ALBERT

Description: Vitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing.

Description: Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p 〈 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Description: 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.

Description: Abstract 1268 Poster Board I-290 Purpose There are reports of an increased risk of skin cancers in patients with B-Chronic Lymphocytic Leukemia (CLL). These skin cancers include basal cell and squamous cell carcinoma. This analysis was performed to more completely define the prevalence of skin cancers in patients in the Mayo Clinic Rochester CLL database and to look for contributing factors. Methods The Mayo Clinic Rochester CLL Database includes all patients with a diagnosis of CLL since January 1995 seen in the Division of Hematology and who have signed institutional review board approved consents for research. For this study, 2240 patients were analyzed to compare differences in characteristics between CLL patients with and without skin cancer. Chi-square statistics were used to compare qualitative variables (age categories, gender, referral status, ALC categories, CD38, ZAP-70, IgVH gene mutation status, FISH categories, Rai stage), and t-tests were used for quantitative variables (age at diagnosis, ALC values). Overall survival (OS) and time to first treatment (TFT) analyses were performed with results being displayed using Kaplan-Meier curves and p-values calculated using a log-rank test. Prevalence of melanoma among CLL patients was compared to the age-adjusted prevalence of melanoma in individuals in the Iowa SEER registry. Results Median follow-up for the 2240 patients diagnosed between 1/1/1995 and 8/11/2009 was 4.6 years. In aggregate, 293 (13.1%) patients were found to have non-melanoma skin cancer (squamous cell carcinoma or basal cell carcinoma) cancer. The diagnosis of non-melanoma skin cancer occurred before the CLL diagnosis in 39% and at or after the diagnosis of CLL in 61%. There were 57 (2.5%) cases of melanoma in association with CLL. The diagnosis of melanoma occurred before the CLL diagnosis in 38% and at or after the diagnosis of CLL in 62%.The prevalence of non-melanoma skin cancer and melanoma skin cancer were both higher in non-referred (geographically regional) CLL patients than referred CLL patients (16.6% vs. 11.4%, p

Description: Abstract 1245 Poster Board I-267 Introduction Chronic lymphocytic leukemia (CLL) patients with del(11q) by fluorescence in situ hybridization (FISH) have a poor prognosis. These patients often have younger age of onset, bulky lymphadenopathy, and short clinical response to treatment with purine analogues. Deletion of 11q in CLL is thought to involve loss-of-function of ataxia-telangiectasia mutated (ATM), a protein kinase central to DNA damage responses. As part of a larger study of early-intermediate stage, high risk CLL patients, we applied whole genome copy number variation (CNV) analysis to characterize the genomic alterations in CLL patients with and without del(11q) by FISH. Patients and Methods We studied 17 patients aged 29-67 with early-intermediate stage, untreated CLL who had high risk for disease progression based on evaluation of molecular and immunophenotypic markers. Of these patients, six had del(11q) by FISH analysis. No patients in this study had del(11q) as a sole FISH abnormality. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 106 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a single nucleotide polymorphism (SNP)-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results CNV analysis reveals hemizygous deletions of 11q in all 6 patients positive for del(11q) by FISH. The size of the deletion varies from ∼980 Kb to ∼44 Mb. All deletions include the region of the ATM gene at 11q22.3. No large homozygous deletions of 11q were detected. The three largest interstitial deletions (∼39 Mb, ∼41 Mb, ∼44 Mb) span 11q14.1-q23.3. A slightly smaller deletion of ∼33 Mb spans 11q14.2-q23.3. One deletion of ∼8.6 Mb covers 11q22.1-q22.3. The smallest deletion of ∼980 Kb is found within 11q22.3, centered on the ATM gene. Three out of 6 patients show complex interstitial deletions of 11q that consist of two or more discontinuous segments of loss of heterozygosity (LOH) separated by short undeleted regions. Five out of 6 patients with 11q deletions also show 13q hemizygous deletion by CNV analysis and by FISH. In 4 of these patients, the 11q and 13q interstitial deletions are the only acquired CNV events detected in the CLL genome. In one patient with 11q and 13q interstitial deletions, CNV analysis also shows chromosome 12 copy number (CN)=3 (also detected by FISH), CN=3 at 3q24-telomere (tel), and LOH events consistent with hemizygous deletion at 11p13 and 7p15.2-tel. Most noteworthy are 2 patients negative for any FISH abnormality who show copy-neutral LOH of most of 11q. The region of copy-neutral LOH includes 11q12-tel in one patient and 11q13.1-tel in the other patient. The copy-neutral LOH event involves close to 100% of the CLL cells in one patient, but only about 50% of the CLL cells in the other patient. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells and provides a powerful tool for CLL clinical research. Applying this new genomic technology to CLL patients with 11q deletions reveals marked complexity in the size and structure of hemizygous deletion events, which include the region of the ATM gene at 11q22.3 and can be discontinuous, indicative of complex genomic events. We identified copy-neutral LOH of 11q in 2 patients, a finding not previously described in CLL cells. Copy-neutral LOH of 11q is a genetic mechanism by which biallelic mutations of ATM, in cooperation with the loss of other tumor suppressor genes on 11q, may contribute to poor outcomes in some CLL patients without detectable FISH abnormalities. Additional studies, including sequencing of the ATM gene in 11q copy-neutral LOH patients, are required to confirm this hypothesis. Disclosures Shanafelt: Genentech, Hospira, Polyphenon E International, Celgene, Cephalon, Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genetech, Genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech: Research Funding; Bayer: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

Description: 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

Description: The standard of care for CLL is to treat only patients with obvious clinical progression because earlier intervention is not of proven benefit. The discovery of more accurate prognostic markers for CLL could change that paradigm. The predictors of more aggressive disease include 17p13 deletion (17p13−), 11q22-3 deletion (11q22−), unmutated (UM) immunoglobulin heavy-chain variable region (IgVH), and expression of ZAP-70 and CD38. In addition, monoclonal antibody (MoAb) therapies provide effective treatment with less toxicity than chemotherapy and are likely to be most efficacious in early stage CLL. The combination of alemtuzumab (ALEM) and rituximab (RIT) is of interest because of non-overlapping mechanisms of action. ALEM is also effective therapy for patients with defects in the p53 apoptotic pathway that are more resistance to purine analogue therapy. We tested the hypothesis that MoAb therapy with ALEM and RIT will eliminate/greatly decrease the high risk clone characterized by 17p13−, 11q22−, or UM IgVH plus either ZAP70+ or CD38+, in early stage CLL. Methods This trial will enroll a maximum of 30 patients and be considered promising if ≥ 19 patients respond. All patients with previously untreated CLL (Rai stage 0 −II) not meeting NCI-WG 1996 treatment criteria and with a high risk CLL clone were evaluated for enrollment. Treatment duration was 30 days (subcutaneous ALEM dose escalation, 3 mg - 10 mg - 30 mg on days 1–3) then 30 mg Monday, Wednesday and Friday for 4 weeks. RIT (375 mg/m2/dose IV x 4) was administered weekly staring on day 8. All patients received PCP and herpes virus prophylaxis and had CMV viral DNA testing for 7 months. Response was evaluated using NCI-WG 1996 criteria and minimal residual disease (MRD) was measured in peripheral blood using sensitive flow cytometry (1:104) for CD19+/CD5+/CD79b− lymphocytes. Results Since January 2005, 17 patients have been enrolled and the interim analyses are for the first 11 patients accrued. Median age was 62 years (29 – 75) with 6 males and 5 females. The qualifying high risk features were 17p13− (n = 4), 11q22− (n = 3), and UM IgVH + CD38+ +/− ZAP-70+ (n = 4). Median time from diagnosis to treatment was 11 months (2–72). Clinical stage (Rai) was 0 in 3 patients, I in 5 patients and II in 3 patients. Median absolute lymphocyte count was 25.6 x 109/L (15.9 – 81.8), Hgb 14.4 g/dL (12 – 15.8), and platelet count 171 x 109/L (125 – 312). Two patients had serious adverse reactions requiring intervention (CMV reactivation responsive to treatment; febrile drug reaction to sulfamethoxazole/trimethoprim). Grade 3–4 adverse reactions not requiring interventions were leukopenia (n = 4), neutropenia (n = 2), anemia (n = 1), elevated ALT (n = 1), and skin reaction to ALEM (n =1). There were no “first dose” reactions. All patients responded to therapy with 5 CR (4 of these MRD negative), 3 nodular PR, and 3 PR. Median duration of response has not yet been reached at median follow up of 11.7 months (6.5 – 14.9). Patients with a MRD negative CR had recurrence of detectable MRD at 120 – 210 days after completing therapy but all remain in CR. One patient died off study of complications of a myeloablative allogeneic transplant for progressive CLL. Discussion ALEM and RIT is effective and tolerable therapy for early stage high risk CLL. All patients responded with 36% achieving a MRD negative CR but serial MRD assays showed that the CLL clone was not deleted. This promising, treatment requires further improvement.

Description: Mutation status of the immunoglobulin heavy chain variable region (IgVH) in B cell chronic lymphocytic leukemia (B-CLL) is a critical prognostic tool. Although patients with unmutated (UM) IgVH genes exhibit an overall shorter survival than those with mutated (M) IgVH genes, considerable heterogeneity in clinical progression exists among UM B-CLL patients. The goal of this study was to evaluate UM CLL patients (n=215) in a large B-CLL cohort for Ig V, D, and J gene usage and relevant clinical parameters to identify Ig molecular features in addition to UM vs. M status that have prognostic value. Consistent with the literature, the most commonly expressed IgVH gene in our UM B-CLL cohort was VH 1–69 (69/215). We first evaluated D and J usage in VH 1-69 vs. non-VH 1–69 UM patients. The factors that were significantly different between VH1–69 vs. non-VH 1–69 cohorts were JH6 usage (p=0.0014), D3–3 usage (p=0.0025), and the combination of JH6 and D3–3 usage (p=0.0002). We then examined potential associations between patient time to treatment (TTT) and specific IgVH molecular features. Although there was a trend that VH 1–69 patients exhibited a shorter TTT than non-VH 1–69 patients, the association did not reach statistical significance (p=0.06). When all UM patients were instead grouped on the basis of D and J usage, JH6 usage was not significantly associated with TTT, but D3–3 usage, irrespective of VH or JH usage, significantly correlated with shorter TTT (p=0.005). Of interest, when JH6 patients were excluded from the analysis, differences in TTT between those with and without D3–3 usage were particularly pronounced (p=0.011). We next explored whether a specific D3–3 reading frame (RF) is associated with TTT. Within the group of D3–3 patients, we evaluated differences in TTT between those with RF 2 (n=38) vs. RF 3 (n=19) but did not study RF1 patients due to small numbers (n=6). Comparison of D3–3/RF 3 patients (n=19) with all other UM patients (n=190), did not reveal a significant difference in TTT, however, there was a significant difference (p=0.012) in TTT between D3–3/RF 2 patients (n=38) and all other UM patients (n=171). Rai risk was still the best overall prognostic factor, and was the only significant factor (p