a 13q translocation). CpG did not reveal a homozygous 13q abnormality in any patient. In total, CpG revealed a 13q abnormality in 15 of 40 (38%) patients (8 had multiple abnormal metaphases and 7 had only one abnormal metaphase). FISH RESULTS: By interphase FISH, 29 of 40 (73%) patients had a 13q–. The deletion was heterozygous in 18 patients, homozygous in 7, and mixed homo– and heterozygous in 4. Of the 18 with a heterozygous 13q loss by FISH, CpG revealed an abnormal 13 in only 8. Of the 11 patients with homozygous or mixed homo– and heterozygous 13q– by FISH, CpG revealed a heterozygous 13q abnormality in only 6. Of the 8 patients with a normal CpG karyotype, by FISH 5 had a 13q– in one or both 13s. Of the 9 patients with heterozygous 13q loss by CpG, the FISH result was 13q– in 4 and homozygous or mixed homo– and heterozygous 13q– in 5. Of the 6 with a chromosome 13 translocation by CpG, FISH revealed heterozygous 13q– in 4, mixed homo– and heterozygous 13q– in one, and in one case the 13q FISH result was within normal limits. In this latter case, CpG stimulation followed by metaphase FISH analysis confirmed that the apparently balanced translocation harbored a microdeletion 13q. Interphase FISH analysis confirmed the presence of a clonal deletion 13q in all 7 of the patients with a CpG metaphase result of a non-clonal (one metaphase cell) 13q abnormality. CONCLUSIONS: Even though CpG induces CLL metaphases in cell culture and reveals many chromosome abnormalities that cannot be identified by interphase FISH, cytogenetic evaluation of CLL by CpG alone significantly underestimates the incidence of 13q deletions. When a 20-cell analysis after CpG stimulation reveals a 13q abnromality in only one (apparently nonclonal) metaphase cell, concurrent interphase FISH analysis often confirms clonality for the 13q defect.

Abstract Abstract 4152 BACKGROUND: Patients with biologically aggressive chronic lymphocytic leukemia (CLL) characterized by being refractory to purine nucleoside analogues and/or possessing the cytogenetic abnormality del (17p13) have poor clinical outcomes with a median survival of <3 years. Several groups have reported that nonmyeloablative allogeneic stem cell transplant (alloSCT) provides long-term disease control in a subset of high-risk CLL patients. The Seattle group also recently reported that alemtuzumab use prior to alloSCT may increase the risk of early relapse/progression (Sorror, ASH 2010). This is a potentially critical observation since alemtuzumab is one of the few active agents in patients with del(17p13) and is frequently used for pre-transplant debulking in patients with this genetic defect. METHODS: We performed retrospective analysis of 38 CLL patients who underwent nonmyeloablative alloSCT for CLL at the Mayo Clinic between 2003 and 2011. The relationship between transplant outcome and debulking regimen used immediately prior to alloSCT, disease status at the time of alloSCT, post transplant engraftment, GVHD development was explored. The degree of marrow involvement prior to alloSCT was assessed using the leukemia index calculated by multiplying the % involvement by CLL cells and the % cellularity. RESULTS: Median age at the time of alloSCT was 55.5 (range, 29–69) and 13 patients (34%) were female. The median duration from CLL diagnosis to alloSCT was 4.7 years. Thirty-two patients (84%) had received more than 3 prior therapies prior to alloSCT. Thirty-seven patients (97%) had received purine analog-based therapies, 18 (47%) patients had received alkylating agents and 25 (66%) had received alemtuzumab. 33 (87%) patients were purine analog refractory. On cytogenetic evaluation by FISH, 17 (45%) patients had del(17p13), 10 (26%) had del(11q23) and 1 (3%) had del(6q23). Nine patients were in complete remission (CR) by IWCLL 2008 criteria at the time of alloSCT and 7 patients were in complete remission with incomplete marrow recovery (CRi). Of the remaining 22 patients, 20 had either a partial remission or stable disease at the time of alloSCT and 2 had progressive disease. Two (5%) patients had bulky lymphadenopathy (≥ 5cm by CT imaging), 11 (29%) patients had splenomegaly, and 8 (21%) patients had more than 30% CLL involvement by the leukemia index. Fludarabine (F) and 200cGy total body irradiation (TBI) was used as the conditioning regimen for 35 patients (92%). 22 patients received unrelated donor grafts (15 matched, 7 mismatched), and 16 received related donor grafts (all matched). Patients received mycophenolate (MMF) and either tacrolimus (unrelated grafts) or cyclosporin (related grafts) for graft versus host disease (GVHD) prophylaxis. 31 out of 37 evaluable patients (84%) achieved initial 100% donor chimerism (22 within 100 days) and 24 (63%) developed ≥grade 2 GVHD. After median follow-up of 1 year (range = 0–5.4 years), 26 of 38 patients (68%) are alive, and the median overall survival is 47 months. To date, 13 (34%) have experienced CLL progression/relapse requiring accelerated taper of GVHD prophylaxis (n = 2), donor lymphocyte infusion (n = 5), or chemo/immunotherapies (n= 6). For the 12 deceased patients, causes of death was CLL relapse/progression (n = 5), GVHD (n = 2) and infectious or other complications without CLL relapse/progression (n = 5). Pre-transplant use of alemtuzumab (A) within 3 months of transplant was the only pre-transplant characteristics associated with overall survival (OS). Median OS of patients receiving A within 3 months of alloSCT was 1 year compared to not reached for those who did not receive A within 3 months of alloSCT (p = 0.011; Figure 1a). A trend was also observed between alemtuzumab use within 12 months of alloSCT; however, it did not reach statistical significance (p=0.069; Figure 1b). No statistically significant associations between residual marrow involvement at time of alloSCT (p = 0.93), type of graft (p = 0.65), or presence of del (17p13) (p = 0.13), number of prior treatments (p = 0.45), patient age (p = 0.80) or disease status at time of alloSCT (p = 0.21) were observed. CONCLUSION: In this cohort of patients with high risk CLL, alemtuzumab use prior to transplant was the greatest predictor of OS after nonmyeloablative alloSCT. Non-alemtuzumab based debulking strategies need to be developed for patients with del (17p13). Disclosures: No relevant conflicts of interest to declare.

Abstract Serum BLyS levels have been found to be elevated in a number of immune disease models and there is increasing evidence that this may correlate with pathogenesis of various B cell related disorders including B cell malignancies. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in these scenarios and definition of the mechanisms that control BLyS expression remain to be fully elucidated. Serum BLyS levels are elevated in a number of B cell malignancies known to have a familial incidence. Therefore, we sought to determine if there was any correlation between serum BLyS levels and family history of B cell cancers. In our initial studies we examined the serum BLyS levels in patients with sporadic vs. familial B-cell chronic lymphocytic leukemia (B-CLL; near relative with B-CLL, multiple myeloma, or non-Hodgkin lymphoma), and normal age-matched controls. In the normal controls (n=50), the mean serum BLyS level was 12.7 ng/ml, and we found that 4/50 (8%) had BLyS levels exceeding 20 ng/ml. In the sporadic CLL cohort (n=52), the mean serum BLyS level was 18.3 ng/ml, and we found that 5/52 (10%) had BLyS levels exceeding 20 ng/ml. In striking contrast, in the familial CLL cohort (n=24), the mean serum BLyS levels was 33.4 ng/ml, and we found that 11/24 (46%) had BLyS levels exceeding 20 ng/ml. The percentage of patients with elevated BLyS levels, as well as the mean BLyS levels in the familial CLL cohort compared to the normal controls, was significantly higher (mean, p=0.002) and suggests that elevated BLyS levels may correlate with familial CLL. Because of the correlation between BLyS levels and family history B cell malignancies, we next wanted to determine if there was a common underlying genetic event influencing BLyS expression in this group of patients. We began by sequencing the BLyS promoter in 19 patients with B-CLL and 11 normal controls. We identified 2 sites that were polymorphic, −661 A/G and −871 C/T. The −871 T/T genotype has been previously reported to be expressed at increased frequency in SLE patients and is associated with elevated BLyS mRNA levels. Using RFLP analysis we examined the presence of the two polymorphisms in our control, sporadic, and familial CLL cohorts. In the control group (n=50) we found that at −871, C/C (wildtype) was expressed in 24%, C/T in 54%, and T/T in 22% of patients. Similarily, in the sporadic CLL group (n=35) we found that at −871, C/C was expressed in 29%, C/T in 57%, and T/T in 14%. In the familial group (n=24) we found that at −871, C/C was expressed in only 8%, C/T in 67%, and T/T in 25%. These data suggest a decreased representation of −871 C/C in the familial group. No significant changes were observed between the three groups at the −661 A/G locus. Additionally, we found that none of the patients with BLyS levels exceeding 20 ng/ml expresses C/C at −871. In summary, our data suggest that BLyS levels are elevated in patients with familial CLL relative to both normal controls and individuals with sporadic CLL and that elevated BLyS levels may correlate with the presence of a T at −871 in the BLyS promoter.

Abstract Abstract 3933 Poster Board III-869 Background Non-Hodgkin Lymphoma (NHL) is a malignancy of lymphocytes with few known risk factors identified to date. Members of the BCL2 and caspase gene families are known regulators of programmed cell death in these cells, and the t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in NHL, a somatic event which results in constitutive BCL2 expression and inhibition of apoptosis. Further, recent pooled analyses of three case-control studies reported gene level associations for both BCL2L11 (Cancer Epidemiol Biomarkers Prev 2009;18:1259) and CASP9 (Blood 2009;114:264) with NHL risk. We therefore evaluated the hypothesis that germline variation in genes from the apoptosis pathway is associated with risk of developing NHL. Methods We genotyped 226 single nucleotide polymorphisms (SNPs) within 36 candidate BCL2-family and capsase-family member genes within the apoptosis pathway in a clinic-based study of 441 newly diagnosed NHL cases (within 9 months of first NHL diagnosis) and 475 frequency matched controls from Minnesota, Iowa, and Wisconsin who were seen at the Mayo Clinic from 2002-2005. Tagging SNPs were selected from HapMap for the following candidate genes: BCL2; BCLAF1; BCL2LA1; BCL2L1, 2, 10, 11, 12, 13, 14; BAD; BAX; BAG1, 3, 4, 5; BAK1; BID; BIK; BNIP2, 3; HRK; CASP1 – CASP10; APAF1; BIRC3; AIF1; and DFFB. Genotyping was completed using the ParAllele (now Affymetrix) Immune and Inflammation SNP panel, supplemented with 9 SNPs from a custom Illumina OPA. SNP call rate and per person call rate were >95% for all SNPs evaluated. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect in a logistic regression model adjusted for age, sex, and residence. We used principal components analysis to assess the association with NHL risk at the gene level. Genes at p<0.05 were declared statistically significant, as were SNPs at p<0.05 from significant genes. Results The mean age at diagnosis for cases was 60.1 years, and 58% were male; among controls, the mean age at enrollment into the study was 61.7 years, and 55% were male. The most common NHL subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 29%), follicular lymphoma (FL, 27%), and diffuse large B-cell lymphoma (DLBCL, 16%). In gene level analyses, BCL2L11 (also know as BIM) (p=0.0019), BLCAF1 (p=0.0097), BAG5 (p=0.026), and CASP9 (p=0.0022) were statistically significant at p<0.05. For BCL2L11, 2 of the 5 tagSNPs (both intronic) were significant at p<0.05 (rs6746608 and rs12613243). Both genotyped BCLAF1 tagSNPs (rs797558 and rs703193, both intronic) were significant, as was the single genotyped BAG5 tagSNP (rs7042474) from the non-coding region interval of an mRNA transcript. Three of the 7 genotyped CASP9 tagSNPs were significant (rs6685648, rs2020902, rs2042370, all intronic). Similar associations (as assessed by ordinal odds ratios) were observed for significant SNPs from BCLAF1, BAG5, BCL2L11 and CASP9 within the subtypes of CLL/SLL, FL, and DLBCL; however, caution is warranted in interpreting the subtype results due to small sample sizes. Conclusions Three genes from the BCL2 family (BCL2L11, BCLAF1, and BAG5) and one gene from the caspase family (CASP9) were associated with risk of NHL. This confirms gene level results for BCL2L11 and CASP9 in NHL overall and suggests a role for additional BCL2 family members in NHL etiology. In addition to extending these results to other populations, investigation of the functional significance of these genes and consideration of their upstream regulators and downstream targets should assist in clarifying the role of this pathway in lymphomagenesis. Furthermore, in the era of targeted therapeutics, identification of genetic variation in the apoptotic pathway may also assist in predicting prognosis as therapies targeting this pathway are developed. Disclosures: No relevant conflicts of interest to declare.