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Abstract Background Although hypogammaglobulinemia is a well-recognized complication in patients with chronic lymphocytic leukemia (CLL), its prevalence at the time of CLL diagnosis and association with novel prognostic markers and clinical outcomes are not well understood. Methods All patients seen at Mayo Clinic between 1/1995 - 4/2013 with newly diagnosed CLL (<12 months diagnosis) and who had baseline assessment of serum immunoglobulin G (IgG) were included in this analysis. Baseline demographic and clinical characteristics as well as serum IgG levels measured by either immunoassay or serum protein electrophoresis analysis were recorded. Based on the normal range for IgG at our laboratory, patients with serum IgG<757 mg/dL were classified as having hypogammaglobulinemia. Patients with IgG<757 were also further stratified by terciles (lower tercile: IgG<585 mg/dL; middle tercile: IgG 585-687 mg/dL, upper tercile: IgG 688-756 mg/dL). The relationship between hypogammaglobulinemia at diagnosis and prognostic parameters including immunoglobulin heavy chain (IGHV) gene mutation status, genetic abnormalities detected by fluorescence in-situ hybridization (FISH), and ZAP-70, CD38 and CD49d expression was assessed. Associations of hypogammaglobulinemia with time to first treatment (TTT) and overall survival (OS) was also evaluated. Results Of 1485 patients who met the eligibility criteria, 382 (26%) had hypogammaglobulinemia (median IgG=624 mg/dL) at the time of CLL diagnosis while the remaining 1103 (74%) had normal serum IgG levels (median IgG=1040 mg/dL). Baseline demographic and clinical characteristics of patients with and without hypogammaglobulinemia are shown in Table 1. When patients with hypogammaglobulinemia (IgG<757 mg/dL) were further stratified into terciles, patients in the lower tercile (n=127) were more likely to have advanced Rai stage (III-IV) at diagnosis (15% vs. 6% vs. 4%, p=0.008), be CD49d positive (56% vs. 43% vs. 32%, p=0.01), and have trisomy 12 on FISH testing (29% vs. 24% vs. 9%, p=0.005) compared to patients in the middle (n=127) and upper (n=128) terciles. After median follow-up of 4.2 years, 479 (32%) patients had received treatment for CLL, and 328 (22%) patients had died. The median TTT for patients with hypogammaglobulinemia was 3.8 years compared to 7.4 years for patients with a normal IgG (p-value<0.001). Among patients with hypogammaglobulinemia, the median TTT for patients in the lower, middle, and upper terciles were 1.7, 2.9, and 6.3 years respectively (p-value<0.001, Figure 1). On multivariable analysis that included age, gender, Rai stage, IGHV mutation status, FISH risk category and hypogammaglobulinemia, the following were independently associated with a shorter TTT: advanced (III-IV) Rai stage (hazard ratio [HR] =15.3, p<0.0001); intermediate (I-II) Rai stage (HR=3.0, p<0.0001); unmutated IGHV (HR=2.6, p=<0.0001); and hypogammaglobulinemia (HR=1.5, p=0.02). Conclusion In this large cohort study, approximately one-quarter of CLL patients had a low serum IgG level at the time of diagnosis. High-risk features, including advanced Rai stage and expression of CD49d, were associated with greater risk of hypogammaglobulinemia at the time of diagnosis. Patients with hypogammaglobulinemia at diagnosis had shorter TTT, a finding that persisted after adjusting for other prognostic factors. Evaluation of acquired hypogammaglobulinemia during the course of follow-up among patients who initially had normal IgG levels is ongoing. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Background Chronic lymphocytic leukemia (CLL) can effect renal function in a variety of ways including direct infiltration of the kidney, ureteral obstruction by lymphadenopathy, and treatment related tumor lysis syndrome (uric acid nephropathy). Rarely, CLL has also been reported to be associated with light chain nephropathy, renal amyloidosis, membranoproliferative glomerulonephritis (MPGN), granulomatous interstitial nephritis (GIN), and minimal change disease (MCD). Nearly all the data on the effects of CLL on renal function is at the case report level. We systematically evaluated the prevalence of renal insufficiency at diagnosis as well the incidence of acquired renal insufficiency during follow-up in a large cohort of patients with newly diagnosed CLL to more accurately define the effects of CLL on the kidney and its impact on clinical outcomes. Methods Between January 1995 -February 2013, previously untreated CLL patients seen in the Division of Hematology at Mayo Clinic at diagnosis (<12 months) and who had baseline assessment of serum creatinine were included in this analysis. Patients with serum creatinine (Cr) ≥1.5 mg/dL at baseline were classified as having renal insufficiency at diagnosis. Patients who initially had baseline creatinine <1.5 mg/dL but who developed a Cr≥1.5 mg/dL during the course of their disease were considered to have acquired renal insufficiency. Results Existing renal insufficiency at the time of CLL diagnosis: Of 2047 patients who met the eligibility criteria, 153 (7.5%) patients had renal insufficiency (Cr≥1.5 mg/dL) at the time of CLL diagnosis including 15 (0.7%) with a Cr≥3 mg/dL. Renal insufficiency was also more common among men (9.3% vs. 3.9%; p<0.00001), those with advanced stage disease (Rai 0=7.0%; Rai I-II=6.4%, Rai III-IV=20.2%; p<0.0001), and CD49d positive patients (6.8% vs. 3.8%; p<0.038). Patients with renal insufficiency at diagnosis were also older (median age 72.2 vs. 63.9; p<0.0001). No difference in the prevalence of renal insufficiency at diagnosis was observed based on cytogenetic abnormalities detected by FISH or CD38, ZAP-70 or IGHV gene mutation status. Although renal insufficiency at diagnosis was strongly associated with OS on univariate analysis (p<0.001), no association was observed between renal insufficiency and TTT or OS on multi-variate analysis adjusting for age, sex, and Rai stage. Acquired renal insufficiency during CLL disease course: Among the 1894 patients with normal renal function at diagnosis, 304 (16.1%) acquired renal insufficiency (Cr≥1.5 mg/dL) during the course of their CLL disease course including 43 (2.3%) with peak Cr≥3 mg/dL. In addition to age (older) and male sex, a number of CLL disease characteristics were associated with a higher likelihood of acquired renal insufficiency including: IGHV UM (OR=2.0; p=0.0001), unfavorable FISH (del17p- or 11q-; OR=2.0; p=0.001), and being CD49d+ (OR=1.8; p=0.002), ZAP-70+ (OR=1.6; p=0.004), or CD38+ (OR=1.4; p=0.0.032),. Shorter TTT (p<0.001) and OS (P<0.001) was observed among patients with initially normal creatinine who acquired renal insufficiency (Figure 1A and 1B). On MV analysis adjusting for age, sex, and stage at diagnosis, acquired renal insufficiency remained an independent predictor of TTT (OR=1.77; p=0.001) and OS (OR=2.67; p<0.001). Renal insufficiency and therapy selection After median follow-up of 4.5 years (range 0-18.0), 620 of 2047 (30.3%) patients have progressed to require treatment. Patients with renal insufficiency prior to treatment were less likely to receive purine nucleoside analogue based therapy and more likely to receive single agent alkylator based treatment. Conclusions Approximately 1 in every 13 patients (7.5%) with CLL has renal insufficiency at the time of diagnosis and an additional 16.1% acquire renal insufficiency during the course of the disease. The risk of developing renal insufficiency is associated with a variety of CLL B-cell characteristics and is associated with TTT and OS. Data on causes of acquired renal insufficiency is being abstracted and will be presented at the meeting. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Off Label Use: MK2206 in a phase 1 trial of CLL.

Abstract Introduction Approximately 2-8% chronic lymphocytic leukemia (CLL) patients develop diffuse large B-cell lymphoma (DLBCL, so-called Richter syndrome [RS]) over long-term follow-up. The outcomes of patients with RS are variable and poorly understood. Methods CLL patients seen at Mayo Clinic from January 1995 to April 2013 who developed biopsy-proven DLBCL during follow-up were included in this analysis. Clinical characteristics, therapy and overall survival (OS) of all patients with RS were analyzed. Results One hundred and twenty patients with biopsy-proven RS were identified in the Mayo Clinic database. The median age at diagnosis of RS was 67 years (range, 29-88 years), 87 (73%) were male, and 83 (69%) had received prior CLL therapy. The median duration between diagnosis of CLL and development of RS was 3.8 years (range, 0-34.5 years). Stereotyped B-cell receptors were found in 9/30 (30%) RS patients compared to 219/1294 (17%) non-RS patients (OR=2.1, p=0.07). IGHV4-39 gene usage was present in 2/26 (8%) RS patients compared to 57/1354 (4%) non-RS patients (p=0.3). Of the 30 RS biopsy samples in which Epstein Barr virus (EBV) in-situ hybridization studies were performed, EBV was detected in 7 (23%) patients. After a median follow-up of 5.2 years, 86 (72%) patients have died. Causes of death were as follows: progressive DLBCL (n=46, 53%), progressive CLL (n=2, 2%), infection (n=8, 9%), unknown (n=20, 23%) and other (n=10, 12%). The median OS of the entire cohort was 0.9 years (95% CI, 0.5-1.3 years). Tsimberidou and colleagues proposed a prognosis score (RS score) based on the following variables at the time of RS diagnosis: Eastern Co-operative Group performance status of 2-4, lactate dehydrogenase >1.5 x normal, platelets<100 x 109/L, tumour size >5 cm and >1 prior therapy for CLL. Each parameter was assigned one point, and patients were then assigned to one of four risk groups based on their calculated RS score: 0 or 1, low risk; 2, low-intermediate risk; 3, high-intermediate risk; 4 or 5, high risk. The OS according to the RS score for our group of patients is shown in Figure 1. Median survival in the low, intermediate-1, intermediate-2 and high-risk groups were 2.4 years, 0.7 years, 0.3 years and 0.2 years, respectively (p<0.001). Fifteen (12%) patients received cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) as initial therapy for RS, and 68 (57%) received rituximab in addition to CHOP (R-CHOP). There was no difference in survival among patients who received CHOP compared to R-CHOP (median, 1.8 years. vs. 1.2 years, p=0.5). Twenty-four patients (20%) received non-CHOP-based chemotherapy, and treatment was unknown in 4 (3%) patients. Nine patients (7%) died before definitive therapy could be instituted. Thirteen (11%) patients were able to proceed to stem cell transplantation (SCT): 10 (8%) autologous SCT, 2 (2%) allogeneic SCT and 1 (1%) autologous SCT followed by an allogeneic SCT. Although RS patients who underwent SCT were younger compared to those who did not undergo SCT (median, 59 years. vs. 69 years, p=0.001), no differences in prior CLL therapy or receipt of R-CHOP therapy for DLBCL was noted between the two groups. The median survival of patients who proceeded to SCT was superior compared to those who did not (5.2 years vs. 0.7 years; p=0.005). Conclusion Stereotyped B-cell receptors but not VH4-39 had a suggested evidence of an increased risk of RS in our cohort. Although 90% RS patients received intensive chemotherapy, only 11% patients were able to proceed to SCT. Median survival of RS patients remains dismal at 1 year, and newer therapies are needed to improve outcomes. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Introduction We have previously developed a primary human bone marrow model in CLL which allows us to study marrow derived mesenchymal stroma cells (MSC) and mimic in vivoMSC-leukemic interactions in CLL (Ding, Blood, 2010) These interactions proved to be bidirectional with functional implications for both cell types. However the biologic and clinical implications of these bilateral cellular interactions in relation to MSC function in CLL patients have not been completely studied. MSC are clearly established as an essential component in forming supportive marrow niches for hematopoiesis. Our focus in this study thus was to further investigate on the status of CLL MSC function in comparison with normal MSC and the impact of the leukemic CLL B cell on MSC function and its role in forming marrow niches in CLL. Methods Bone marrow (BM) aspirates of CLL patients or normal age-matched subjects were used to generate MSC and these MSC were then assessed for various functions. Specifically we assessed the growth, differentiation capacity, gene expression, cytokine secretion profile and transcriptome analysis of CLL and normal MSC using colony forming unit (CFU) assay, MSC differentiation assay, Affymetrix U133 2.0 plus array and RNA sequencing respectively. To determine MSC CFU frequency, fixed number of nucleated cells (5 or 10 million) from BM aspirates were added in MSC growth medium for 10 days to form CFU. CFU frequency was then calculated by dividing the number of MSC colonies by the fixed number of inputted nucleated cells. In order to test the specific gene changes that occurred in MSC after their interaction with CLL leukemic cells, normal MSC were co-cultured with CLL cells for 1 week and subsequently followed by cell growth, gene expression analysis with RNA sequencing and cytokine profiles using a multiplex assay. Results CLL MSC generated significantly lower CFU colonies compared to normal subjects (CLL [n=24] vs normal [n=6] mean: 2.2 vs. 22 CFU per million nucleated cells, p=0.01). In order to test if the lower number of CLL MSC CFU was due to their reduced growth capacity or lower frequency, we then performed the CFU assay using identical numbers of CLL and normal MSC at the start of culture. CLL MSC was found to have significantly lower CFU capacity compared to normal MSC (mean: 2.1 [CLL] vs 4.5 [normal] CFU/10 input cell, n=7 in each group, p=0.03). To investigate the mechanism(s) for the reduced growth capacity of CLL MSC, we compared the gene expression profiles of non-stimulated CLL vs. normal MSC (n=10 for each group) with the following outcomes: CLL MSC had increased expression of cell cycle inhibitor: p16(Ink4a) and p57, both key markers of cell senescence (Baker, Nature, 2011); CLL MSC overexpressed homeobox B cluster of transcription factors, key regulator for cell development and differentiation. Wnt inhibitors DKK1/DKK2 and non-canonical Wnt 5b known to be associated with aging were found to be 10 and 2 fold more elevated in CLL MSC. In addition, MSC abundantly produced a milieu of the cytokines MCP-1/IL-8/IL-6/IL-1Ra after interaction with CLL leukemic cells. These cytokines are associated with a senescent associated secretory phenotype (SASP). Co-culture experiments of normal MSC with CLL Leukemic cells revealed significant impact of leukemic cells on normal MSC functions. Co-culture with a low density of CLL leukemic cells (CLL to MSC ratio: ≤10,000/1) increased MSC growth (1.2 fold). However, high density co-culture of CLL cells with MSC (CLL to MSC ratio of 100,000/1) inhibited MSC growth (0.8 fold). Transcriptome analysis of normal MSC before and after one week of co-culture with CLL leukemic cells showed that normal MSC had alteration of ~1500 genes. Many of the modified genes (CDKN2B, DKK2, LIF, HGF, FOXQ1 etc) are known to be involved in regulation of cell growth and senescence. Conclusion Collectively, these results demonstrated that bone marrow derived CLL MSC have decreased proliferative potential and possess senescent phenotypes. Importantly these senescent features were inducible in normal MSC after their interaction with CLL leukemic cells implying that leukemic B cells can educate MSC to become senescent. The implications of the CLL MSC senescence status are multiple and include: defective hematopoietic niches due to dysfunctional MSC, immune dysregulation and the enhanced nurturing of CLL B cell by these MSC. We are now investigating several of these latter features in our current work. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract BACKGROUND: Although chemoimmunotherapy (CIT) has improved response rates, treatment free survival, and overall survival in patients with chronic lymphocytic leukemia (CLL), only 40-50% of patients achieve a complete remission and the majority have residual disease when evaluated using sensitive assays. Interactions with nurturing environments can enhance CLL B-cell resistance to apoptosis. These interactions include cytokine mediated prosurvival signals by angiogenic molecules, such as VEGF and bFGF that nurture CLL B-cells in an autocrine fashion and promote CLL cell survival partly through up regulation of anti-apoptotic proteins. These findings provide a strong rationale for testing anti-VEGF therapy in combination with a purine nucleoside analogue CIT regimen for upfront treatment. We conducted a randomized phase 2 CIT trial using pentostatin, cyclophosphamide, and rituximab with (PCR-B) or without (PCR) bevacizumab (B), an anti-VEGF monoclonal anti-body. METHODS: Eligible patients were previously untreated and had CLL in need of treatment by NCI-WG criteria (Blood 111:5446). Patients were randomized using a dynamic allocation procedure stratifying for stage (0-II vs. III-IV) and FISH (17p or 11q deletion vs. other) to receive either 6 cycles of rituximab (100 mg on day 1 of cycle 1; 375 mg/m2on day 2 of cycle 1 and day 1 of cycles 2-6) followed by pentostatin (2 mg/m2) and cyclophosphamide (600 mg/m2) (PCR) administered every 21 days. Patients in the PCR-B cohort also received bevacizumab 15mg/kg on day 1 of cycles 1-5 and days 1, 22, & 43 of cycle 6. All patients underwent complete response evaluation 3 months after day 1 of cycle 6 (or last cycle of treatment for those completing < 6 cycles). MRD was assessed using 6-color flow cytometry (Leukemia 21:956) at the completion of treatment. RESULTS: 68 patients were enrolled through the Mayo Clinic Cancer Research Consortium between 1/2009 and 1/2013. Three patients were excluded from analysis: 1 patient canceled prior to treatment, 1 was dosed incorrectly, and 1 was ineligible due to immunophenotyping inconsistent with CLL. Median age of eligible patients was 63 years (range 43-81) and 43 (66%) were men. With respect to disease stage, 3 (5%), 38 (58%), and 24 (37%) had low, intermediate and high Rai stage disease. Eleven (17%) patients had deletion 17p or 11q & 29 (45%) had unmutated IGHV. No statistically significant differences were observed in these variables by treatment arm. All 65 evaluable patients have completed active treatment, with 54 (83.1%) completing the intended 6 cycles (PCR group 27/32 [84.4%] and PCR-B 27/33 [81.8%]). Hematologic grade 3+ adverse events deemed at least possibly related to treatment were observed in 10 (31.3%) patients on PCR and 12 (36.4%) on PCR-B (p=0.79). Non-hematologic grade 3+ adverse events deemed at least possibly related to treatment were observed in 9 (28.1%) patients on PCR and 18 (54.4%) on PCR-B (p=0.04). The most common such events were hypertension (PCR: 3.1% vs. PCR-B: 21.2%), proteinuria (0% vs. 6.1%) and creatinine increase (3.1% vs. 6.1%). Across both arms, 64/65 (98.5%) patients achieved a response including 31/32 (96.9%) treated with PCR and 33/33 (100%) treated with PCR-B (p=0.49). CR/CRi was achieved in 10/32 (31.3%) patients treated with PCR & 18/33 (54.5%) treated with PCR-B (p=0.08). Of the 28 who achieved a CR/CRi, MRD analysis was completed on 26, of whom 12 (46%) were MRD negative. With respect to treatment arm, 5/32 (16%) patients on PCR and 7/33 (21%) on PCR-B achieved an MRD negative CR. Median time to retreatment for all 65 patients was 44.8 (95% CI: 34.6 – NA) months. Median overall survival has not yet been reached. With current follow-up no differences between treatment-free survival (p=0.38), progression-free survival (p=0.23), or overall survival (p=0.45) are observed by treatment arm. Plasma levels of angiogenic cytokines VEGF, bFGF, thrombospondin (TSP) and the chemokines CCL3 and CCL4 were measured prior to treatment and at the time of the post treatment response evaluation. Correlations of these cytokines with clinical outcome will be presented. CONCLUSION: The addition of bevacizumab to purine analogue-based CIT was generally well-tolerated and may increase complete remission rates in patients with CLL. No clear improvement in treatment free survival has been observed to date. Disclosures Shanafelt: Hospiria: Research Funding; Pharmacyclics/Jannsen: Research Funding; Cephalon: Research Funding; Celgene: Research Funding; glaxoSmithKline: Research Funding; Genetech: Research Funding; Polyphenon E Int'l: Research Funding. Off Label Use: Off label use of pentostatin for treatment of CLL. Off label use of bevacizumab for treatment of CLL. . Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Several B-cell malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are known to have a familial component to disease risk. Although several common inherited genetic variants have been found to be associated with risk of these malignancies, a large proportion of the heritability remains unexplained. Here we set out to identify rare high-penetrance susceptibility variants for CLL and MM using the established Mayo Clinic family study of B-cell malignancies. Methods: We performed exome sequencing on germ line DNA of 106 families with either two or more members with CLL (n=93 families) or two or more members with MM or monoclonal gammopathy of undetermined significance (MGUS) (n=13 families) using Aglient capture kits. All samples were sequenced on an Illumina HiSeq2000. Bioinformatics analyses leveraged the following software packages: Novoalign, Picard, The Genome Analysis Toolkit (GATK), and the Biological Reference Repository (bioR). Quality control filters were implemented; subjects with mis-specified relationships were removed as were variants with <95% call rate or <20x coverage, and those variants identified as sequencing artifacts. To identify potentially functional single nucleotide variants, the following was required a) uncommon in public databases, b) cosegregating variants in multiple families, c) variants to be highly conserved and in coding regions, and d) functional prediction status of deleterious (SIFT score), damaging (PolyPhen score), and a moderate, or high variant impact (SNPEffect). The association of these variants with risk was then evaluated in large samples of cases and controls in genome-wide association studies (GWAS). Results: In the CLL familial cohort, there were 443 individuals (160 with CLL, 73 with monoclonal B cell lymphocytosis (MBL), and 210 relatives without CLL or MBL) sequenced; and in the MM cohort, there were 39 individuals (13 with MM, 13 with MGUS and 13 without MM or MGUS) sequenced. A total of 61,992 single nucleotide variants passed quality control filters in the CLL cohort and 39,456 single nucleotide variants in the MM cohort. From these, we identified 32 variants in the CLL cohort and 39 variants in the MM cohort that were cosegregating in multiple CLL or MM families, respectively, and were predicted to affect protein structure or function. Of these, one CLL variant was within 1 Mb of a known GWAS-discovered locus. Moreover, there were seven CLL and four MM variants that were within 1 Mb of single nucleotide polymorphisms (SNPs) that had statistical significance between p<10-4 and p<10-6 in GWAS. Finally, there were 3 variants found to be common between CLL and MM families (see Table). Conclusions: Through whole exome sequencing, we identified a number of rare, highly penetrant predisposition variants for CLL and MM. Several of these variants were common to both B-cell malignancies providing evidence of shared genetic components; one variant may be a functional driver of the GWAS-discovered loci. These identified variants provide a list for future validation studies. Table: Exome variants common in both Chronic Lymphocytic Leukemia and Multiple Myeloma SNP ID rs41314099 rs61754114 rs143025033 Chromosome 17 6 7 Gene WSCD1 BACH2 TNS3 Penetrance (CLL) 0.70 0.75 0.80 Penetrance (MM) 1.00 0.80 0.75 Allele Frequency (CLL) 0.015 0.025 0.017 Allele Frequency (MM) 0.077 0.077 0.058 1000 Genome Frequency (European) 0.010 0.020 0.010 SIFT Term Deleterious Deleterious Deleterious PolyPhen Term Probably Damaging Probably Damaging Probably Damaging SNP: Single Nucleotide Polymorphism; CLL: Chronic Lymphocytic Leukemia; MM: Multiple Myeloma; Penetrance is defined as proportion of individuals carrying the alternate allele that are also affected; SIFT: Sorting Intolerant From Tolerant, predicts whether an amino acid substitution affects protein function; PolyPhen: Polymorphism Phenotyping, predicts possible impact of an amino acid substitution on the structure of a human protein Disclosures No relevant conflicts of interest to declare.

Background: Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder of mature cytotoxic T- or Natural Killer (NK)-cells. T-cell LGL (T-LGL) leukemia is characterized by accumulation of cytotoxic T-cells in blood and infiltration of the bone marrow, liver, or spleen. T-LGL leukemia may manifest as asymptomatic persistent lymphocytosis or present with a myriad of autoimmune diseases including autoimmune cytopenias. Population-based studies have not been reported in LGL leukemia. Here, we present clinical characteristics, natural history, and risk factors for poor survival in patients with T-LGL leukemia using the Surveillance, Epidemiology, and End Results Program (SEER) and the National Cancer Data Base (NCDB), the two largest national cancer registries in the US. Methods: We queried the SEER and NCDB databases using ICD-O histology code 9831 corresponding to LGL leukemia. The query was further refined to include patients with T-LGL leukemia only. Incidence rates (case/1,000,000) were calculated using 2000-2011 data from SEER and age-adjusted to the US 2000 standard population. Patient level data were obtained from the NCDB and used to describe patient characteristics such as demographics, year of diagnosis, treatment, and survival. Results: The incidence of LGL leukemia was 0.2 cases/1,000,000 individuals. The incidence did not change significantly over the period of time studied. The incidences according to racial groups were (mean, 95%CI): White 0.2 (0.18-0.24), Black 0.14 (0.07-0.24), American Indian/Alaska native 0.24 (0.03-0.8), and Asian/Pacific Islander 0.15 (0.07-0.25). The incidences among Blacks, American Indians/Alaska Natives, or Asian/Pacific Islanders were not significantly different compared to Whites. Patient-level data was available for 978 patients with T-LGL leukemia. The median age at diagnosis was 66.5 years (range, 19-90) with a male to female ratio of 1.05. Females were diagnosed at a younger age compared to males (65 vs. 68 years, p<0.05). Forty-five percent of the patients with T-LGL leukemia required some form of systemic treatment at the time of diagnosis. The vital status of 760 (77.7%) patients was known at the time of last follow up with a median follow up of 32 months (range, 0-152). At the time of last follow up, 75.5% were alive with the median overall survival (OS) of 108.4 months (Figure 1). Multivariate analysis that included age at diagnosis, sex, race, year of diagnosis, and Charlson-Deyo co-morbidity score, showed that older age (>60 years) and higher co-morbidity score (≥2) were independent predictors of poorer survival (Table 1). Conclusions: We present the first population-based study of patients with LGL leukemia, an extremely rare disease. The incidence has remained stable over the last decade and does not vary significantly among races. Most patients with T-LGL leukemia do not require treatment and the overall prognosis is relatively good. Table 1. Multivariable analysis showing predictors of mortality in patients with T-LGL leukemia Parameter Hazard Ratio Lower CI Upper CI P -Value Age <60 vs ≥ 60 years 2.73 1.90 4.04 <.0001 Male vs Female 0.82 0.61 1.09 0.19 White vs Other 0.99 0.97 1.00 0.23 Charlson-Deyo score <2 vs ≥2 2.03 1.29 3.06 0.003 Year of Diagnosis (2001-2006 vs. 2007-2012) 1.11 0.76 1.69 0.58 Figure 1. Overall survival in patients with T-LGL leukemia Figure 1. Overall survival in patients with T-LGL leukemia Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 4252 Background: Multiple recent studies have demonstrated the survival benefits of reduced intensity allogeneic stem cell transplant for high risk CLL patients. These transplant series, however, represent highly selected cases where age, co-morbidities and performance status preclude transplant in many patients with CLL. The portion of newly diagnosed CLL patients that are eventually considered for transplant is unknown, as well as the proportion that are referred for transplant evaluation; the proportion that actually receive stem cell transplant, and the reasons for not pursuing transplant in referred patients. We conducted a cohort study of newly diagnosed CLL patients to address this gap in knowledge. Methods: We used the Mayo Clinic database to identify all patients with newly diagnosed CLL seen at Mayo Clinic within 1 year of diagnosis between July of 2003 and May of 2011 (time interval during which reduced intensity allogeneic stem cell transplantation was part of routine care at Mayo Clinic). Electronic medical record and laboratory studies were reviewed to identify all patients referred for stem cell transplant evaluation (either allogeneic transplant for progressive CLL or autologous stem cell transplant for Richter's transformation). Patients referred for transplant were analyzed to determine what proportion completed transplant and, for those who were not transplanted, the reason they were not transplanted (e.g. lack of insurance approval, co-morbidity, lack of a donor, inadequate disease control, death prior to transplant or patient preference). Results: A total of 1250 patients with newly diagnosed CLL between July 2003 and May 2011 who were seen at Mayo Clinic within 1 year of diagnosis were identified. The mean age at diagnosis was 63.6 yrs and 34.2% were women. With respect to disease stage, 48.7% were Rai stage 0, 45.5% Rai stage I/II, and 5.8% Rai stage III/IV. CLL FISH analysis revealed 126 (13.6%) patients who had either del (17p) or del (11q) and 801 (86.4%) patients who had other FISH abnormalities or normal FISH results. After a median follow-up of 41 months, 425 (34%) of patients have received treatment. A total of 54 patients (4.3%) were referred for a transplant evaluation during follow-up. Of these 54 patients, 48 (3.8% overall cohort) were referred for consideration of allogenic transplant for treatment of progressive CLL and 6 (0.5%) were referred for autologous transplant for treatment of Richter's transformation. Baseline characteristics associated with subsequent transplant referral included age at diagnosis (median: 56 yrs) (p &lt; 0.0001, Odds Ratio (OR) for each year older = 0.91, 95% Confidence Interval (CI)=(0.88, 0.95)), Rai stage (III/IV vs. 0) (p = 0.0002, OR = 22.7, 95% CI=(5.1, 102.4)) and FISH (del(11q), del(17p) vs Normal, del(13q) or Trisomy 12) (p &lt; 0.0001, OR = 7.3, 95% CI=(3.3, 16.1)). Gender, Rai stage (I/II vs. 0), and patient residence (local vs. not local) were not significant. Among the 54 patients referred for transplant, 28 (51.9%) completed a transplant. This included 22 (45.8%) patients referred for allogenic transplant and 6 (100%) patients referred for autologous transplant. Among the 26 patients referred who did not undergo transplant, the principal reasons were patient preference (n = 6, 23.1%), patient still being evaluated for transplant as of the study end date (n = 6, 23.1%), alternative therapy chosen (n = 5, 19.2%), inadequate disease control (n = 3, 11.5%), death prior to transplant (n = 3, 11.5%), lack of insurance approval (n = 2, 7.7%), and lack of a donor (n = 1, 3.8%). Conclusion: 4.3% of the newly diagnosed CLL patients in this series were considered for stem cell transplant at some point during follow-up. Young age, high Rai stage and high risk FISH abnormalities are principal reasons for transplant referral. Only 51.9% of those considered for transplant completed transplant. The principal reasons for referral patients not completing transplant who had adequate time to follow were patient preference, alternative therapy, inadequate disease control and death prior to transplant. The remaining 6 patients (23%) are in process of transplant evaluation with longer follow required. These analyses provide first incidence data for CLL transplant referral in a cohort of newly diagnosed patients. Disclosures: Zent: Biothera: Research Funding; Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; GlaxoSmithKlime: Research Funding. Shanafelt:Celgene: Research Funding; Teva/Cephalon: Research Funding; GlaxoSmith Klein: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Polyphenon E International: Research Funding.

Abstract Introduction T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-Cell malignancy typically associated with aggressive clinical course. Leukemic T-cells demonstrate post-thymic T-cell phenotypes (Tdt-, CD1a-, CD5+, CD2+ and CD7+) and commonly are CD4+/CD8- T-cells, but co-expression of CD4+/CD8+ or CD8+/CD4- have also been observed. Rearrangement of chromosome 14 involving TCL1 locus is the cornerstone for T-PLL diagnosis which leads to overexpression of oncogene TCL1. As a binding partner for TCL1, AKT expression correlates with worse prognosis. Despite advances in identification of novel mutations, therapeutic option is limited with most patients having very short survival. Herein, we performed a detailed analysis of specific T-cell subsets affected in T-PLL and a comprehensive genomic analysis by whole exome sequencing (WES) and RNA sequencing. We also explored the preclinical efficacy of targeting AKT activation by AKT inhibitor MK2206 on leukemic T-cells isolated from T-PLL patients. Methods T-PLL leukemic cells were isolated from blood or marrow samples of T-PLL patients (n=9) and were tested for involved T-cell subsets by multicolor flow cytometric analysis. Leukemic T-cells were treated with escalating doses of MK2206 (0 to 10 µM) for about 80 hours and were assessed for their apoptosis levels using Annexin V flow cytometric analysis. Whole exome sequencing (WES) and RNA sequencing were conducted using genomic DNA and RNA isolated from purified leukemic T cells from T-PLL patients. Results Phenotypic analysis of T-cell subsets in T-PLL patients showed that leukemic T-cells in 4 untreated T-PLL patients are consistent with naïve T-cell phenotype (CD45RA+, CD45RO-, CCR7+). Leukemic T-cells in 3 of the untreated patients and in 2 of the treated patients have central memory T (CD45RA-, CD45RO+, CCR7+) and effector memory T phenotypes (CD45RA-, CD45RO+,CCR7-) respectively. MK2206 treatment was able to induce dose-dependent apoptosis on the isolated PBMC (containing > 90% leukemic T-cells) of T-PLL patients (n=4) (IC50: 5 µM). FISH analysis found a rearrangement of TCL1 locus on chromosome 14 in all T-PLL cases (n=9). Three cases have also been detected to have del (11q) involving ATM locus and one patient has both del(11q) and del(17p). Subsequent WES (n=7) and RNA sequencing (n=6) analysis revealed recently reported mutations in ATM (frame shift mutation and early stop mutation W579*) in 2 cases, JAK3 (M511I) in 2 cases and STAT5B (T628S and N642H) in 2 cases. Importantly, novel somatic mutations in gene IKZF1 (N159S), NTRK1 (R33W), AP2A2 (P514L), HDAC8 (I115R), RARB (G90W) and TNIP2 (K104Q) were detected by WES. Mutations in EML4 (L548W and F304S) and VAV3 (C282Y and splice site mutation) were also identified in 2 different T-PLL cases. RNA sequencing revealed several fusion transcripts resulting in early stop of several different genes including PTPRT, L3MBTL1, UCKL1 in one T-PLL case. Conclusion T-PLL leukemic T-cells are consistent with either naïve or central memory T-cell subsets in untreated patients. The AKT inhibitor MK-2206 was capable of inducing apoptosis and could be a potential therapeutic agent for T-PLL patients. In addition, we detected known mutations in ATM and JAK-STAT pathways. Novel mutations in genes involving DNA binding and chromatin remodeling (IKZF1, HDAC8) or kinase signal pathway (NTRK1, TNIP2, VAV3, EML4) were uncovered. These results suggest that further therapeutic approaches could be developed to target DNA binding factors or JAK-STAT or AKT-TCL1 signal pathway with an ultimate goal to improve survival of T-PLL patients. Disclosures Off Label Use: Off label use of MK2206: MK-2206 is an AKT inhibitor obtained from Selleck Chemicals and is used to treat leukemic cells in vitro to test the leukemic sensitivity to AKT inhibition. .

Abstract INTRODUCTION: Erdheim-Chester Disease (ECD) is a rare hematologic disease manifesting as granulomatous or fibrotic infiltration of long bones and non-skeletal tissues by non-Langerhans foamy histiocytic (CD68+, CD1a-, S100-) cells. The clinical presentation can be diverse and any organ can be affected. It is estimated that less than 500 cases have been reported in the literature. METHODS: We retrospectively reviewed the medical records of patients evaluated at Mayo Clinic from January 2001 to May 2014. A tissue biopsy review by pathologists at Mayo Clinic was necessary for inclusion in the study. In all cases, the diagnosis of ECD was confirmed using clinical criteria in conjunction with histopathologic findings. Clinical data such as patient characteristics, disease presentation, management strategies, treatment responses, and survival time were collected. RESULTS: Forty-five patients with confirmed ECD were included in our study. The median age at diagnosis was 53 years (range, 32-78), while the median age of onset of symptoms prior to diagnosis was 24 months (range, 1-180). There was a male predilection with a male to female ratio of 2.4. The duration of follow-up ranged from 1-168 months (median, 20). At the time of last follow-up, 38 patients were alive and 5 were dead, while the vital statuses of 2 patients were unknown. The median and mean survival times from the time of diagnosis were 150 and 130 months respectively. The median survival time from the time of symptom onset was not reached and mean time was 154 months (Figures 1A and 1B). Bone pain was the most common presenting symptom (27%). Other common presentations included diabetes insipidus (24%), abdominal pain (22%), B symptoms (13%), visual changes (13%), renal failure/obstruction (11%), pleural effusion (9%), other neurologic symptoms (9%), ataxia (7%), pericardial effusion (7%), edema (7%), dyspnea (7%), and hypertension (7%). The organs/sites involved were bone (80%), central nervous system (59%), kidneys (56%), lungs (38%), retroperitoneum (33%), heart (22%), sinuses (22%), bone marrow (14%), adrenal glands (13%), liver (11%), spleen (11%), and skin (16%). Only 18 (40%) patients had the diagnosis established by the first biopsy. The rest required multiple biopsies before the diagnosis was confirmed (median, 2; range, 1-5). Testing for BRAF V600Ewas performed in 12 patients and 8 (67%) tested positive for the mutation. Eight patients have not required ECD-specific treatment so far. Of the 37 patients who required treatment, the median number of treatments received was 2 (range, 1-7). The systemic treatments received and their response rates are summarized in Table 1. Four patients received radiotherapy but none responded. At the time of the last follow up, 5 patients with BRAFV600E were receiving treatment with vemurafenib or dabrafenib. However, follow-up is insufficient for proper assessment of response. CONCLUSIONS: ECD is an extremely rare diagnosis as exemplified in our retrospective analysis. The diagnosis is often challenging due to protean and diverse clinical presentation as well as frequent inconclusive initial biopsies. There is usually a long-latency period of smoldering symptoms leading to definitive diagnosis. A high level of suspicion is essential in order to make the diagnosis and BRAFmutation analysis should be considered an integral part of work up even if biopsy is non-diagnostic. Traditional oncologic systemic therapies and even novel rheumatologic anti-inflammatory agents generally have limited objective activity. BRAF inhibitors offer a novel treatment option and results of prospective studies are eagerly awaited. Abstract 1405. Table 1: Summary of Response to Various Treatment Modalities in Patients with ECD Therapeutic Agent Number of Patients Treated Response (%) Complete Response Partial Response Stable Disease No response Corticosteroids 11 0 0 9 91 Methotrexate 9 0 0 11 89 Cyclophosphamide 4 0 0 25 75 2-CDA 12 8 17 25 50 Interferon-α 8 0 29 14 57 Vinorelbine 4 0 50 0 50 Tumor necrosis-α factor Inhibitors 7 0 0 14 86 Interleukin-1 Receptor Antagonists 6 17 17 0 67 Tamoxifen 2 0 50 0 50 Radiotherapy 4 0 0 0 100 Figure 1 Figure 1. Disclosures Off Label Use: We report among all treatments utilized at our institution for ECD including the use of steroids, immunosupressants, TNF blockers, interleukin-1 receptor antagonists, and BRAF inhibitors in patients with Erdheim Chester Disease. These treatments, though supported by other reports in the literature, have not been explicitly approved by the FDA for ECD..