Call, Timothy G.

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.

Abstract Background: Over the last decade, increasing numbers of families with at least 2 cases of CLL or other B-cell lymphoproliferative disorders have been described. Methods: Families seen at Mayo Clinic with at least one individual diagnosed with CLL and one or more first or second degree relative diagnosed with CLL or a B-cell lymphoproliferative disorder(LPD) were identified. Clinical characteristics and risk stratification test results (cytogenetic abnormalities by FISH, level of CD38 expression, and IgVH gene mutation status) were extracted and reviewed. Serum, intracellular and CLL B cell secreted levels of pro- and anti-angiogenic cytokines (VEGF, BFGF, and TSP) were also compared between cases of familial and sporadic CLL. Results: Seventy-one families seen at Mayo Clinic were identified meeting criteria for familial CLL. Of the 71 index CLL families, 78.9% (n=56) had at least one other first or second-degree relative with CLL or a B cell LPD, 15.5% (n=11) had 2 other affected members, and 5.6% (n=4) had 3 or more affected members. Of affected family members with B-cell LPDs other than CLL, 64% (n=25) had non-Hodgkin lymphoma, 21% (n=8) had Hodgkin disease, 10% (n=4) had multiple myeloma or Waldenstrom macroglobulinemia, and 5% (n=2) had another lymphoid leukemia (1 ALL, 1 hairy cell leukemia). The median age was 62, with 63% males and 37% females. The majority of individuals had an early Rai stage at diagnosis (stage 0–66%, stage I-19%). Results of cytogenetic testing by FISH, CD38 status, IgVH gene mutation status appear similar in familial and sporadic CLL cases seen at Mayo clinic during the same time interval. No clear pattern of IgVH gene mutation status, IgVH gene family usage, level of CD38 expression, or cytogenetic abnormalities by FISH was seen among multiple affected members from the same family. There were also no significant differences noted in serum, intracellular, and CLL B cell secreted levels of VEGF, BFGF or TSP in the familial samples when compared to a cohort of non-familial samples. Conclusions: Expression of cytogenetics by FISH, immunophenotype, IgVH mutation status and levels of serum, intracellular and cytoplasmic pro- and anti-angiogenic factors(VEGF, BFGF, and TSP) were similar in individuals with familial and sporadic CLL.

Abstract Abstract 2409 Background: Serum free light chain (FLC) quantitation has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and amyloid light chain (AL) amyloidosis, and it has been incorporated in response criteria for both MM and AL amyloidosis. Two recent studies have shown FLC abnormalities are present in 39–54% of CLL patients and are associated with shorter time to treatment (TTT) and inferior overall survival (OS) in CLL. Here we report the largest study to date of FLC abnormalities in a prospective cohort of CLL patients and explore the effects of both monoclonal and polyclonal FLC elevations. Methods: Newly diagnosed CLL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum FLC was quantitated from stored research serum samples using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Elevated FLC was defined as either kappa or lambda above the reference range (k>19.4 mg/L, l>26.3 mg/L). Monoclonal FLC elevation was defined as elevated FLC with an abnormal FLC ratio. Polyclonal FLC elevation was defined as elevated FLC with a normal FLC ratio (0.26–1.65). Patients with normal FLC but abnormal FLC ratio were evaluated as a distinct group (Abnormal FLC Ratio). Clinical data was abstracted using a standard protocol for the MER and patients were followed systematically for TTT and OS. Cox proportional hazards models were used to assess the association of FLC with TTT and OS. Results: 385 CLL patients diagnosed between September 2002 and February 2008 with available serum were assayed. At a median follow-up of 47 months (range 1–94), 135 patients (35%) have been treated and 43 patients have died (11%). Abnormalities in FLC were present in nearly half of the CLL patients (48.8%). Although 32.4% had an elevated kappa or lambda, only 14.0% had a monoclonal FLC elevation while the remaining 18.4% had a polyclonal FLC elevation. An additional 16.4% had an abnormal ratio without elevated FLC. Monoclonal FLC elevation was associated with high risk features of CD38+, CD49D+, ZAP70+, IGHV unmutated and having high risk FISH (del 17p13; del 11q22). Monoclonal FLC elevation was highly associated with shorter TTT (HR=3.24, p=0.0007) and poor overall survival (HR=4.14, p=0.0006). These associations remained statistically significant after adjusting for stage and prognostic factors. Abnormal FLC ratio without elevated FLC was also associated with shorter TTT (HR=1.74, p=0.03), though this association did not hold after adjustment for stage and prognostic factors. Polyclonal FLC elevations were associated high risk FISH and poor overall survival (HR=6.41, p<0.0001, adjusted HR = 4.82, p=0.0002) but not with TTT. Conclusions: In the largest series of newly diagnosed CLL patients reported to date, nearly half of CLL patients have abnormal FLC concentrations. Monoclonal FLC elevation is an independent predictor of shorter TTT and inferior overall survival in CLL, is associated with high risk markers, and may be a marker of an aggressive tumor. Polyclonal FLC elevation without abnormal FLC ratio is independently associated with poor overall survival but not TTT suggesting it may be a non-specific marker of host frailty/comorbidity rather than disease specific outcomes. Regardless of cause, CLL patients with elevated FLC have poor outcome and FLC may have utility as a biomarker in CLL. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 2443 Background: There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association (GWA) study of CLL identified genetic variants located on chromosomes 2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13 that increased the risk of CLL within a European population. We replicated 5 of these 6 loci in an independent sample of CLL cases and controls from the United States. We now investigate whether these loci also influences MBL, a reported precursor condition of CLL. In addition, a follow-up analysis of the initial GWA study identified four more CLL-susceptibility loci on 2q37.3, 8q24.21, 15q21.3, and 16q24.1. Herein, we also evaluate the association of these four loci with risk of CLL. Methods: Peripheral blood samples were obtained from three ongoing studies: the Genetic Epidemiology CLL (GEC) Consortium, the Mayo Clinic non-Hodgkin lymphoma (NHL)/ CLL study, and the Mayo Clinic Biobank. We implemented rigorous genotyping quality-control measures, and successfully genotyped a total of 407 CLL patients, 965 controls, and 60 MBLs from these studies. Within each locus, the previously reported single nucleotide variants (SNPs) or variants in high linkage disequilibrium (LD) with the previously reported SNPs were evaluated with risk of MBL or CLL. Tests for association was done using the Cochran-Armitage trend test, and unconditional logistic regression was used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for CLL or MBL risk Results: In our evaluation of the six initially reported CLL-susceptibility loci (2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13) with MBL risk, we found three of the six had suggestive associations (p-value < 0.20) with ORs comparable to and in the same direction as those observed from CLL risk. Our strongest finding was with rs13397985 at locus 2q37.1 (OR= 1.56; 95% CI: 1.05, 2.31; p-trend = 0.041), followed by rs17483466 at locus 2q13 (OR= 1.49; 95% CI: 0.99, 2.24; p-trend = 0.074). As expected given our previously reported findings with CLL risk, the association between rs11083846 on chromosome 19q13 and MBL risk was not significant (p-trend = 0.70). Of the four recently reported CLL-susceptibility loci SNPs located on 2q37.3, 8q24.21, 15q21.3, and 16q24.1, we found all to be associated with CLL risk but one. Specifically, the strongest association was seen for locus 8q24.21 (best tagged SNP rs1021955; OR = 1.37; 95% CI: 1.10, 1.70; p-trend = 0.005), followed by locus 16q24.1 (best tagged SNP rs305065; OR= 0.77; 95% CI: 0.61, 0.97; p-trend = 0.024). However, we found no associations for locus 15q21.3 for the previously reported SNP nor for any SNPs in LD with the previously reported SNP. Conclusions: Our MBL results provide additional robust genetic evidence that MBL is a precursor to CLL and that it shares similar underlying genetic predisposition. Also our results confirm three of the four recently reported CLL-susceptibility loci and further support the role of a genetic basis in the etiology of CLL. More research is needed to elucidate the potential manner in which these genetic loci function in CLL or MBL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4610 Introduction Infections are a major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). Up to 50% of CLL patients develop infectious complications, and 30–50% of all deaths in CLL patients are attributed to infections. Despite these data, the available research on infectious complications in CLL is primarily limited to patients participating in clinical trials of chemotherapy, obscuring the distinction between the risk of infection due to the disease’s natural history relative to the risk associated with therapy related immune suppression. METHODS: The Mayo Clinic Rochester (MCR), a tertiary referral center in rural southeastern Minnesota, is also the primary hematology center for southeastern Minnesota, northern Iowa, and western Wisconsin. There are no metropolitan areas or hematology specialty centers within a ∼50 mile radius. To explore the natural history of infection in a community-based cohort of CLL patients, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed CLL who resided within 50 miles of MCR and diagnosed between January, 1999 and December, 2009. The comparison cohort consisted of 689 adult patients who also resided within 50 miles of MCR who were seen for a general medical examination between September, 2002 and December, 2009 and who were enrolled as controls in a case-control study of non-Hodgkin lymphoma. All hospitalizations at MCR among both cohorts were audited to document hospitalization with infection and cross-referenced with the Mayo Clinic infection database, which includes all culture results obtained from hospitalized MCR patients. Serologies and viral assays were also reviewed. Patients whose cultures were negative but who were given a clinical diagnosis of infection (e.g. pneumonia) and who were treated with a full course of antibiotic therapy were considered to have a culture negative infection. RESULTS: In May 2010, there were 2022 CLL patients in the Mayo Clinic CLL Database diagnosed between 1999 and 2009. Of these, 174 (9%) were local CLL patients residing within 50 miles of MCR. Median age at diagnosis for these non-referred CLL patients was 69 years (range 27–97). Most had early stage disease (60% stage 0; 32% stage I-II). On prognostic evaluation, 121 (75%) patients were CD38 negative, 66 (61%) ZAP-70 negative, 47 (50%) IGHV mutated, and 56 (62%) had either no abnormality or 13q14- as a sole abnormality on FISH analysis. 64 (37%) patients progressed to require treatment for CLL during follow-up. After median follow-up of 4.0 years (range 0–11), 32 (18.4%) CLL patients were hospitalized with infection at least once as compared to 20/689 (2.9%) individuals in the control cohort (odds ratio=7.5; p<0.0001) Figure 1A. Even prior to receiving treatment for CLL, the rate of hospitalization for infection remained higher than the control cohort (p=0.002; Figure 1B). The 32 CLL patients hospitalized with infection were admitted with infection a total of 89 times (median 1 hospitalization for infection per patient). A specific infectious organism was identified by cultures or serologies in 60 (63%) of the 89 infections in CLL cases and 10/27 (37%) infections among controls. Hospitalization for an opportunistic infection (e.g. cytomegalovirus, cryptococcus, filamentous fungi, mycobacteria, Pneumocystis) occurred in 5/174 (3%) CLL patients during follow-up compared to 0/689 controls (p<0.001). Finally, we performed a pooled multivariable analysis of all patients (n=863) to identify factors (age, sex, CLL diagnosis, CLL treatment) associated with infection risk. Most factors were independently associated with hospitalization for infection [Age (per year, OR=1.03, p=0.02), sex (male, OR=4.0, p<0.001), diagnosis of CLL (OR=2.1, p=0.09), and treatment for CLL (OR=5.9, p<0.001)]. In univariate analysis of CLL patients [sex (OR=4.2, p=0.01), higher Rai stage (p=0.046), unfavorable (del 17p13; del 11q23) FISH [OR=4.6, p=0.02] and unmutated IGHV [OR=2.8, p=0.07]) were associated with risk of infection. CONCLUSIONS: In this cohort study, patients with newly diagnosed CLL had a 7.5-fold risk of hospitalization for infection relative to the control cohort. After 4 year follow-up, ∼1 in 5 CLL patients required hospitalization for infection. Although CLL treatment was a substantial risk factor for infection, the risk of infection among untreated patients remained higher than controls. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity’s Board of Directors or advisory committees. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract Genetic analysis has an increasing role in the diagnosis, classification, and management of patients with CLL. Although translocations involving the IgH locus are well characterized in B-cell malignancies such as follicular cell lymphoma, their frequency and significance in CLL are less well defined. We studied a series of patients with a clinical diagnosis of CLL using a FISH probe set including IgH. Methods: FISH was performed on blood using a DNA probe set designed to detect common chromosome anomalies associated with CLL and translocations involving IgH. Patients with an IgH abnormality were further studied using FISH probes sets for IgH and cyclin D1, BCL-2, BCL-3, BCL-6, BCL-11a, c-MYC, MALT-1, c-MAF, FGFR-3 or PAX-5. The clinical presentation, pathology, and flow cytometric immunophenotype was reviewed in patients with various translocations involving IgH. Results: Between 1/1/03 and 6/15/04, samples from 1032 patients with a clinical diagnosis of CLL were analyzed by FISH. Of these, 76 patients (7%) had translocations involving the IgH locus. Sixty one patients had a single translocation partner gene (cyclin D1 45%, BCL-2 24%, BCL-3 8%, c-MYC 2.5%, or BCL-11a 1.3%). Two patients had two independent clones (cyclin D1 and BCL-2; BCL-3 and BCL-11a). In 13 pts (17%) we could not identify the translocation partner. Thirty five (46%) of the 76 patients with IgH translocations had been seen at Mayo Clinic Rochester and had clinical and laboratory data for detailed review. Among these 35 patients, 16 (45%) had fusion of IgH and cyclin D1 and were diagnosed with mantle cell lymphoma (MCL). The immunophenotype of the monoclonal B cells was typical of MCL (CD5+, CD23-) in 6 patients and typical of CLL (CD5+, CD23+) in 4 patients. Six MCL patients had non-diagnostic immunophenotypes: CD5-, CD 23+ (n = 3) and CD5-, CD23- (n = 3). Twelve (34%) of the 35 patients had the IGH/BCL-2 fusion gene. Of these, 3 patients had leukemic phase follicular lymphoma, one had both CLL and MCL, and 8 had the diagnostic immunophenotype of CLL. The remaining 7 patients (19%) had a variety of translocation partners: BCL-3 (n = 1), BCL-3 and BCL-11a (n = 1), c-MYC (n = 2), and unknown (n = 3). The patients with a translocation involving BCL-3 had a typical CLL immunophenotype. Discussion: The IgH FISH probe detected a molecular defect in 7% of patients with a circulating monoclonal B cell population. In patients with a CLL immunophenotype, use of the IgH probe identified the IgH/cyclin D1 translocation characteristic of MCL in 4 patients, a finding of major clinical significance. Interestingly, after exclusion of MCL patients, more than 4% patients with CLL had other translocations involving IgH, a proportion comparable with other better defined chromosomal abnormalities in CLL. Although IgH/BCL-2 translocation is usually associated with follicular lymphoma, we identified 8 patients with CLL and this translocation. Translocations involving BCL-3 had been described before in CLL with an “atypical” immunophenotype. In contrast both of our patients with BCL-3 had typical phenotypes. Translocations involving IgH and BCL-11a have not been previously described. We conclude that FISH studies using the IgH probe improve the precision of diagnosis in CLL and could also have prognostic value.

Abstract BACKGROUND: CD49d (a.k.a. alpha 4 integrin) plays a critical role in leukocyte trafficking, activation, survival, and facilitates interactions between leukocytes and stromal cells via VCAM-1 and fibronectin. We and others have previously reported that CD49d gene expression in CLL B-cells correlates with CD38 expression (Durig Blood101:2748; Pittner Leukemia19:2264). We have also confirmed CD49d protein expression correlates with CD38 protein expression on CLL B cells (Pittner Leukemia 19:2264). Notably, one small study reported that CD49d expression may relate to overall survival among CLL patients independent of CD38 status (Zucchetto, Leukemia 20:523), although the relationship to other prognostic markers, such as ZAP-70 and cytogenetic abnormalities, was not evaluated. METHODS: We measured CD49d expression by flow cytometry in 130 patients with CLL (NCI 1996 criteria), accrued to observational studies at Mayo Clinic, between 1994 – 2006. CD49d expression was measured by 2 color flow cytometry using antibodies specific for CD19 (BD Biosciences) and CD49d (BD Pharmingen). CD49d expression was compared to level of CD38 expression, ZAP-70 expression, and degree of IgVH gene mutation (all treated as continuous variables). We also evaluated the relationship between CD49d expression and time to treatment (TTT) and other prognostic parameters, using the previously published 30% threshold to classify CD49d expression (Zucchetto, Leukemia 20:523). Finally, we evaluated the relationship between CD49d expression and in vitro sensitivity to fludarabine (1 um x 24 hrs) and chlorambucil (1um x 24 hrs) in a subset of 70 patients. RESULTS: The percent of B-cells expressing CD49d varied from 0.3 to 99.4% among the 130 patients tested (median expression= 6.1). The level of CD49d strongly correlated with the expression of ZAP-70 (r=0.34; p<0.0001) and CD38 (r=0.65; p<0.0001), as well as the degree of IgVH gene mutation (r=−0.29; p=0.006). The relationship between CD49d expression and other prognostic parameters using the previously published 30% threshold to classify CD49d expression is shown in Table 1. The median time to treatment for patients with low CD49d expression was 14.9 years compared to 5.4 years for those with high CD49d expression (p=0.008) High CD49d expression was correlated with in vitro resistance to chlorambucil (r=0.27, p=0.03) but not fludarabine (r=0.20, p=0.09). CONCLUSION: CD49d expression varies on CLL B-cells and relates to other prognostic parameters and TTT as well as in vitro sensitivity to chlorambucil. Since CD49d facilitates interactions between CLL B-cells and stromal cells, the association of this marker with poor prognostic features and in vitro drug resistance may relate to enhanced stromal nuturing of leukemic cells with higher CD49d expression. Additional studies are needed to determine whether CD49d expression may be clinically useful as a prognostic test or to predict response to treatment in patients with CLL. CD49D low CD49D high P value % ZAP70+ 33.0 75.0 <0.0001 % IgVH unmutated 24.3 55.6 0.02 %CD38+ 12.8 63.9 <0.0001 FISH <0.0001 %with 17p- or 11q- or 6q− 9.2 25.0 % with +12 5.8 43.8 %with normal or 13q− 85.1 31.3 Current Stage 0.17 0 30.9 16.7 I-II 67.0 77.8 III-IV 2.1 5.6

Abstract Background Although its prevalence is not well defined, transformation to Hodgkin lymphoma (HL) is a rare but well recognized complication in patients with CLL. Prior studies have reported a median survival of 0.8-1.7 years in these patients. Prior therapy of CLL, especially with purine analogs, has been shown to have adverse impact on survival after development of HL. We used the Mayo Clinic CLL database to describe outcomes of CLL patients seen at our institution who developed HL. Methods The Mayo Clinic CLL database includes all consenting patients with a pathologic diagnosis of CLL. Patients who developed biopsy-proven HL were included in this analysis. For patients referred from an outside institution, pathology review at Mayo Clinic was mandatory to be included. Clinical characteristics, therapy and overall survival (OS) were analyzed for all HL patients. Results Among 3124 CLL patients seen at Mayo Clinic from 1/1995 to 7/2011, 26 developed HL. In a nested cohort of 1644 patients who were seen within 12 months of CLL diagnosis and followed prospectively, the prevalence of HL was 0.05%/year (10 year risk=0.5%). All 26 patients had classical HL. The median duration between CLL diagnosis and development of HL was 6.2 years (range, 0-24.5 years). The median age at diagnosis of HL was 68 years (range, 45-88 years) and 21 (81%) were male. At the time of HL diagnosis, the median WBC count was 7.8 x 109/L (range, 1.3-87.3), median hemoglobin was 11 gm/dL (range, 7.2-13), median platelet count was 220x109/L (range, 53-627), median serum lactate dehydrogenase was 249 U/L (range, 133-658), and median serum albumin was 3.4 gm/dL (range, 2.6-4.2). According to the Ann Arbor staging system for HL, 10 (38%) were Stage II, 2 (8%) were Stage III, 11 (42%) were Stage IV, and undetermined in 3 (12%) patients. Sixteen patients (61%) received HL therapy with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD), seven (27%) received other chemotherapy, and treatment was unknown in 3 (12%) patients. No patient underwent stem cell transplantation as therapy for HL. After a median follow-up of 8.7 years from CLL diagnosis date, 16 (61%) patients have died. The median OS from date of HL diagnosis was 3.9 years (95% CI, 1.0 – 5.0 years). The International Prognostic score (IPS) for HL, originally proposed by Hasenclever in 1998, identified seven prognostic factors: serum albumin <4 gm/dL, hemoglobin <10.5 gm/dL, male sex, age >45 years, stage IV disease, WBC≥15 x 109/L, and lymphocytopenia in patients with newly diagnosed HL. When we applied these prognostic parameters (available in 22/26 patients, 85% in our cohort), the IPS score predicted median survival as follows: score of ≤2 (18% of the patients), median survival not reached; score of 3 (23% of the patients), 6.2 years; score of 4 (36% of the patients), 2.4 years; and score ≥5 (23% of the patients), 0.3 years (p-value=0.005, Figure 1). In contrast, the Richter syndrome prognostic score developed for CLL patients who transform to diffuse large B-cell lymphoma failed to predict survival in these patients (Tsimberidou, JCO, 2006). Of the 26 patients with HL, 16 (61%) had received therapy for their CLL prior to developing HL, including 12 (46%) who had received more than one line of therapy. Purine analogs had been administered for treatment of CLL prior to development of HL in 13/26 (50%) patients. The median survival of HL patients who had previously received CLL therapy was significantly worse compared to those who were untreated for CLL (2.8 years vs. 7.6 years, p-value=0.02). There was a trend toward shorter survival for HL patients who had received previous purine analog therapy for CLL compared to those who received alternate therapies (1.8 years vs. 5 years, p-value: p-value=0.07). Of the 9 HL biopsy samples in which Epstein Barr virus (EBV) in-situ hybridization studies were performed, EBV was detected in 6 (67%) patients. No association was observed between prior purine analog therapy and EBV status (p-value=1.0). There was no difference in survival according to the EBV status of HL (p-value=0.33). Conclusion The 10 year incidence of transformation to Hodgkin transformation among patients with newly diagnosed CLL is 0.5% (∼1/200 CLL patients). In this single-institution analysis, median survival of CLL patients who developed Hodgkin transformation of CLL was approximately 4 years after HL diagnosis. The IPS accurately predicted survival in our cohort of HL patients. Disclosures: Zent: Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Shanafelt:Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding; Genentech: Research Funding.