Citations

Abstract Abstract 4610 Introduction: Infections are a major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). Up to 50% of patients with CLL will develop infectious complications during the course of their disease, and 30–50% of all deaths from CLL are attributed to infections and infectious complications. The risk of infection in CLL is related to immune deficiencies associated with the primary disease process, as well as immunosuppression secondary to treatment. Available research on infectious complications of CLL has focused on patients in clinical trials receiving chemotherapy. Little is known about the natural history of infection in community dwelling patients with newly diagnosed CLL. Method: To explore the natural history of infection in a local, community dwelling cohort of patients diagnosed with CLL, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed CLL seen in the Mayo Clinic Division of Hematology after January 1, 1999 and who resided within 50 mi of Mayo Clinic (Rochester, MN). All patient hospitalizations were reviewed to document infection including cross-referencing with the electronic Mayo Clinic infection database which includes all culture and serology results obtained from hospitalized Mayo Clinic patients. Patients who had positive blood, sputum, cerebrospinal fluid, pleural fluid, or paracentesis fluid culture results were identified. Patients whose cultures were negative were considered to have culture negative infection. Serologies and viral assays (e.g. CMV viral load) were also reviewed to identify infections. Results: In May 2010, there were 2022 patients in the Mayo Clinic CLL Database with the diagnosis of CLL/SLL made between Jan 1999 – Dec 2009. Of these, 336 (17%) patients resided within a 50 mi radius of Mayo Clinic and were considered non-referred, community-based CLL patients. Median age at diagnosis for these 336 patients was 69 yrs (range 27–97) and most had early stage CLL (57% stage 0; 34% stage I-II). With respect to prognostic markers, 206 (70%) patients were CD38 negative, 125 (64%) ZAP-70 negative, 85 (53%) IgVH mutated, and 101 (63%) had either no abnormality or 13q14- as a sole abnormality on FISH analysis. Consistent with being a community dwelling cohort, ~90% (152/165) of patients had co-morbid health problems and ~50% (86/165) had a major comorbidity (e.g. coronary artery disease, cerebral vascular disease, CHF, diabetes, COPD, other malignancy). Although ~17% (43/249) of patients had gamma-globulin levels below the reference range at diagnosis, only 2% (6/249) had an IgG level <500 mg/dL. Median follow-up for all patients was 49 months (range 0–121) and 104 (31%) patients progressed to require treatment for CLL. Although 222 (66%) patients required hospitalization during follow-up, only 38 (11.3%) were hospitalized with infection. Collectively, these 38 patients were hospitalized for infection 139 times (median 2 hospitalizations for infection per patient). Among these 38 patients, 13 had no organism identified on cultures (culture negative infection) while the remaining 25 (66%) had an organism identified during at least hospitalization for infection. Of the 38 patients hospitalized with infection, 18/38 (47%) had a gram positive organism (8 strep pneumonia, 11 staphylococcus Aureus, 2 nocardia), 11/38 (29%) had a gram negative organism (3 E.Coli, 4 Pseudomonas, 4 Hemophilus influenzae), 5/38 (13%) a mycobacterial infection (4 MAC, 1 M. gordonae), 15/38 (40%) a cytomegalovirus infection, 19/38 (50%), cryptococcus and/or cryptosporidium (18 cryptococcus AG, 4 cryptosporidium), and 12/38 (32%) a filamentous fungi infections (12 aspergillous). In aggregate, hospitalization for an opportunistic infection (e.g. cytomegalovirus, cryptococcus, filamentous fungi, myocobacteria, pneumocystis) occurred in 26/336 (8%) CLL patients during follow-up. Being male (OR 2.7; p=0.02), ZAP-70+ (OR 3.9; p=0.004), IGHV unmutated (OR 5.5; p=0.002), and receiving treatment (OR 4.7, p<0.001; Figure) were associated with increased risk of hospitalization for infection. Conclusions: Approximately 8% of newly diagnosed, community dwelling CLL patients will require hospitalization for opportunistic infection at some point during the course of their disease. Gender, disease factors (e.g. ZAP-70, IGHV status), and treatment characteristics are associated with infection risk. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding. Shanafelt:Celgene: Research Funding; Hospira: Research Funding; Genentech: Research Funding.

Abstract Abstract 2408 Purpose: Vitamin D insufficiency is common globally and in the United States. Approximately 25–50% of patients seen in routine clinical practice have vitamin D levels below the optimal range with low levels linked to higher cancer incidence. Although vitamin D insufficiency is also related to inferior prognosis in some cancers, no data exists for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL). Patients and Methods: To test the hypothesis that vitamin D levels predict outcome in patients with CLL, we evaluated the relationship of 25 (OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in 390 newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and separate cohort of 153 previously untreated patients participating in an observational study (confirmation cohort). 25 (OH)D levels were measured by liquid chromatography-tandem mass spectrometry. Intra- and inter-assay coefficients of variation were all <7% (data not shown). All vitamin D assays were performed on stored research samples; results were not included in the clinical record and were not known to the treating physicians in either study. Vitamin D insufficiency was defined as a serum 25 (OH)D level <25 ng/mL (62.5 nmol/L), an accepted level3 for the establishment of hypovitaminosis D and the threshold used in routine clinical practice to identify individuals with 25 (OH)D insufficiency by our laboratory in routine clinical practice (http://www.mayomedicallaboratories.com). Results: 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. 25 (OH)D insufficiency had no relationship with CD38, ZAP-70, CD49d or IGHV mutation status and had no relationship with FISH risk category as classified using the Dohner system. After median follow-up of 3 years, TTT (hazard ratio [HR]=1.66; p=0.005) and OS (HR=2.39; p=0.01) were shorter for 25 (OH)D insufficient patients (figure). We next attempted to validate these findings in an independent group of previously untreated CLL patients. In the validation cohort, 61 of 153 patients (39.9%) were 25 (OH)D insufficient. After median follow-up of 9.9 years, TTT (HR=1.59; p=0.05) and OS (HR 1.63; p=0.06) were again shorter for 25 (OH)D insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, stage, CD38, ZAP-70, IGHV, CD49d, and FISH, 25 (OH)D insufficiency remained an independent predictor of TTT (HR=1.47; p=0.008). On multivariable analysis for OS, the HR for death associated with 25 (OH)D insufficiency was similar to univariate findings although the p value did not reach the threshold of statistical significance (HR=1.47; 95% CI=0.97–2.23; p=0.07). Conclusion: 25 (OH)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. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract 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 (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 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. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

Abstract 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.

Abstract Background: PRCA is characterized by severe normochromic normocytic anemia, reticulocytopenia, and markedly reduced bone marrow erythroid precursors with intact leukocytic and megakaryocytic lineages. There is limited information about the prevalence and outcomes of PRCA in CLL, with the vast majority of information derived from case reports and small series comprising <10 patients. Here, we describe the clinical features, therapy, and outcomes of CLL patients with PRCA seen at our institution. Methods: The Mayo Clinic CLL Database includes all patients with a diagnosis of CLL evaluated at Mayo Clinic Rochester, MN between January 1995 and December 2014. We identified patients diagnosed with PRCA per the following diagnostic criteria: those who presented with normocytic anemia, had decreased erythroid precursors on bone marrow examination, and reticulocytopenia that could not be explained by any other cause besides PRCA. The baseline demographics, clinical characteristics, therapy, and outcomes were abstracted from the electronic medical records. We generated descriptive statistics and calculated overall survival (OS) from diagnosis date (PRCA, autoimmune cytopenia [AID], or CLL) to last known alive or death date. OS was plotted using Kaplan Meier curves and compared between groups using log-rank statistic. The Mayo Clinic IRB approved this study. Results: Of the 3945 CLL patients seen during the study interval, we identified 30 patients who met the diagnostic criteria of PRCA as described in the Methods section. The baseline characteristics, etiology and therapy for PRCA are shown in Table 1. The median time to PRCA from CLL diagnosis was 4.5 years (range-0.1-14 years). Of the 13 patients with IGHV results available, 12 (92%) were IGHV unmutated. Twenty-one (70%) were male and their median age at PRCA diagnosis was 67 years (range 40-81 years). Parvovirus testing was positive in 6 of 24 (25%) patients who were tested; 3 of these patients were treated with IVIG, 2 were treated with prednisone and IVIg, and 1 was treated with prednisone and rituximab. Eight patients (27%) had both PRCA and autoimmune hemolytic anemia (AIHA), and 1 patient (3%) had concomitant PRCA and immune thrombocytopenia (ITP). Of the 27 patients for whom we have treatment data, 10 required only one treatment regimen and 17 required 2-6 treatment regimens, with two patients requiring splenectomy for concomitant AIHA. The median survival of CLL patients after a diagnosis of PRCA was 4.6 years (Figure 1). There was no difference in survival between PRCA cases with and without parvovirus (p=0.92). Compared to newly diagnosed CLL Rai stage III/IV patients, there was no difference in OS (p=0.53). There was no statistically significant difference in survival from an AID cytopenia diagnosis when comparing PRCA, ITP, AIHA, and Evans syndrome (Figure 1; median 5 years for PRCA and 6 years for other groups; p=0.17). Conclusion: PRCA is a rare complication in CLL, occurring in <1% CLL patients seen at our institution over the past 20 years. Parvovirus was the etiologic factor in ~25% of patients and did not influence outcomes. There appears to be no significant difference in outcomes of PRCA patients compared to patients with other AID in CLL. Table 1. Patient Characteristics Characteristic N [range] or (%) Number of patients 30 Males 21 (70) IGHV status* · Unmutated · Mutated 12 (92) 1 (8) FISH results at CLL diagnosis* · 17p- · 11q- · Trisomy 12 · 13q- · Negative 0 (0) 1 (10) 3 (30) 3 (30) 3 (30) At PRCA Diagnosis Median age 67 [40 - 81] Median hemoglobin (gm/dL)* [normal range 12-15.5 gm/dL] 8.2 [3.5 - 10.8] Median absolute reticulocyte count (x 109/L)* [normal range: 38 - 113 x 109/L] 3.5 [0.0 - 25.3] Patients treated for CLL prior to PRCA diagnosis 25 (83) Etiology of PRCA Parvovirus (n = 24) 6 (25) Therapy-related: · Fludarabine · Alemtuzumab/rituximab · Pentostatin/cyclophosphamide/rituximab 3 (10) 1 (3) 1 (3) Autoimmune (neither therapy nor parvovirus related) 19 (63) Initial Therapy of Non-Parvovirus Related PRCA Steroids alone 2 (8) Steroids and Rituximab 3 (13) Steroids in combination with: · IVIG, Chlorambucil · Cyclosporine · Rituximab, IVIg · IVIg, splenectomy 1 (4) 2 (8) 1 (4) 1 (4) Monoclonal Ab alone · Alemtuzumab · Rituximab 1 (4) 4 (17) Cyclosporine 1 (4) Cyclophosphamide 2 (8) RCP (Rituxan, Cyclophosphamide, and prednisone) 2 (8) IVIg 1 (4) Not available 3 (13) *Not available for all patients Figure 1. Figure 1. Disclosures Ding: Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Tolero Pharma: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding. Shanafelt:Cephalon: Research Funding; Hospira: Research Funding; Glaxo-Smith-Kline: Research Funding; Genentech: Research Funding; Jannsen: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding.

Abstract Abstract 1246 Poster Board I-268 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a variable clinical course. Genomic aberrations as detected by FISH analysis and the mutational status of the IgVH genes have been found to have prognostic clinical significance with unmutated IgVH genes associated with clinically more aggressive disease. Previous studies have suggested that unmutated IgVH B-CLL is associated with increased chromosomal instability and increased chromosomal aberrations leading to accelerated disease progression. We recently performed a high-throughput, high-resolution array-based comparative genomic hybridization (aCGH) in a cohort of B-CLL patients. Fifty-eight patients have been analyzed by using the Sureprint G3 microarray (one million probes, Agilent). Our working hypothesis was that we would see increased genomic aberrations and instability in the unmutated IgVH group. The total number of copy number aberrations and total megabases (Mb) deleted or gained per genome were used as a surrogate marker for genomic instability. Variations in chromosomal aberrations in various VH families were also evaluated. B-CLL with unmutated IgVH genes utilized members of the VH1, VH3, VH4 and VH5 families with VH1-69 the most prevalent. B-CLL with mutated IgVH genes utilized VH families VH1, VH2, VH3, VH4, and VH6. Considerable variation was seen in the number of genomic abnormalities between cases. Overall, 315 abnormalities have been identified (236 deletions and 79 gains), with a median of 4 copy-number abnormalities per patient (range 0-32). In patients with IgVH mutational status available the median number of chromosomal aberrations in the mutated group was 5 (range 1-17) and unmutated group was 4 (range 0-20). Copy number deletions were significantly more common than chromosomal gains for the total group and in both the unmutated and mutated IgVH gene groups. Mean number of megabases (Mb) of DNA deleted in the unmutated IgVH group was 17.80Mb (standard deviation 24.17 Mb, median 6.63 Mb, range 0 - 90.55Mb) and 40.65 Mb in the mutated IgVH group (standard deviation 86.04 Mb, median 15.35Mb, range 0 – 307.57Mb). Mean number of Mb gained in the unmutated group was 37.74Mb (standard deviation 57.58Mb, median 0.44Mb, range 0 – 132.47Mb) and 32.99Mb (standard deviation 59.76Mb, median 0.14Mb, range 0 – 172.50Mb) in the mutated group. Nine patients had B-CLL with VH1-69 genes and 8 of them were unmutated. Median number of chromosomal deletions was 2 and gains also 2 (ranges 0 – 8 deletions and 0 – 4 gains) for this group. Similar findings were seen for the other VH families. These results initially suggest that total copy number variations do not account for the observed clinical differences between mutated and unmutated IgVH B-CLL. The catalogue of potentially clinically relevant chromosomal abnormalities found in this aCGH study is being currently evaluated. Disclosures Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Fonseca:BMS: Consultancy; Amgen: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Abstract Abstract 1232 Poster Board I-254 B-Chronic Lymphocytic Leukemia (CLL) shows a strong familial risk and also co-aggregates with other indolent lymphomas. Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic hematologic condition characterized by small B-cell clones, most with a surface phenotype similar to that of chronic lymphocytic leukemia (CLL). These clones are detectable at low absolute lymphocyte cell (ALC) numbers in otherwise healthy individuals using sensitive 6 or 8 color flow cytometry analysis and can be a precursor to CLL. In the general population, MBL increases with age with a prevalence of 5% in individuals over age 65. In contrast, the rate of MBL is 13-18% in first degree relatives of CLL patients in high risk families. In the largest study to date, we report the characteristics of MBL among 430 first-degree relatives of CLL patients (with no associated lymphoproliferative diseases [LPD]) in 132 high risk families. Patients and Methods Individuals studied came from “high risk” families (defined as those with two or more confirmed cases of CLL) that are participating in the Genetic Epidemiology of CLL Consortium. Multi-parameter flow cytometry analysis with a detection sensitivity of 0.02% was performed on either fresh or cryopreserved PBMC and MBL was classified according to previously published criteria (Marti et al, Br. J Haematol 130:325, 2005). Both the ALC and the absolute B cell count (B-ALC) were calculated for each individual. Survival analysis was used to compute the probability of developing MBL with age using the life table method. Results The overall rate of MBL was 17% (73/430) among first–degree relatives but the probability for developing MBL by age 90 was 51%. Males had a slightly higher (but non-significant) risk for MBL than females. MBL patients had significantly higher ALC and B-ALC than did those with normal immunophenotype. The mean ALC was 2.5×109/L among MBL patients, and 20% had an ALC greater than 3 ×109/L. The B-ALC count averaged 0.51 ×109/L. Ninety percent of the MBL cases had a CLL-like phenotype (CD20dim, CD5+, CD23+). Conclusions MBL is found at a very high rate in families selected for having 2 or more patients with CLL suggesting that MBL reflects inherited predisposition to CLL. Although most of the MBL cases in these families had low cell counts and thus have a presumed low likelihood of progressing to CLL or other LPD, a higher proportion of them had lymphocytosis compared to those with normal immunophenotype. We hypothesize that if MBL is an early step in the process of development of CLL, then germ line genes are likely to be acting early in carcinogenesis with more “hits” required before CLL develops. By looking for genes associated with MBL or CLL in families or in the population, we could substantially increase our power to identify germ line genes predisposing to CLL. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Abstract Background Severe cytopenias in CLL are typically associated with bone marrow failure due to massive infiltration of the bone marrow by leukemic cells and negatively impact patient morbidity/mortality. Although near complete marrow replacement by CLL is typically observed in patients whose degree of cytopenia meet criteria for advanced Rai stage disease (Hg<11g/dL; platelet <100 x10^9/L), profound variability in the degree of cytopenia and the extent of marrow infiltration exists, and the relationship between these dimensions is not well characterized. Methods We used the Mayo Clinic CLL database to identify all previously untreated CLL/SLL patients who underwent bone marrow biopsies. CLL patients known to have autoimmune-based cytopenias were excluded from this study. Hemoglobin, platelets, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and absolute monocyte (AMC) at the time of bone marrow biopsy were collected. Characteristics from bone marrow biopsy including marrow cellularity, % CLL involvement, CLL leukemic index (calculated as % cellularity times % CLL), and non-CLL leukemic index (calculated as % cellularity times (1-% CLL)) were also collected. Bone marrow cellularity and % CLL involvement were recorded in the Mayo Clinic CLL database based on clinical estimates made by Mayo hematopathologists. Clinical characteristics and CLL prognostic factors (e.g., Rai stage, CLL FISH, IGHV, ZAP-70, CD38 and CD49d) were also obtained from the database. Age- and sex-adjusted Pearson correlations between the bone marrow measurements and hemoglobin and platelet levels were computed, and scatterplots were created. Results A total of 695 untreated CLL patients diagnosed between 1972 and 2015 who had a CBC test at the time of bone marrow biopsy performed at Mayo Clinic were identified. Median age at diagnosis was 61 years, and 487 (70%) patients were men. Mean time from CLL diagnosis to bone marrow biopsy was 2.2 years. At the time bone marrow biopsy was performed, 20% of patients had advanced stage disease (Rai III/IV) disease, while 44% and 36% had intermediate (Rai I/II) and early stage (Rai 0), respectively. With respect to prognostic parameters, 53% had unmutated IGHV and 20% had high risk CLL FISH (11q- and 17p-). The distribution of other prognostic factors included 43% CD49d+, 35% CD38+, and 42% ZAP-70+ CLL diseases. Median peripheral blood counts were: hemoglobin 13g/dL, platelet 167,000/mL, ALC 26,600/mL, ANC 4100/mL, AMC 700/mL. The median bone marrow cellularity and percent CLL involvement were 70% and 70%, respectively. The median CLL leukemic index was 42%, and median non-CLL leukemic index was 21%. Pearson correlation analysis of bone marrow measurements with peripheral blood hemoglobin found the strongest correlation with the CLL leukemic index (r= -0.49, p<0.001) (Figure), followed by the % marrow CLL involvement (r= -0.45, p<0.001), cellularity (r= -0.40, p<0.001), and non-CLL leukemic index (r= 0.37, p<0.001). Pearson correlation analysis of the bone marrow measurements with platelet count also found the strongest correlation with the CLL leukemic index (r= -0.39, p<0.001), followed by % marrow CLL involvement (r= -0.38, p<0.001), non-CLL leukemic index (r= 0.35, p<0.001), and cellularity (r= -0.28, p<0.001). The peripheral blood ANC levels did not correlate with the four bone marrow measurements. Age- and sex-adjusted Pearson correlations between the bone marrow measurements and hemoglobin and platelet levels were similar to the above unadjusted results. Conclusion To our knowledge, this is the first study to examine the relationship between peripheral blood counts and the extent of bone marrow infiltration in patients with CLL. Although the bone marrow leukemic index had the strongest correlation with peripheral blood Hg and platelet counts, the correlation was modest with an r value of -0.49. These findings suggest that the extent of infiltration of the bone marrow by CLL cells only partially explains of the anemia and thrombocytopenia observed in CLL, indicating other biologic interactions between CLL B-cells and hematopoietic cells contribute to anemia and thrombocytopenia in these patients. More research is needed to determine the nature of these interactions. Disclosures Ding: Merek: Research Funding. Kay:Genentech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: 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, Research Funding; Tolero Pharm: Research Funding. Shanafelt:genentech: Research Funding; Glaxo-Smith-knine: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding.

Abstract 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<0.001 for non melanoma; 2.0% vs. 3.6%, p=0.03 for melanoma). The prevalence of melanoma in CLL patients was higher than that of age-adjusted prevalence for individuals in the Iowa SEER registry (2.5% vs. 0.03%; p<0.001). We next evaluated the relationship between CLL patients with skin cancer and demographic characteristics, prognostic parameters, and CLL related treatment. The risk of non-melanoma skin cancer in CLL was found to be associated with age (median age at diagnosis with skin cancer 67.6 vs. without skin cancer 63.3, p<0.001) and with sex (males 15.1% vs females 8.9%, p <0.001). There was no statistical significant difference in frequency of non-melanoma skin cancer associated with absolute lymphocyte count (ALC), Rai stage, CD 38, Zap 70, IgVH gene mutation status, FISH, or treatment history. The risk of melanoma in CLL was found to be associated with age at diagnosis (median age with melanoma 69.9 vs. without melanoma 63.8, p=0.002) and CD38 (positive 1.3% vs negative 3.1%, p=0.03). There was no statistically significant difference associated with gender, ALC, Rai stage, Zap 70, IgVH gene mutation status, FISH, or treatment history. Since the presence of skin cancer could be a marker of immune dysregulation we hypothesized skin cancer may be associated with clinical outcome of CLL. Accordingly, we evaluated the relationship between non-melanoma skin cancer and melanoma skin cancer and TFT and OS. Contrary to our hypothesis, TFT (median 6.0 years vs. 4.9; p=0.04) and OS (median 10.8 years vs. 9.7; p=0.02) of patients with non-melanoma skin cancer were both longer than those without non-melanoma skin cancer. No differences in TFT (p=0.06) or OS (p=0.66) were observed among patients with melanoma compared to those without melanoma. Conclusions We find in our cohort of CLL patients a higher prevalence of melanoma than the general population. Risk of melanoma and non-melanoma skin cancer among patients with CLL does not appear to be related to CLL characteristics, with the exception of CD 38, or CLL outcome. The diagnosis of melanoma and non-melanoma skin cancer in patients with CLL does not appear to be a risk factor for either CLL specific outcomes (TFT) or shorter survival. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding.

Abstract Patients with very-high risk (purine analogue refractory and TP53 defective) CLL have limited treatment options. In these patients alemtuzumab can be effective against CLL cells in the circulation and bone marrow, and in combination therapy with fludarabine can be active in patients with bulky disease, but these regimens have a high risk of serious infections. Addition of rituximab to alemtuzumab can also improve efficacy but has limited activity against bulky disease. We conducted a phase II clinical trial to determine the efficacy and toxicity of therapy with pentostatin, alemtuzumab, and low dose higher frequency rituximab (PAR) in patients with relapsed/refractory or progressive CLL with 17p13 deletion. The rituximab schedule was designed to decrease the loss of CD20 expression by circulating CLL cells. Methods This two-stage phase II trial study (NCT00669318) conducted at the Mayo Clinic Rochester and University of Iowa with IRB approval had an accrual goal of 38 evaluable patients. Eligibility required a diagnosis of progressive CLL by standard criteria and either previous treatment for CLL (<4 purine analogue regimens) or 17p13 deletion (17p13-). Exclusion criteria were organ failure, poor performance status (ECOG >3), infection with HIV, hepatitis B, hepatitis C, active autoimmune cytopenia, or alemtuzumab therapy within the past 2 months. Rituximab 20 mg/m2 IV M-W-F started on day 1, alemtuzumab started on day 3 with an escalation of 3-10-30 mg/d SQ and then 30 mg M-W-F from day 8, and pentostatin 2 mg/m2 IV every 2 weeks started on day 8. Peg-G-CSF or GM-CSF was used after each dose of pentostatin and patients received Pneumocystis and Varicella prophylaxis. CMV PCR assays were done weekly during treatment and viremia was treated with either valganciclovir or ganciclovir. Cycle 1 was 5 weeks and subsequent cycles were 4 weeks. At the end of cycle 2 patients with a clinical CR had a CT scan and a bone marrow study with immunohistochemical (IHC) staining for residual CLL cells, and therapy was stopped if there was no radiological or IHC evidence of residual CLL (stringent CR). Patients with residual disease received a 3rd cycle of therapy. Results Forty-one patients were enrolled (July 2008 - February 2013) and all 39 who started therapy were evaluable for response: Median age 61 years (range 47-78), 30 (77%) males, 36 (92%) relapsed/refractory CLL (median prior regimens = 2, range 1-10), 3 (8%) previously untreated, 23 (59%) advanced stage (Rai III-IV), 16 (41%) intermediate stage (Rai I-II). Prognostic factors: FISH (hierarchical classification) 15 (38%) 17p13-, 6 (15%) 11q22-, 5 (13%) 12+, 3 (8%) no defects, 8 (21%) 13q14-, and 2 (5%) other abnormalities, IGHV analysis (n=38) 27 (71%) unmutated (<2%), ZAP-70 (n=37) 28 (76%) positive (>20%). Thirty (77%) patients completed planned therapy (28 had 3 cycles, 2 had 2 cycles with stringent CR). Nine patients received one (n=4) or two (n=5) cycles of therapy because of disease progression or complications. Grade 3-4 hematological adverse events (n=37) at least possibly related to treatment included neutropenia (n=22), thrombocytopenia (n=11), anemia (n=2) and hemolysis (n=2). Non-hematological adverse events (n=17) included infections/neutropenic fever (n=8), fatigue (n=3), and hemorrhage (n=2). CMV reactivation was detected and treated in 14 patients (grade 1-2). No patients died during treatment or from treatment related complications. The overall response rate was 56% (95% CI 40-72) with 4 (10%) CR, 7 (18%) CRi, 11 (28%) PR, 7 (18%) SD, and 10 (26%) PD. Four patients (3 CR and 1 CRi) had IHC negative bone marrow studies. Thirteen (33%) patients have died due to progressive CLL (n = 11), sepsis (n=1), and pneumonia (n=1). Median follow up for surviving patients is 23 months (range 3-55). Seven (18%) patients proceeding to RIC allogeneic transplant were censored for time to next treatment. Twenty-one (54%) patients required therapy for progressive CLL and 7 (18%) have required no further therapy. Median progression free survival was 7 months (95% CI: 5-16), time to next treatment 9 months (95% CI: 6-27) and median overall survival has not been reached. Discussion PAR was effective and tolerable therapy for high-risk CLL. This study suggests that alemtuzumab can be used safely in combination with a purine analogue in a short-duration regimen. Disclosures: Zent: Genentech : Research Funding; Genzyme: Research Funding; Biothera: Research Funding; GlaxSmithKline: Research Funding; Novartis: Research Funding. Off Label Use: Pentostatin therapy for CLL, use of lower doses of rituximab.