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Nucleotide insertions/duplications or deletions in immunoglobulin heavy chain genes have been found in 24/760 patients (3.15%) with chronic lymphocytic leukemia (CLL). In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. The pattern and location of insertions/duplications or deletions in CLL and their restriction to mutated IGHV rearranged genes strongly suggests that they resulted from somatic hypermutation. Their incidence in CLL is consistent with previous reports in normal, auto-reactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease-specific associations. A striking exception to this rule was identified in CLL IGHV3-21-expressing cases: one amino acid was deleted from the CDR2 region in 16/63 (25.4%) mutated CLL IGHV3-21 sequences (including public database-derived IGHV3-21 CLL cases + the present series) vs. only 2/257 (0.78%) public database-derived mutated non-CLL IGHV3-21 sequences; 15/16 CLL IGHV3-21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3 and light chain CDR3 motifs. This finding further supports the idea of selective antigenic pressures playing a pathogenetic role in some CLL cases.

Background. Fludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant advancement in the treatment of patients with chronic lymphocytic leukemia (CLL) and has the potential of inducing durable remissions of the disease. In the new scenario of targeted agents for CLL, there is an increasing interest in identifying patients who may gain the maximum benefit in terms of disease control by a single shot of FCR chemo-immunotherapy. Purpose. Here we aimed at identifying predictors of durable remission after first line FCR treatment. Methods. The study was based on 405 progressive and previously untreated CLL patients who consecutively received standard FCR (up to 6 cycles) as first line therapy in 19 hematologic centers between 2001 and 2010. According to an intention to treat approach, all patients who received at least one FCR cycle were registered in the study. This series was representative of a FCR treated CLL cohort with respect to baseline characteristics, including age (median: 61 years; >65 years in 33% of patients), gender (male in 68% of patients), stage (progressive Binet A in 11% of patients; Binet B in 59%; Binet C in 30%) and number of FCR courses (median: 6; Results. After a median follow-up of five years, 159 patients have progressed and 72 have died, accounting for a 5-year progression free survival (PFS) according to the IWCLL criteria of 47% (median: 58 months) and for a 5-year overall survival (OS) of 81% (median: not reached). When the demographic effects of age, gender and year of treatment were compensated, the 5-year and 10-year survival of the whole CLL cohort were 85% and 68%, respectively, of those expected in the matched normal general population (p Conclusions. The combination of three biomarkers that are routinely tested at treatment allows to segregate a subgroup of CLL (IGHV mutated without 17p or 11q deletion) that may achieve a durable remission after first line FCR treatment and experience an expected survival similar to that of the general population. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

BACKGROUND. The detection of minimal residual disease (MRD) at the level of 0.01%/10-4 or above is a strong independent predictor of reduced progression-free (PFS) and overall survival (OS) in patients with CLL treated with chemoimmunotherapy. Although newer agents such as B-cell receptor pathway inhibitors can result in prolonged survival without achieving complete response, there remains a important role for MRD analysis in assessing therapeutic strategies aimed at disease eradication and cure. This is particularly important in front-line trials for fit patients which now require at least five years of follow-up if PFS is used as an endpoint. The feasibility of using MRD as a surrogate or intermediate endpoint for accelerated approval of new treatments is under review by regulatory agencies but further prospective validation is required. At the same time technology is rapidly evolving and high-throughput sequencing (HTS) technologies now detect MRD at the 0.0001%/10-6 level. It is therefore important to determine the most effective approaches for quantifying MRD that are compatible with previous studies but sufficiently sensitive for current treatments. AIMS. This collaborative project had two objectives. First, to identify the simplest and most flexible flow cytometry panel capable of detecting MRD at the 0.01%/10-4 or lower, that is compatible with published data and independent of instrument/reagent manufacturer. Second, to compare the flow cytometry approach with HTS analysis using the ClonoSEQ assay (Adaptive Biotechnologies, Seattle, WA). METHODS AND RESULTS. A core panel of antibodies for MRD detection was identified by testing an 8-marker combination in 52 samples (27 post-treatment and 25 dilution study) and re-analysing data with serial exclusion of single markers to determine redundancy. A 1-tube core panel of CD19, CD20, CD5, CD43, CD79b, and CD81 was identified and validated against the previously published 2-tube 6-marker and 4-tube 4-marker ERIC panels in 76 samples (19 post-treatment and 57 dilution study). The results showed good concordance (for log-transformed data above the LoQ, linearity=0.977, Pearson correlation co-efficient=0.983, average difference=0.026 log, 95% limit of agreement 0.20log) and a limit of detection of 0.001%/10-5 for the 1-tube core panel. Inter-operator variation was similar to CML MRD monitoring with both experienced operators, or inexperienced cytometrists after ~1 hour of specific education, achieving a 95% limit of agreement less than 0.3log in samples with MRD levels ranging from 0.0001 – 100%. The flow cytometry approach was compared with the ClonoSEQ HTS assay in 109 samples (21 dilution study and 88 post-treatment samples, complete data currently available on 13/88). The assay was applicable to the vast majority CLL patients, often with two clonal markers. There was 94% concordance at the 0.01% (10-4) threshold (15 samples with ≥0.01% CLL by both methods, 14 samples with CONCLUSIONS. The 1-tube 6-marker flow cytometry core panel is compatible with published studies, manufacturer-independent and flexible, providing directly quantitative results to 0.001%/10-5 without requiring a pre-treatment sample. HTS requires further work to standardise the quantitative analysis and prospective validation but the ClonoSEQ assay is applicable to >95% of CLL patients, does not require viable cells and is extremely sensitive, detecting residual disease in a significant proportion of cases with Disclosures Rawstron: Roche: Honoraria; Biogen Idec: Consultancy; Gilead: Consultancy, Honoraria; Abbvie: Honoraria; BD Biosciences: Intrasure reagent Patents & Royalties; Celgene: Honoraria; GSK: Honoraria. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Sanders:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Hallek:Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; GSK: Honoraria; Gilead: Honoraria. Hillmen:Roche: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

Introduction: The role of various prognostic factors in CLL/SLL is not yet fully understood, including the implications of new genetic markers associated with high risk. Ibrutinib (Imbruvica®), a first-in-class Bruton’s tyrosine kinase inhibitor, is a once-daily single agent approved by the US FDA for CLL patients (pts) who have received ≥ 1 prior therapy, and for pts with 17p deletion CLL. We report updated efficacy results for the phase 3 RESONATETM(PCYC-1112) study of ibrutinib (ibr) vs ofatumumab (ofa), relative to genetic features and prior treatment exposure, and provide updated adverse event (AE) data. Methods: Pts received 420 mg oral ibr daily until disease progression or unacceptable toxicity or IV ofa for up to 24 weeks. Primary endpoint was IRC-assessed PFS with secondary endpoints OS and IRC ORR (Byrd et al, NEJM 2014). At interim analysis, with median follow-up of 9.4 months, the Independent Data Monitoring Committee recommended pts in the ofa arm be provided access to ibr. Additional endpoints including investigator-assessed efficacy, investigation of potential predictive biomarkers, and safety with longer follow-up are reported. Results: The 391 pts were randomized to ibr (n=195) or ofa (n=196). Median age was 67 years, with 40% ≥70 years, 57% Rai stage III/IV disease. Median number of prior therapies was 3 (ibr) vs 2 (ofa) with 23% having 1, 28% with 2, and 49% ≥ 3 prior therapies. Approximately 32% had del(17p) and 32% (122/381) del(11q). 72 of 300 pts had complex cytogenetics (≥3 abnormalities) and 68% (181/266) unmutated IGHV. With outcome analyses pending, preliminary analysis revealed that 19% of pts had NOTCH1, 23% SF3B1, 36% TP53, and 1% MYD88 gene mutations at baseline. With median follow-up of 16 months, investigator-assessed PFS was significantly longer for ibr vs ofa (median NR vs 8.1 months, [HR 0.106, 95%CI 0.073-0.153, P Conclusions: Ibr significantly improved investigator-assessed PFS, OS, and ORR relative to ofa in pts with CLL/SLL who had received ≥ 1 prior therapy. The updated results are consistent with previously published phase 2 single-agent results (Byrd et al. NEJM 2013). Pts in the ibr arm treated in the second line experienced better outcomes than those salvaged in later lines of therapy. These results provide further evidence of ibrutinib’s robust clinical activity in CLL pts regardless of high-risk baseline genetics or number of prior therapies. Figure 1 Progression-Free Survival Figure 1. Progression-Free Survival Table. Investigator-assessed Efficacy Outcomes Table. Investigator-assessed Efficacy Outcomes Disclosures Brown: Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Hillmen:Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding. O'Brien:Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding. Barrientos:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Coutre:Janssen, Pharmacyclics: Honoraria, Research Funding. Tam:Pharmacyclics and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mulligan:Roche, Abbvie: Consultancy, Honoraria. Jaeger:Janssen Cilag: Honoraria. Barr:Pharmacyclics: Research Funding. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Kipps:Pharmacyclics: Research Funding. Cymbalista:Janssen, Roche, GSK, Gilead, Mundipharma: Honoraria. Delgado:Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria; Celgene: Honoraria; Novartis: Consultancy, Honoraria. Montillo:Janssen: Honoraria. Burger:Pharmacyclics: Consultancy, Honoraria, Research Funding. Chung:Pharmacyclics: Employment. Lin:Pharmacyclics: Employment. Gau:Pharmacyclics: Employment. Chang:Pharmacyclics: Employment. McGreivy:Pharmacyclics: Employment. James:Pharmacyclics: Employment. Byrd:Pharmacyclics: Research Funding.

Introduction : Ibrutinib (Imbruvica®), an oral, first-in-class covalent BTK inhibitor, is a new treatment option approved by FDA for chronic lymphocytic leukemia (CLL) patients (pts) with ≥1 prior therapy. We previously reported results from the phase III trial, in which ibrutinib (ibr) significantly improved progression-free and overall survival vs ofatumumab (ofa) in pts with relapsed/refractory CLL or small lymphocytic lymphoma (SLL) (Byrd et al. NEJM, 2014). Here, we report measures of patient well-being, including hematologic, immunologic, and quality of-life parameters, at interim analysis (IA). Methods : Pts with CLL/SLL after ≥1 prior therapy were randomized 1:1 to ibr 420 mg/day until progression or unacceptable toxicity, or to ofa for up to 24 weeks. At IA, secondary efficacy endpoints of hematologic improvement (sustained improvement ≥56 days without transfusions or growth factors) and FACiT-Fatigue (FACiT-F) outcomes were analyzed, along with exploratory endpoints assessing disease-related symptoms (DRS), serum immunoglobulin, patient-reported outcomes (PROs) by EORTC QLQ-C30, and medical resource utilization (MRU). Results : Of 391 enrolled pts (ibr n=195; ofa n=196), 63% had cytopenia(s) at baseline: 45% had anemia (hemoglobin ≤11 g/dL), 35% thrombocytopenia (platelets ≤100×109/L), and 20% neutropenia (absolute neutrophil counts [ANC] ≤1.5×109/L). On the ibr arm, 69% of pts with baseline cytopenias experienced sustained improvement in blood counts compared to 43% on ofa ( P

Background Gene expression profiling using microarrays has become an important genetic tool. Spotted arrays prepared in academic labs have the advantage of low cost and high design and content flexibility, but are often limited by their susceptibility to quality control (QC) issues. Previously, we have reported a novel 3-color microarray technology that enabled array fabrication QC. In this report we further investigated its advantage in spot-level data QC. Results We found that inadequate amount of bound probes available for hybridization led to significant, gene-specific compression in ratio measurements, increased data variability, and printing pin dependent heterogeneities. The impact of such problems can be captured through the definition of quality scores, and efficiently controlled through quality-dependent filtering and normalization. We compared gene expression measurements derived using our data processing pipeline with the known input ratios of spiked in control clones, and with the measurements by quantitative real time RT-PCR. In each case, highly linear relationships (R2>0.94) were observed, with modest compression in the microarray measurements (correction factor Conclusion Our microarray analytical and technical advancements enabled a better dissection of the sources of data variability and hence a more efficient QC. With that highly accurate gene expression measurements can be achieved using the cDNA microarray technology.

Genome-wide linkage analysis is a powerful tool for the identification of genes underlying single gene and complex genetic disorders. The most commonly used technique for performing genome wide scans for genetic studies in the rat is by analysis of simple sequence length polymorphism (SSLPs) or microsatellite markers. A sensitive and flexible method for high-throughput genotyping is described. Addition of an M13 tail to the SSLP primer eliminates the need for direct conjugation of the fluorescent dye to the primers, allowing for any combination of primer and fluorophor, and therefore for easy multiplexing of primers in the same reaction. With the use of three different dyes, it is possible to run more than five hundred genotypes in each run of the automatic sequencer. Automation in the fluorescent detection system and data tracking software for processing genotypes, contributes to the ability to genotype large number of samples rapidly and accurately.

Genome-wide linkage analysis is a powerful tool for the identification of genes underlying single gene and complex genetic disorders. The most commonly used technique for performing genome wide scans for genetic studies in the rat is by analysis of simple sequence length polymorphism (SSLPs) or microsatellite markers. A sensitive and flexible method for high-throughput genotyping is described. Addition of an M13 tail to the SSLP primer eliminates the need for direct conjugation of the fluorescent dye to the primers, allowing for any combination of primer and fluorophor, and therefore for easy multiplexing of primers in the same reaction. With the use of three different dyes, it is possible to run more than five hundred genotypes in each run of the automatic sequencer. Automation in the fluorescent detection system and data tracking software for processing genotypes, contributes to the ability to genotype large number of samples rapidly and accurately.

Purpose To investigate whether allogeneic stem-cell transplantation (allo-SCT) may overcome the negative impact of unmutated VH genes in the outcome of patients with chronic lymphocytic leukemia (CLL). Patients and Methods We analyzed the outcome of patients who underwent SCT according to their VH mutational status. Results Thirty-four patients (14 allo-SCT and 20 autologous SCT [auto-SCT]) presented unmutated VH genes and 16 patients presented mutated VH genes (nine allo-SCT and seven auto-SCT). Tumoral burden pre-SCT was significantly higher in the allo-SCT patients independent of the VH mutational status. The risk of relapse was significantly higher after auto-SCT (5-year risk, 61%; 95% CI, 44% to 84%) than after allo-SCT (5-year risk 12%, 95% CI, 3% to 44%; P < .05). In the unmutated group, 13 of 20 auto-SCT and two of 14 allo-SCT patients experienced disease progression, with a risk of relapse at 5 years of 66% (95% CI, 48% to 93%) v 17% (95% CI, 5% to 60%), respectively (P = .01). Conclusion These results show that allo-SCT may overcome the unfavorable effect of unmutated VH genes in patients with CLL.

ZAP-70 is a tyrosine kinase that participates in early B-cell differentiation and is a prognostic factor in chronic lymphocytic leukaemia (CLL), where it is associated with an unmutated configuration of the IgV(H) genes. In this study ZAP-70 expression was studied by immunohistochemistry in a spectrum of B-cell lymphoid neoplasms; this staining method was compared with flow cytometry, and the relationship of ZAP-70 expression to mutational status and prognosis was assessed. 242 tissue samples from 225 patients with B-cell lymphoid neoplasms arising at different maturational stages were included. Flow cytometry was performed in all CLL cases (n = 52). IgV(H) mutational status was determined in 25 CLL and 12 mantle cell lymphoma (MCL) patients. ZAP-70 was positive in 34/52 (65%) CLL, 9/31 (31%) Burkitt's lymphoma, 2/7 (29%) lymphoblastic lymphomas, 3/36 (8%) MCL, 1/23 (4%) marginal zone lymphoma, and 1/45 (2%) diffuse large B-cell lymphomas, but in none of the 19 follicular lymphomas or the 14 Hodgkin lymphomas. An identical ZAP-70 pattern was obtained in six patients with simultaneous biopsies from different sites and in 12 patients with sequential biopsies. Immunohistochemistry and flow cytometry gave identical results in 48 the 52 CLLs. All but one ZAP-70-positive CLL had IgV(H) gene in an unmutated configuration, whereas all but one ZAP-70-negative CLL had somatically hypermutated IgV(H). The 12 MCLs analysed were ZAP-70 negative regardless of IgV(H) mutational status (4 mutated, 8 unmutated). ZAP-70 positive CLL was associated with a shorter overall survival (median time 103 months vs. 293 months, p = 0.01) and a shorter time to disease progression (median time 26 months vs. 60 months, p = 0.01). In conclusion, ZAP-70 is expressed in several types of B-cell neoplasm and is easily detected by immunohistochemistry, providing a useful prognostic marker in patients with CLL from whom no other material is available or when other techniques for its assessment cannot be performed.

We report that Rab38 , a gene within the Rf-2 locus appears to influence the development of proteinuria (UPV) and albuminuria (UAV) in fawn-hooded hypertensive rats (FHH). Using congenic animals, we narrowed the region to eight genes; however, only one gene had a sequence variant. Rab38 has a

We studied immunoglobulin variable heavychain (IGHV) repertoire and mutational status in 553 patients with chronic lymphocytic leukemia (CLL) from the Mediterranean area to gain insight into the potential pathogenetic role of antigenic stimulation. The most commonly represented IGHV genes mirrored the usage of normal B cells, with the exception of IGHV1-18, IGHV3-30.3, and IGHV4-59 that were underrepresented. The IGHV3-21 gene, frequently expressed in Northern European CLL, was present only in 16 cases (2.9%). Based on HCDR3 cluster analysis, cases using IGHV3-21 could be grouped in 2 subsets of similar frequency. The first one (7 of 16 cases) carried a similar HCDR3 amino acid sequence (common-HCDR3 subset), virtually identical to the Scandinavian IGHV3-21 CLL. These cases used the IGHJ6 gene; 4 of 7 were unmutated; 6 of 7 carried the V2(lambda)-14 (IGLV3-21) light-chain gene with a similar LCDR3. All expressed CD38 and had a progressive disease. The second subset (9 of 16) was characterized by heterogeneous HCDR3 rearrangements (nonhomogeneous-HCDR3 subset), diverse IGHJ and lGV light-chain gene usage, variable IGHV mutational status (5 of 9 unmutated), variable CD38 expression, and variable clinical course (4 of 9 progressed). The first subset suggests a potential antigenic element rarely encountered in the Mediterranean area, possibly responsible for a negative outcome. The second subset may reflect the physiologic heterogeneity of expression of IGHV3-21 rearrangements in the normal repertoire and is characterized by a variable clinical outcome. (C) 2005 by The American Society of Hematology

Genetic linkage analyses in human populations have traditionally combined male and female progeny for determination of quantitative trait loci (QTL). In contrast, most rodent studies have focused primarily on males. This study represents an extensive female-specific linkage analysis in which 236 neuroendocrine, renal, and cardiovascular traits related to arterial pressure (BP) were determined in 99 female F2 rats derived from a cross of Dahl salt-sensitive SS/JrHsdMcwi (SS) and Brown Norway normotensive BN/SsNHsdMcwi (BN) rats. We identified 126 QTL for 96 traits on 19 of the 20 autosomal chromosomes of the female progeny. Four chromosomes (3, 6, 7, and 11) were identified as especially important in regulation of arterial pressure and renal function, since aggregates of 8–11 QTL mapped together on these chromosomes. BP QTL in this female population differed considerably from those previously found in male, other female, or mixed sex population linkage analysis studies using SS rats. Kidney weight divided by body weight was identified as an intermediate phenotype that mapped to the same region of the genome as resting diastolic blood pressure and was correlated with that same BP phenotype. Seven other phenotypes were considered as “potential intermediate phenotypes, ” which mapped to the same region of the genome as a BP QTL but were not correlated with BP. These included renal vascular responses to ANG II and ACh and indices of baroreceptor responsiveness. Secondary traits were also identified that were likely to be consequences of hypertension (correlated with BP but not mapped to a BP QTL). Seven such traits were found, notably heart rate, plasma cholesterol, and renal glomerular injury. The development of a female rat systems biology map of cardiovascular function represents the first attempt to prioritize those regions of the genome important for development of hypertension and end organ damage in female rats.

Dahl salt-sensitive (Dahl S) rats exhibit many phenotypic traits associated with salt-sensitive hypertension in man. Specifically, they are salt-sensitive, insulin-resistant and hyperlipidemic. They also develop endothelial dysfunction, cardiac injury and glomerulosclerosis. Insulin resistance is linked to hypertension, renal and cardiac damage and endothelial dysfunction. Thus, an agent that has diuretic action and can improve insulin resistance, like recombinant glucagon-like peptide-1(7-36)amide (rGLP-1), may have an antihypertensive effect.To determine whether chronic administration of rGLP-1 attenuates the development of hypertension, endothelial dysfunction and/or hypertension-induced renal and cardiac end organ damage in Dahl S rats.Mean arterial pressure (MAP) and urinary excretion of protein and albumin were measured in Dahl S rats before and after they were fed a 8% NaCl diet and infused with rGLP-1 (1 micro g/kg per min, i.v.) or vehicle for 14 days. At the end of the study, the degree of renal and cardiac injury was histologically assessed and endothelium-dependent relaxing function was studied using aortic rings. In other rats, the effects of rGLP-1 on sodium and water balance and plasma glucose and insulin levels for the first 3 days following a step change in sodium intake from a 0.1% NaCl diet to 7.5 mEq/day were determined.rGLP-1 significantly attenuated the development of hypertension in Dahl S rats (136 +/- 7 versus 174 +/- 6 mmHg). This was associated with reduction in proteinuria (46 +/- 7 versus 128 +/- 15 mg/day) and albuminuria (46 +/- 7 versus 86 +/- 18 mg/day) and improvement of endothelial function and renal and cardiac damage. rGLP-1 markedly increased urine flow and sodium excretion for the first 3 days following elevation in sodium intake. It had no significant effects on plasma glucose and insulin concentrations.rGLP-1 has antihypertensive and cardiac and renoprotective effects in Dahl S rats fed a high salt diet. The antihypertensive effect of rGLP-1 in Dahl S rats is due mainly to its diuretic and natriuretic effects, rather than an effect to improve insulin-resistance.

Background Despite intense investigation, the mechanism of isoflurane-induced cerebral hyperemia is unclear. The current study was designed to determine the contributions of neuronal nitric oxide synthase, prostaglandins, and epoxyeicosatrienoic acids to isoflurane-induced cerebral hyperemia. Methods Regional cerebral cortical blood flow was measured with laser Doppler flowmetry during stepwise increases of isoflurane from 0.0 to 1.2, 1.8, and 2.4 vol% end-tidal concentration in alpha-chloralose-urethane-anesthetized, C57BL/6 mice before and 45 min after administration of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI, 40 mg/kg, intraperitoneal), the cyclooxygenase inhibitor indomethacin (INDO, 10 mg/kg, intravenous), and the cytochrome P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-proparglyoxyphenyl)hexanoic acid (PPOH, 20 mg/kg, intravenous). Results Isoflurane increased regional cerebral cortical blood flow by 9 +/- 3, 46 +/- 21, and 101 +/- 26% (SD) at 1.2, 1.8, and 2.4 vol%, respectively. The increases in regional cerebral cortical blood flow were significantly (*P < 0.05) smaller after 7-NI (5 +/- 6, 29 +/- 19*, 68 +/- 15%*) or PPOH (4 +/- 8, 27 +/- 17*, 67 +/- 30%*), but not after administration of INDO (4 +/- 4, 33 +/- 18 [NS], 107 +/- 35% [NS]). The effect of combined treatment with 7-NI, PPOH, and INDO was not additive and was equal to that of either 7-NI or PPOH alone (5 +/- 5, 30 +/- 12*, 76 +/- 24%*). Chronic treatment of mice for 5 days with 7-NI (2 x 40 mg/kg, intraperitoneal) produced similar decreases in regional cerebral cortical blood flow as those seen with acute administration. Neither PPOH nor INDO conferred a significant additional block of the hyperemia in these animals. Conclusions Nitric oxide and epoxyeicosatrienoic acids contribute to isoflurane-induced hyperemia. However, only approximately one third of the cerebral hyperemic response to isoflurane is mediated by autacoids. The remaining part of this response appears to be mediated by a direct action of isoflurane on smooth muscle by some yet-unknown mechanism.

Abstract 3897 Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia (AIHA). However, the mechanisms governing the association between CLL and AIHA are poorly understood. MicroRNAs (miRNAs) are small 18–22 nucleotide long RNA molecules that regulate gene expression and play a key role in several biological processes. Importantly, deregulated miRNA expression has been implicated both in CLL and autoimmunity. This led us to speculate that patients with CLL who develop AIHA might have a different miRNA expression pattern as compared to those in whom this complication is not observed. We report here the first results of this study. We have evaluated the miRNA expression in purified CLL cells (CD19+, CD5+) from 14 patients who developed AIHA over the course of their disease and 19 sex-, age-, and clinical stage-matched controls who, after a comparable follow up time, did not develop this complication. The expression of 377 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays A v2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2–ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB-ArrayTools and R software. The unsupervised hierarchical cluster analysis identified two groups (CLL AIHA+ and CLL AIHA-). The supervised analysis revealed 7 miRNAs that were down-regulated in CLL AIHA+ patients compared to CLL AIHA- patients: miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-324-3p, miR-340, miR-660. Interestingly, miR-146, which has previously been related to autoimmunity, regulates IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of Toll-like and cytokine receptors. The prediction analysis of microarrays (PAM) was used to determine a set of miRNAs able to classify the samples into CLL AIHA+ and CLL AIHA-. We obtained a 122-miRNA model that classified CLL AIHA+ patients with a sensitivity of 69.4% and specificity of 68.4%. This model correctly classified 67% of the analyzed samples. In summary, CLL AIHA+ samples were characterized by a distinctive signature of 7 down-regulated miRNAs, one of which (miR-146) has previously been related to autoimmunity. Moreover, we identified 122 miRNAs that are able to predict AIHA in CLL patients. This is the first study establishing a relationship with CLL associated with AIHA and a distinctive miRNA signature, which should be useful for further studies aimed at unfolding the biologic complexity of AIHA in CLL. Disclosures: No relevant conflicts of interest to declare.

3880 The two TNF family proteins, B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL], and their three receptors, transmembrane activator and CAML interactor [TACI], B-cell maturation antigen [BCMA], and BAFF receptor [BAFF-R] are critical regulators of normal B-cell development and survival. In CLL, both proteins can rescue CLL cells from apoptosis as shown in in vitro studies. We and others have previously shown that patients with CLL and other B-cell chronic lymphoproliferative disorders show abnormal BAFF and APRIL serum levels. Additionally, a few reports indicate that CLL cells can express BAFF and APRIL receptors. Nevertheless, there is no a meaningful and comparative analysis of BAFF-R, TACI and BCMA levels in CLL. We therefore quantitatively assessed BAFF-R, TACI and BCMA on B-cells from patients with CLL and healthy controls. The expression of BAFF-R, TACI and BCMA was analyzed by flow cytometry in purified peripheral blood B cells from 42 patients with CLL and 13 healthy controls. BAFF receptor was the most highly expressed receptor in both CLL and normal B cells (MFI ratios, 161.5 and 179.1, respectively). TACI was heterogeneously expressed and almost undetectable in 15 CLL and 5 normal B cells. BCMA was expressed on all CLL and normal B cells. Furthermore, the expression of TACI and BCMA was significantly higher on CLL cells than on normal B cells (p=0.034 and p IGHV mutational status and cytogenetics). Higher BCMA expression on leukemic cells were detected in patients with unmutated IGHV gene and high-risk genetics (17p-, 11q-, trisomy 12 or 3 or more cytogenetics abnormalities) (p=0.045 and p=0.068). Additionally, patients with higher levels of BCMA on the CLL cells also had a significantly higher risk of disease progression, with a median progression-free interval of 57 vs. 206 months (p=0.021). We further examined whether activation signals can modulate BAFF-R, TACI and BCMA expression and thereby the sensitivity of CLL cells to respond to BAFF and APRIL. In 10 CLL patients (5 mutated, 5 unmutated) and six healthy controls, receptors expression was measured at 48 hours after stimulation with F(ab9) 2 antihuman IgM (10 μg/ml) and CD40L (500ng/ml) plus IL-4 (20ng/ml). Cell activation and viability, as assessed by labeling of CD69 and Annexin V/TO-PRO-3, were evaluated at 48 and 72 hours after co-stimulation with either soluble BAFF (100ng/ml) or APRIL (500ng/ml). After 48h culture, an increase of all three receptors was observed in normal B-cells in response to either BCR stimulation or CD40 ligation. In CLL cells, CD40 ligation induced a significant up-regulation of TACI expression (p=0.007) and a significant reduction of BCMA expression (p=0.007). In contrast, BCR stimulation induced almost no variation in CLL receptors expression. This was accompanied by a failure of cell activation and a significant decreased viability of CLL cells (from 36% to 24% p=0.013). Of note, no differences were observed in the regulation of BAFF and APRIL receptors nor in the activation or viability on CLL cells according to the IGHV mutational status. The addition of exogenous soluble BAFF or APRIL resulted in an increase in the viability of normal B-cells at 72 hours independently of cells stimulation through BCR or CD40 ligation. The viability of CLL cells was significantly increased upon CD40 stimulation whereas in non-stimulated or BCR-stimulated CLL cells the addition of BAFF and APRIL had a modest effect on their viability. Altogether, these results indicate that BAFF and APRIL receptors are differentially expressed on CLL and normal B-cells, with TACI and BCMA being highly expressed on leukemic cells. In addition, higher BCMA expression was significantly associated with poor prognostic variables (i.e, unmutated IGHV genes, poor cytogenetics) and higher risk of disease progression. Interestingly, activatory signals including BCR and CD40 ligation influenced BAFF and APRIL receptor expression and responsiveness to either BAFF or APRIL. CD40 ligation on CLL cells induced TACI up regulation by positively impacting on the survival promoting effect of BAFF and APRIL. Collectively, these findings link positive regulatory signals of BCR and CD40 ligation with the pro-survival effect of BAFF and APRIL and their receptors on leukemic CLL cells. Disclosures: No relevant conflicts of interest to declare.

Recent studies indicate that in the kidney arachi-donic acid (AA) is metabolized by cytochrome P450(CYP450) enzymes to produce epoxyeicosatrie-noic acids (EETs), dihydroxyeicosatetraenoic acids(DiHETEs), and 19- and 20-hydroxyeicosatetrienoicacids (19- and 20-HETE). CYP450 metabolites of AAplay a major role in the regulation of renal vasculartone, tubuloglomerular feedback (TGF), and sodiumtransport w1,2x. Enzymes of the CYP450 4A and 4Ffamilies catalyse the formation of 20-HETE. CYP4504A mRNA and protein are expressed in the renalarterioles, glomerulus, proximal tubule (PT), corticaland medullary thick ascending limb of the loop ofHenle (TALH), and in the pericytes surroundingvasa recta capillaries w2,3x. The renal arterioles, PTs,and glomeruli avidly produce 20-HETE, EETs, andDiHETEs when incubated with AA, while in theTALH, 20-HETE is the primary metabolite of AA w2x.Enzymes of the CYP450 1A, 2B, 2C, 2D 2E, and2J families catalyse the formation of EETs w1,4x.Inthe kidney, protein for each of these enzymes isexpressed; however, the specific enzymes primarilyresponsible for EETs production in different nephronsegments have not been determined.Numerous factors influence the expression ofCYP450 enzymes. High levels of CYP450 4A1 and4A3 mRNA and protein are detected in neonatal ratkidney, but the levels of these enzymes diminish intoadulthood w5,6x. In contrast, CYP450 4A2 proteinis the major isoform expressed in the adult rat kidneyw5,6x. In PT cells, angiotensin II (Ang II) stimulatesformation of EETs w7x, whereas epidermal growthfactor w8x, dopamine w9x, and parathyroid hormone(PTH) w10x stimulate the formation of 20-HETE.Vasopressin, Ang II, bradykinin, and calcium stimulate20-HETE formation in the TALH w2,4x. The expres-sion of CYP450 4A protein is downregulated in thekidney and renal vasculature when rats are fed a highsalt diet w11–13x and this can be prevented whencirculating Ang II concentrations are maintained atnormal levels via an i.v. infusion of Ang II w2x. On theother hand, EETs formation has been reported to beelevated by increased dietary salt intake in some strainsof rats w13–15x. Upregulated epoxygenase activityhas been postulated to play a role in the chronicadaptation of animals to elevations in salt intake byenhancing sodium excretion.Renal CYP450 4A activity is increased by minera-locorticoids, glucocorticoids, and progesterone w2x.Antilipidaemic agents like clofibrate induce the expres-sion of CYP450 4A1 and 4A3 protein and increase theformation of 20-HETE in the kidney. A variety ofvasoconstrictor agents, including Ang II, norepineph-rine, vasopressin, and endothelin, stimulate phospho-lipases and increase 20-HETE formation in the kidneyand peripheral vasculature w4x. In contrast, NO inhibitsthe formation of EETs and 20-HETE w16x.The expression of CYP450 4A enzymes in the kidneyis altered in diabetes, pregnancy, hepatorenal syn-drome, cyclosporin-induced nephrotoxicity, alcohol-induced liver disease, and in various animal models ofhypertension w2x. However, the role that 20-HETEplays in mediating the changes in renal function asso-ciated with these conditions has not yet been defined.In rats, enzymes of the CYP450 4A and 2C familiesexhibit sexual dimorphism w17x, but the physiologicalsignificance of this observation remains unknown.

We have previously demonstrated that nitric oxide (NO) exerts a greater modulatory influence on renal cortical blood flow in ANG II-infused hypertensive rats compared with normotensive rats. In the present study, we determined nitric oxide synthase (NOS) activities and protein levels in the renal cortex and medulla of normotensive and ANG II-infused hypertensive rats. Enzyme activity was determined by measuring the rate of formation ofl-[14C]citrulline froml-[14C]arginine. Western blot analysis was performed to determine the regional expression of endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms in the renal cortex and medulla of control and ANG II-infused rats. Male Sprague-Dawley rats were prepared by the infusion of ANG II at a rate of 65 ng/min via osmotic minipumps implanted subcutaneously for 13 days and compared with sham-operated rats. Systolic arterial pressures were 127 ± 2 and 182 ± 3 mmHg in control ( n = 13) and ANG II-infused rats ( n = 13), respectively. The Ca2+-dependent NOS activity, expressed as picomoles of citrulline formed per minute per gram wet weight, was higher in the renal cortex of ANG II-infused rats (91 ± 11) than in control rats (42 ± 12). Likewise, both eNOS and nNOS were markedly elevated in the renal cortex of the ANG II-treated rats. In both groups of rats, Ca2+-dependent NOS activity was higher in the renal medulla than in the cortex; however, no differences in medullary NOS activity were observed between the groups. Also, no differences in medullary eNOS levels were observed between the groups; however, medullary nNOS was decreased by 45% in the ANG II-infused rats. For the Ca2+-independent NOS activities, the renal cortex exhibited a greater activity in the control rats (174 ± 23) than in ANG II-infused rats (101 ± 10). Similarly, cortical iNOS was greater by 47% in the control rats than in ANG II-treated rats. No differences in the activity were found for the renal medulla between the groups. There was no detectable signal for iNOS in the renal medulla for both groups. These data indicate that there is a differential distribution of NOS activity, with the Ca2+-dependent activity and protein expression higher in the renal cortex of ANG II-infused rats compared with control rats, and support the hypothesis that increased constitutive NOS activity exerts a protective effect in ANG II-induced hypertension to maintain adequate renal cortical blood flow.

Introduction Follicular lymphoma (FL) is the most frequent indolent lymphoma. Autologous hematopoietic stem cell transplantation (ASCT) allows durable progression-free survival in selected relapsed patients. Recently, the EBMT lymphoma working party has published the recommendations for ASCT and allogeneic hematopoietic transplantation (alloTPH) in FL. For those high-risk young patients relapsing after ASCT, alloTPH is a valid therapeutic option; the majority being done with reduced intensity conditioning (alloRIC). Aims Our aim is to describe the outcome of patients with FL who underwent an alloTPH in our cente. Factors potentially influencing outcome such us age, sex, FLIPI at diagnosis, disease status prior alloTPH, EBMT risk score, type of donor, graft vs host disease (GVHD) prophylaxis and development of GVHD were also analized. Methods Actuarial estimates of OS and PFS rates were calculated using the Kaplan-Meier method. OS was estimated from the date of transplantation to the date of death or last follow-up. PFS was defined as the time from transplantation until progression or death from any cause. Patients We retrospectively found 26 alloTPH in the period 1999 - 2013. Two alloTPH were conditioned with a myeloablative regimen. Patients who underwent an alloRIC (n=24) were all conditioned with Fludarabine 30 mg/m2/5 days and Melphalan 70 mg/m2/1 day. Results AlloTPH was from a matched related donor in 70.8% of cases, from a mismatched related donor in 4.2%, matched unrelated donor in 8.3% and from a mismatched unrelated donor in 16.7%. The median age was 49.5 years (range 35-61). The median time from diagnosis to alloTPH was 48 months (range 9 – 190), male: female ratio was 2:1. FLIPI at diagnosis was 0 in 16.7%, FLIPI 1 in 25%, FLIPI 2 in 33.3%, FLIPI 3 in 8.3% and FLIPI 4 in 16.7. Mean number of treatment pre alloTPH was 4.5 (range 2-6). Ten patients (41.7%) had previously received an ASTC. Response before alloTPH was complete response in 54.2%, partial response in 37.5% and stable disease in 8.4%. The majority of patients (95.8%) had an EBMT risk score higher than 3. Graft vs host disease prophylaxis consisted on cyclosporine + short course of methotrexate in 54.2%, cyclosporine + micophenolate in 41.7% and sirolimus + tacrolimus in 4.2%. In vivo T-cell depletion was done in 4 patients receiving grafts from a mismatched unrelated donor (ATG or alemtuzumab, 2 patients each). Mean CD34 cells/Kg infused was 6.45 x106/kg (range 1.92 – 15.6). The median time to granulocyte recovery (>0.5x109/L) was 15 days (range 11-19) and the median time to platelets recovery (>50x109/L) was 14 days (range 6-56). Acute GVHD was developed in 45.8% of patients; it was global grade 1-2 in 36.4% ,and grade 3-4 in 63.6%. Chronic GVHD was present in 58.3% of patients. The median time of chronic GVHD presentation was 196 days after alloTPH (range 101 – 569 days). Regarding opportunistic infections, 20.8% of patients reactivated CMV with no disease, 16.7% had intestinal disease due to CMV. Thirteen per cent of patients developed pulmonary fungal infection (i.e Aspergillus sp). With a median follow up of 48.50 months (range 0-161), the 5 years OS was 66.1% (CI 95% 43.76 - 88.44) and the 5 years PFS was 63.8% (CI 95% 43.22 - 84.4). Overall morbidity at 3, 6 and 12 months after alloTPH was 5%, 5% and 10.3% respectively. Only 1 patient relapsed at 38 months after alloTPH but is still alive. Causes of death were refractory GVHD (n=5), sepsis (n=3) and acute renal failure due to microangiopathy (n=1). Survival was influenced by GVHD prophylaxis. At the median time of follow up (48.50 months) patients receiving cyclosporine-methotrexate had an OS of 82.5% vs 30% for patients receiving cyclosporine-MMF (p Other variables (FLIPI at diagnosis, sex, age older than 60 years, the EBMT risk score pre alloTPH and disease status) had no influence on survival. Conclusions AlloTPH for high risk FL patients seems to overcome the negative effect of FL prognostic factors at diagnosis. This strategy shows an excellent disease control with a low transplant-related mortality. Disclosures: No relevant conflicts of interest to declare.

Background Although recommendations for assessing prognosis in patients with CLL have been formulated (IWCLL, 2008; ESMO, 2013; NCCN, 2013), the actual use of prognostic and predictive markers in patients with CLL seen in daily practice is unknown. Study objectives To ascertain the use of prognostic and predictive markers in patients with CLL not included in trials. Methods Three-hundred and eighty-two members included in the ERIC database were invited to participate in a survey which included a comprehensive list of parameters potentially useful to evaluate patients with CLL at three different steps: 1. At diagnosis; 2. During clinical course; 3. Before therapy. Results One-hundred sixteen out of the 382 ERIC members (30%), mainly from academic institutions (91%), participated in the survey. Here we present a summary of this study which can be seen at its full length at www.ericll.org Prognostic factors at diagnosis The most frequently evaluated parameters (by > 80% of the participants) are: age, disease stage, ECOG, B-symptoms, WBC count, LDT, tumor bulk, DAT, LDH and B2M; CIRS is evaluated by 45% and only 7% measure sTK routinely. CD38 is assessed by virtually all investigators, but not ZAP-70 (58%) nor CD49d (24%). Imaging studies are frequently employed: ultrasonography (US) (51%), CT (21%). PET/CT is never investigated at diagnosis. FISH for 13q-, 11q-, 17p-, and +12 is required by > 70%. Among biomarkers, those more frequently used are IGHV mutational status (54%) and TP53 mutations (25%). As per the newly described gene mutations around 20% of the participants would like to determine mutations of NOTCH1, SF3B1, MYD88, and BIRC3 should they be available. Prognostic factors over the course of the disease Sixty percent of participants re-assess prognostic parameters over the course of the disease. Bone marrow (aspirate/biopsy), lymph node biopsy and imaging studies are performed in subjects with progressive disease. However, 72% of the participants never use PET/CT, which does not seem to be related to the technique availability since only 3% of the participants would use PET/CT if available. Parameters studied before initiating treatment Beside standard clinical parameters, other features frequently evaluated include ECOG (96%), DAT (77%), CIRS (66%) and bone marrow (45%). US, CT scans, and PET/CT are routinely (or occasionally) assessed by 45% (52%), 39% (42%), and 3% (42%) of the investigators, respectively. IGHV mutational status is repeated by a few participants (19%). 17p- and 11q- are studied by 79% and 68% of the investigators, respectively. Likewise, TP53 mutations are routinely (or occasionally) investigated by 40% (27%), NOTCH1 by 6% (13%), SF3B1 by 6% (12%), MYD88 by 4% (10%), and BIRC3 by 4% (9%). Most (89%) investigators consider pre-treatment findings to select therapy, the clinical parameters frequently considered toward that end being age (80% of investigators), ECOG (43%) and CIRS (37%). Biologically, 17p- (79%) and TP53 mutations (40%) along with 11q- (48%) and 13q- (26%) are the most frequently investigated markers. Novel mutations are investigated by 9%-15% of participants and around 20% would use them if possible. Conclusions Although international recommendations are generally followed, the evaluation of patients with CLL at diagnosis includes in many cases parameters (e.g., 17p-, TP53 mutations) more useful as predictive (i.e., predicting response to therapy) than prognostic (i.e., predicting disease progression) factors, as well as ancillary studies (e.g., imaging studies) only recommended within clinical studies. In many instances prognostic markers are repeated over the course of the disease. Finally, treatment decisions are mainly based on clinical features and consolidated biomarkers (i.e., FISH cytogenetics, TP53 mutations). The results of this study need to be taken cautiously because of potential investigator-related biases and its relatively small sample size. However, it could be useful as a basis for refining, based on what is actually done in daily practice, recommendations about evaluation and prognostication of patients with CLL. Disclosures: Montillo: Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

as part of a continuing effort to integrate physiology and genomics, the American Physiological Society sponsored a meeting at the Riverfront Augusta Hotel in Augusta, Georgia, October 1–4, 2003, titled “Understanding Renal and Cardiovascular Function through Physiological Genomics.” Organized

2744 The current standard of care for fit patients (pts) with chronic lymphocytic leukemia (CLL) is rituximab (R) in combination with fludarabine and cyclophosphamide; however, many pts with CLL are elderly and have comorbidities that render them ineligible for fludarabine treatment. Two common treatment options for fludarabine-ineligible pts are bendamustine (B) or chlorambucil (Clb). A further promising treatment option, following the success of the combination of R with fludarabine and cyclophosphamide, might be to combine B or Clb with R. The following study aims to assess the responses of pts to a combination of R and either B or Clb in first-line (1L) or second-line (2L) pts with CLL, with the primary objective being to compare the confirmed complete response (CR) rate (assessed according to Hallek et al. Blood 2008) after 6 cycles of treatment between the two treatment arms for the pooled 1L and 2L pts. Pts (aged ≥ 18 years) who were ineligible for fludarabine treatment, as a result of age or a greater number of comorbidities, were randomized to either the R-B or R-Clb arm. Pts were 1L or 2L, where relapse had occurred no earlier than 12 months since their last dose of 1L treatment. Pts in the R-B arm were treated with six 28-day cycles of B (1L: 90 mg/m2 Days 1 and 2; 2L: 70 mg/m2 Days 1 and 2) with R administered on Day 1 of cycle 1 (375 mg/m2) and cycles 2–6 (500 mg/m2). Pts in the R-Clb arm received the same dose of R but in place of B they received Clb (10 mg/m2Days 1–7, cycles 1–6), and those pts in the R-Clb arm that had not achieved a CR after 6 cycles continued to receive Clb monotherapy for up to 6 further cycles. Tumor assessments were made after cycles 3, 6 and 12 and then 3-monthly for at least a year. Enrollment and randomization are ongoing and, at present, a total of 339 pts have been randomized between the two treatment arms, 126 of whom are included in this interim analysis with the remaining pts continuing on the study. Of these 126 pts, 58 were randomized into the R-B arm and 68 the R-Clb arm. Patient characteristics between the two treatment arms were well balanced (Table). The median age of pts was 74 years (75 years for the R-B arm and 73 years for the R-Clb arm) and the majority of pts were taking concomitant medication (57/58 pts [98%] in the R-B arm; 64/68 pts [94%] in the R-Clb arm). Compared with previous clinical trials in CLL where pts are usually younger and fitter, this patient population is closer in age and fitness to pts presenting in the clinic. A total of 85 pts were previously untreated, with the remaining 41 having received one line of previous treatment. 2L pts had received a median of 6 prior cycles of treatment. The safety population was made up of 124 pts (R-B: n = 57; R-Clb: n = 67) who had received at least one dose of the study medication; 104/124 pts completed all 6 cycles of rituximab treatment. After 6 cycles of treatment, 14/58 pts (24%) in the R-B arm had a confirmed CR compared with 7/68 pts (10%) in the R-Clb arm (p = 0.033). For 1L pts the corresponding CR rates were 30% in the R-B arm vs 13% in the R-Clb arm (p = 0.054) and 2L pts exhibited CR rates of 11% in the R-B arm vs 4% in the R-Clb arm (p = 0.413). At the end of treatment, the overall response rate (ORR) was not different between the R-B and the R-Clb arms (88% and 81%, respectively [p = 0.404]). ORRs for 1L pts were 88% in the R-B arm vs 80% in the R-Clb arm and for 2L pts were 89% in the R-B arm vs 83% in the R-Clb arm. Safety was similar between the two treatment arms with the most common adverse events (AEs) (any grade) being neutropenia (R-B: 42% vs R-Clb: 46%) followed by nausea (R-B: 26% vs R-Clb: 22%). The most common AE at ≥ grade 3 was neutropenia (R-B: 32% vs R-Clb: 34%). Serious AEs were experienced by 20 pts (35%) in the R-B arm and 23 pts (34%) in the R-Clb arm; the most frequent serious AE was pneumonia (R-B: 7% vs R-Clb: 2%). 5/57 pts (9%) in the R-B arm and 8/67 pts (12%) in the R-Clb arm withdrew from the study prematurely due to AEs. R-B or R-Clb could provide useful treatment options for pts with CLL who are ineligible for the current fludarabine-containing standard of care. Interim results from this study have indicated that R-B shows a trend towards higher CR rates compared with R-Clb. | Characteristic | R-bendamustine (n = 58) | R-chlorambucil (n = 68) | |:-------------------------:| ----------------------- | ----------------------- | | Median age, years (range) | 75 (49–87) | 73 (44–91) | | Sex, % | - | - | | Male | 60 | 65 | | Female | 40 | 35 | | Binet stage, % | - | - | | A | 5 | | | B | 55 | 59 | | C | 33 | 37 | | Not reported | 7 | 4 | | 17p/11q del, % | 12 | 4 | | IGVH status, % | - | - | | Mutated | 41 | 52 | | Unmutated | 53 | 38 | | Other | 5 | 10 | | Line of treatment, % | - | - | | 1L | 69 | 66 | | 2L | 31 | 34 | Table: Baseline patient characteristics Disclosures: Leblond: Roche: Advisory Board Other, Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Janssen-Cilag: Honoraria. Off Label Use: Rituximab in Combination with Bendamustine or Chlorambucil for Treating Patients with Chronic Lymphocytic Leukemia. Rassam: Johnson and Johnson: Unrestricted research grant Other; ROCHE: Honoraria; BMS: Honoraria. Raposo: Roche: Consultancy. Oertel: Roche: Employment, Equity Ownership.

not Available.

Abstract 3897 Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia (AIHA). However, the mechanisms governing the association between CLL and AIHA are poorly understood. MicroRNAs (miRNAs) are small 18–22 nucleotide long RNA molecules that regulate gene expression and play a key role in several biological processes. Importantly, deregulated miRNA expression has been implicated both in CLL and autoimmunity. This led us to speculate that patients with CLL who develop AIHA might have a different miRNA expression pattern as compared to those in whom this complication is not observed. We report here the first results of this study. We have evaluated the miRNA expression in purified CLL cells (CD19+, CD5+) from 14 patients who developed AIHA over the course of their disease and 19 sex-, age-, and clinical stage-matched controls who, after a comparable follow up time, did not develop this complication. The expression of 377 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays A v2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2–ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB-ArrayTools and R software. The unsupervised hierarchical cluster analysis identified two groups (CLL AIHA+ and CLL AIHA-). The supervised analysis revealed 7 miRNAs that were down-regulated in CLL AIHA+ patients compared to CLL AIHA- patients: miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-324-3p, miR-340, miR-660. Interestingly, miR-146, which has previously been related to autoimmunity, regulates IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of Toll-like and cytokine receptors. The prediction analysis of microarrays (PAM) was used to determine a set of miRNAs able to classify the samples into CLL AIHA+ and CLL AIHA-. We obtained a 122-miRNA model that classified CLL AIHA+ patients with a sensitivity of 69.4% and specificity of 68.4%. This model correctly classified 67% of the analyzed samples. In summary, CLL AIHA+ samples were characterized by a distinctive signature of 7 down-regulated miRNAs, one of which (miR-146) has previously been related to autoimmunity. Moreover, we identified 122 miRNAs that are able to predict AIHA in CLL patients. This is the first study establishing a relationship with CLL associated with AIHA and a distinctive miRNA signature, which should be useful for further studies aimed at unfolding the biologic complexity of AIHA in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1366 Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune phenomena, particularly autoimmune hemolytic anemia (AIHA). However, there are very few studies analyzing autoimmune cytopenias and its clinical implications in large, unselected series of patients with CLL. We investigated the prevalence, characteristics, and prognostic significance of autoimmune cytopenias in 960 patients with CLL diagnosed at the Hospital Clínic of Barcelona between January 1980 and December 2008. Seventy of 960 (7%) patients presented autoimmune cytopenias, of whom nineteen were detected at diagnosis, three prior to diagnosis and 48 during the course of the disease. Forty-nine patients had AIHA, 20 immune thrombocytopenic purpura (ITP) and one both AIHA and ITP. A clear association was observed between autoimmune cytopenias and poor prognostic variables such as high blood lymphocyte count (p= 0.004), blood lymphocyte doubling time less than 12 months (p= 0.01), increased serum beta-2 microglobulin (p= 0.02), and high expression of ZAP-70 (p=0.02) and CD38 (p=0.07). No statistically significant differences were found in the incidence of AIHA based on treatment modality: fludarabine-based 8/204 (4%) vs. alkylating agents-based 12/231 (5%). The outcome of patients with autoimmune cytopenias was not significantly different from that of patients without this complication (median survival: 8 years vs. 9 years; p=ns). Furthermore, no significant differences were observed according to the time at which cytopenia was detected (i.e., before, at diagnosis or after the diagnosis of CLL). Importantly, significant differences in the response rate were observed in the 73 patients with advanced (Binet C stage) disease based on the origin of cytopenia. Whereas 16 of the 19 patients with stage C “immune” disease responded to corticosteroids and as a result switched from stage C to stage A, only 9 of 54 patients with stage C “infiltrative” responded to therapy (p In conclusion, in this large, unselected and single institution series of patients with CLL autoimmune cytopenias correlated with well-known poor prognostic variables but not with treatment modality (i.e. purine analogs vs. alkylating agents). From the prognostic point of view, autoimmune cytopenias did not significantly influenced prognosis in the whole group of patients. Moreover, patients presenting with advanced disease related to an immune mechanism had better prognosis than those in whom advanced stage reflected a high tumor burden only. These results emphasize the importance of determining the origin of cytopenias in patients with CLL for both treatment and prognostic purposes. Disclosures: No relevant conflicts of interest to declare.

Abstract 1389 Although chronic lymphocytic leukemia (CLL) is considered incurable, a proportion of patients treated with modern therapy achieve complete remission (CR) with minimal residual disease (MRD) negative status, which translates into a better outcome. In addition, survival curves for allogeneic stem cell transplant (alloSCT) appear to plateau, suggesting possible cure. Profound immune disturbances are a major cause of morbidity and mortality in CLL and can be exacerbated by therapy. We studied the immune status of 26 patients with CLL with a sustained (median 8 years, range 2.2 to 17) CR following treatment (4 patients received fludarabine, cyclophosphamide and mitoxantrone [FCM], 6 autologous SCT [autoSCT]) and 16 allogeneic stem cell transplantation [alloSCT]. Lymphocyte subsets were studied using multiparameter flow cytometry and compared to healthy controls. CD8+ T cell response to cytomegalovirus (CMV) was assessed using a pentameric HLA-A2 binding CMV pp65-derived peptide and functionality was confirmed by interferon gamma (IFNγ) ELISPOT. Immunoglobulin subtypes, complement proteins, and β2-microglobulin (B2M) were quantified by standard techniques, interleukin 10 (IL-10) and vascular endothelial growth factor (VEGF) by flow-based cytometric bead array technology, and B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) serum levels by commercial ELISA. Median age at the time of study was 57 years (55 for the alloSCT, 54 for the autoSCT and 63 for the FCM patients). Two alloSCT patients had chronic graft-versus host disease (cGVHD) at sample collection and were receiving immunosuppression. In addition, three patients experienced autoimmune cytopenia after alloSCT, but had responded to therapy at the time of the study. Detectable residual CLL cells (> 10-4) in peripheral blood were found in six patients (3 alloSCT, 3 FCM). The 20 patients in MRD negative CR had a significantly higher absolute B cell count compared to normal controls comprised of both CD19+CD5- cells (154 cells/mm3vs. 76 cells/mm3, p=0.005) and CD19+CD5+ cells (22 cells/mm3vs. 1 cell/mm3, p=0.002). Normal serum levels of immunoglobulin were present in 17 patients whereas 9 had persistent hypogammaglobulinemia (4 were MRD positive, 2 were on immunosuppression for cGVHD and 1 patient had received rituximab). Thus only 2 patients had unexplained hypogammaglobulinemia despite being in MRD negative-CR. T cell abnormalities were also common in these patients. An abnormal CD4:CD8 ratio was present in 9 patients, and CD4+ cells had failed to recover to >400 cells/mm3 in an additional 5 patients. An increase in CD8+ cells (620 cells/mm3vs. 379 cells/mm3 in normal controls) was mostly comprised of cells with a chronically activated phenotype, CD8+DR+ (220 cells/mm3vs. 78 cells/mm3 in normal controls, p=0.062). Furthermore, in CMV+/HLA-A2 patients, an elevated cytotoxic CD3+CD8+CD45RA population was seen (200 cells/mm3vs. 51 cells/mm3, p=0.037). In all cases, cytotoxic T cells expansions secrete IFNγ in response to pp65. As regards T regulatory cells, CD4+CD25+FOXP3+ cells were significantly increased in patients after alloSCT compared to normal controls (4.1% of CD4+ cells vs. 2.05% in normal, p=0.023). In contrast, patients who received either FCM or autoSCT had Treg levels equivalent to our normal samples (2.45% of CD4+ cells), including the three patients who were MRD positive after FCM. B2M was elevated in 7 patients. No differences were found in levels of complement proteins C3 and C4, direct Coombs test, IL-10 and VEGF. Nevertheless, patients showed median BAFF serum levels significantly higher than healthy controls, even when those patients with conditions considered likely to elevate BAFF and APRIL were excluded (cGVHD, autoimmune cytopenia) (p Disclosures: No relevant conflicts of interest to declare.

Abstract 4649 Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B lymphocytes. In addition, immune disturbances are commonly present. Although CLL is still considered incurable, long term remissions can be observed after allogeneic stem cell transplantation (SCT). Whether immune function is restored in patients responding to treatment is largely unknown. We report on the immune status in 19 patients with CLL in long-lasting complete remission (CR) after SCT (13 allogeneic, 6 autologous). Median age was 51 (range, 33- 64) and median follow-up since transplantation was 6 years (range, 2-17). Three patients had chronic graft-versus host disease at sample collection, two of whom were receiving immunosuppression. Lymphocyte subsets were studied using multiparameter flow cytometry and compared to healthy controls. We quantified immunoglobulin subtypes, complement proteins, and β2-microglobulin (B2M) by standard techniques and IL-10 and VEGF by using flow-based cytometric bead array technology. CD8+ T cell response to CMV was assessed using a pentameric HLA-A2 binding CMV pp65-derived peptide. Three patients (all following allogeneic SCT) had detectable residual CLL cells (> 10-4) in peripheral blood at the time of the analysis. In the remaining 16 patients with no detectable minimal residual disease (MRD) normal CD19+CD5- and CD19+CD5+ B cell populations were lower than in healthy individuals (10.5% vs. 14.5%; p=0.02 and 1.6% vs. 3.9 %; p=0.002 respectively). A significant increase in the proportion of CD8+ T cells (median 28.1% vs. 18.7%, p=0.03), particularly those with a chronically activated phenotype CD3+CD8+DR+ (10.2% of CD3+ cells vs. 4.9% in controls), was observed (p=0.013). The nine CMV+/HLA-A2 patients all showed specific cytotoxic CD8+ T cells which exhibit predominantly a CD45RA+CD27- phenotype, being better preserved in autologous SCT than in allogeneic SCT patients. Also, higher numbers of CD8+CD45RA+CD27- T cells were observed in patients with a longer follow-up. CD4+ T cell count was < 400/mm3 in 4 patients. An abnormal CD4:CD8 ratio was seen in 7 out of 19 patients. Interestingly, a significant increase of double positive CD4 and CD8 T cells was detected in most patients comprising 2.6% vs.1.6% of lymphocytes in normal subjects (p=0.02). There were no quantitative abnormalities in CD3-CD56+ cells. Not surprisingly, hypogammaglobulinemia was present in all but four patients immediately prior to transplant. Whereas IgM levels normalized in all patients, 4 and 6 patients respectively still had low IgG and IgA levels more than two years after transplantation. Of note, low serum immunoglobulin levels were seen in 6 out of 14 MRD negative-CR patients and all three MRD positive-CR patients, including one who had normal immunoglobulin levels at the time of transplant. Complement proteins C3 and C4 were within the normal range in all cases. The direct Coombs test (DCT) was also negative in all patients although one patient had indirect signs of hemolysis. Regarding serum markers, B2M was increased (>2.5mg/dl) in 5 out of 19 patients. No significant differences were found in IL-10 and VEGF levels between patients and normal controls (median levels, 2.14 vs. 1.06 pg/ml and 156.88 vs. 104.14 pg/ml), but there were 2 patients with markedly elevated IL-10. In summary, these data demonstrate that immune defects persist over time in CLL patients with a long lasting CR (including MRD-negative CRs) after SCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 1235 Poster Board I-257 BAFF (B-cell activating factor) and APRIL (a proliferation-inducing ligand) are TNF family proteins that upregulate anti-apoptotic genes through the NF-kB pathway. Studies in vitro suggest that BAFF and APRIL protect neoplastic B cells from apoptosis in chronic lymphoproliferative disorders (CLPD) including chronic lymphocytic leukemia (CLL). Serum BAFF levels have been previously shown to be lower in CLL than in other CLPD or normal subjects. To contribute to a better understanding of their role in CLL, we analyzed BAFF and APRIL at mRNA and protein serum levels and their receptors [transmembrane activator and CAML interactor (TACI), B-cell maturation antigen (BCMA) and BAFF receptor (BAFF-R)] by flow cytometry, in 82 patients with CLL, 36 with other CLPD and 35 age- and sex-matched controls. mRNA BAFF and APRIL levels were calculated as a the percentage of expression referred to an internal control and the receptor expression as the ratio between the mean fluorescence intensity (MFI) of the receptor antibody and the MFI of the isotype control. Patients with CLL showed significantly lower median BAFF and APRIL levels (0.63 μg/ml and 3.18 μg/ml) than those with other CLPD (1.27 μg/ml and 5.51 μg/ml) (p Disclosures No relevant conflicts of interest to declare.

Abstract 1244 Poster Board I-266 Allogeneic stem cell transplantation (SCT) is the only curative treatment for chronic lymphocytic leukemia (CLL). Advanced age, extensive prior therapy, lack of response to treatment, and T-cell depletion of the graft are poor prognostic factors which have been identified in many studies. Beta-2 microglobulin (B2M) has important prognostic value in patients treated with chemotherapy or chemoimmunotherapy, but has been scarcely investigated in the context of allogeneic SCT. In two studies (Khouri et al. Cytotherapy 2002; Sorror et al. J Clin Oncol 2008) no correlation was found between B2M and transplant outcome. Against this background, we analyzed the influence of B2M and other prognostic parameters in 32 patients (median age 50 yrs [range, 29-63], 20 males) who received an allogeneic SCT in our institution between 1991 and 2006. Interval between diagnosis and transplantation was 44 months (range, 6-116). Median number of prior therapies was 2 (range, 1-6). Six patients had previously received an autologous SCT. Most patients had adverse biologic features (high ZAP-70 expression, unmutated IGHV, poor cytogenetics). Serum B2M was increased (≥2.5 mg/L) in 13 out of 29 patients prior to transplant. Creatinine levels and glomerular filtration rate were normal. Median follow-up after transplantation was 7 years (range, 1.8- 16.9). The relapse risk (RR) at 5 and 10 years was 5 % (95% CI, 0-14%) and 23% (95% CI, 2-44), respectively. At one and 10 years the cumulative non-relapse mortality (NRM) was 34% (95% CI, 17-51) and 38% (95% CI, 20-55), respectively. Five and 10-year progression free survival (PFS), event free survival (EFS) and overall survival (OS) were 85% (CI, 66-100) and 65% (CI, 35-94), 58% (CI, 40-76) and 40% (CI, 19-62), and 62% (CI, 45-79) and 57% (CI, 38-75). In the univariate analysis, factors associated with a higher NRM were prior autologous SCT (p=0.006), chemorefractory disease (p=0.04), and high serum B2M levels at the time of SCT (p=0.03). Parameters associated with EFS and OS were high B2M levels (p=0.001 and p=0.002), prior autologous SCT (p In contrast, IGHV mutational status, high ZAP-70 expression, > 30% bone marrow infiltration, and disease status (CR vs. no CR) at the time of SCT were not associated with outcome. In summary, this study indicates that, as in patients treated with chemo or chemoimmunotherapy, B2M is a strong predictor of clinical outcome in patients with CLL submitted to allogeneic SCT. Disclosures No relevant conflicts of interest to declare.

Abstract 2361 Poster Board II-338 Clinical staging systems are the backbone for assessing prognosis in patients with chronic lymphocytic leukemia (CLL). Clinical stages, however, are assigned without taking into consideration the mechanisms of the disease. In this regard, the prognosis of patients with advanced (Binet C, Rai III, IV) stage due to immune cytopenia is controversial. To address the prognosis of patients with CLL in advanced clinical stage due to immune mechanisms, we studied two groups of patients with and without autoimmune cytopenia. The first group consisted of 62 patients (39 men, median age 65 yrs; range 33-89) with advanced stage due to autoimmune cytopenia (stage C “immune”). Autoimmune hemolytic anemia (AIHA) was defined as a hemoglobin level Disclosures: No relevant conflicts of interest to declare.

The immunoglobulin heavy chain variable (IGHV) gene repertoire in CLL is biased and uniquely characterized by the existence of subsets of cases with “stereotyped” heavy chain complementarity-determining region 3 (HCDR3) sequences. As previously shown, HCDR3 stereotypy may have important pathogenetic and clinical implications, at least for certain subsets of CLL patients. However, the detection of stereotypy has so far been hindered, mainly due to the lack of suitable computational tools. We developed new methods for identification of sequence patterns within HCDR3 amino acid (AA) sequences using a sophisticated combinatorial pattern discovery algorithm. Included in the analysis were HCDR3 sequences from 2,845 CLL patients from our collaborating institutions, as well as 5,344 non-CLL sequences from public databases, for a total of 8,189. The identified patterns were subjected to a multiple and strict filtering process, based on the following criteria: sequence relatedness; location (offset) within HCDR3; and HCDR3 AA length. Clustering of sequences based on the filtered HCDR3 patterns revealed that the CLL IG repertoire can be distinguished in two broad categories: the first includes cases with heterogeneous B cell receptors (BCRs) (non-clustered cases) while the second is characterized by remarkable BCR stereotypy (clustered cases). In particular, 783/2,845 CLL sequences (27.5%) were placed in 339 ground-level clusters. Common sequences among these ground-level clusters allowed their progressive grouping in clusters at higher levels of hierarchy. High-level clusters were characterized by striking IGHV repertoire restriction, with only six IGHV genes (1–69, 1–3, 1–2, 3–21, 4–34, 4–39) accounting for >80% (382/459) of cases. While ground-level clusters provided a high-resolution picture of HCDR3 stereotypy, high-level clusters were considerably larger in size (up to 86 sequences each) and able to capture and describe more distant sequence relationships in the form of more widely shared sequence patterns. In particular, they were defined by patterns characterized by just a few critically positioned residues, reminiscent of the receptors expressed by cells participating in innate immune responses: due to this remarkable structural conservation, we consider them to be BCR “archetypes”. To test the hypothesis that sequence relatedness between IGHV genes may have structural and functional meaning for the IGHV repertoire in CLL, we also constructed sequence distance trees of functional human IGHV genes. Examination of the tree for the IGHV3 gene subgroup revealed a branching that was reflected in the repertoires of clustered vs. non-clustered CLL sequences. In particular, IGHV3-21, the foremost example of a gene with a propensity to be used in clustered rearrangements in CLL, belongs to a branch clearly distinct from other branches that include, for instance, the IGHV3-23, IGHV3-30, IGHV3-33 and IGHV3-7 genes, which were essentially absent from the repertoire of CLL sequences in high-level clusters; a similar case was also evident among IGHV1 subgroup genes. On these grounds, we argue that CLL cases with clustered (i.e. stereotyped) vs. non-clustered (i.e. heterogeneous) BCRs could derive from different progenitor cell populations evolutionarily adapted to particular antigenic challenges. In particular, prompted by the fact that clustered cases were found to express a limited set of highly conserved BCRs, we propose that in such cases the clonogenic progenitors may originate from a B cell population intermediate between a true innate immune system and the conventional adaptive B cell immune system, similar to what has been previously suggested for mouse B1 cells.

The Fcgamma receptors (FcγRs) are a family of molecules that modulate immune responses. FcγRIIb is an inhibitory FcγR that bears immunoreceptor tyrosine-based inhibitory motifs which transduce inhibitory signals on coligation with the surface membrane Ig of the B-cell antigen receptor (BCR). The role of FcγRIIb in controlling B cell activation through inhibition of BCR signaling has been extensively studied in animal models. Nevertheless, data on FcγRIIb are scant in human normal and neoplastic B cells, this being due to the lack of a specific antibody for human FcγRIIb. Consequently, there is little information on this receptor in chronic lymphocytic leukemia (CLL). Considering the activated nature of CLL cells and the central role of the BCR in the biology of the disease, studies of FcγRs are warranted. We used a novel specific mAb directly conjugated with Alexa 488 fluorophore that solely reacts with the human FcγRIIb (MacroGenics, Inc.) to investigate the receptors expression on CLL and normal human B cells. The study population included 84 patients with CLL and 24 age- and sex-matched controls. FcγRIIb expression was assessed as the mean fluorescence intensity (MFI) of surface membrane staining. In CLL cells, FcγRIIb was measured on CD19+CD5+ cells in combination with CD38, CD49d or CD69. Normal B cells were immunostained for CD19, CD5, IgD and CD38 expression and B cell subsets: naïve (IgD+CD38−), activated (IgD+CD38+) and memory B cells (IgD−CD38−) were studied for their relative expression of FcγRIIb. FcγRIIb expression was found significantly higher in naïve B cells compared to activated and memory B cells [median MFI: 17420 (11960–21180) vs. 11.140 (7899–16970) and 11.830 (6984–17100); p Although CD69 and CD38 expression was significantly higher on unmutated IGHV cases, no correlation was found between FcγRIIb levels and IGHV mutational status. Similarly, there was no correlation between FcγRIIb and other poor prognostic variables such as ZAP-70 (≥20%), CD38 (≥ 30%) or high risk cytogenetics. Nevertheless, cases with ≥ 30% CD49d+ cells had higher FcγRIIb expression than those with circulating normal CD5+ B cells > circulating CD5+ CLL B cells. In addition, although FcγRIIb is present on all normal B cell subsets its expression is higher in naïve B cells. Furthermore, in CLL FcγRIIb density is greater in CD38+ and CD49d+ cells within the clone. Although CD49d and FcγRIIb on CLL clones is linked in a direct manner, there is no relationship with FcγRIIb density and IGHV mutations, ZAP-70, CD38 and unfavorable cytogenetic markers. Finally, the relationship between FcγRIIb expression on CLL cells and functional responses to BCR and other receptor-mediated signals deserve further investigation.

Whether recent advances in the understanding and treatment of CLL are changing referral patterns of patients with this disease to specialized centers and their outcome is largely unknown. We analyzed demographics, disease characteristics, treatments administered, and survival over a period of 28 years in a series of 900 patients with CLL separated in three different cohorts based on the year of referral (table). As shown in the table, one of the main differences observed over the years relies on the stage of the disease, with most patients being currently seen in early phase of the disease, reflecting an earlier diagnosis because of analyses performed for routine reasons. In contrast, no differences were observed in patients’ age over the years, in agreement with the stable incidence of CLL in different age groups (SEER). No differences were detected in the proportion of patients developing autoimmune cytopenias, Richter’s syndrome or second neoplasias (data not shown). Not unexpectedly, since 1990 most patients receive purine analogs-based therapies. Importantly, the increasing proportion of patients who remain alive in each time period not only reflects an earlier diagnosis but also improvement in clinical management. Thus, median survivals of patients < 65 with Binet B or C disease are 3.9 yrs (1980–1989), 7.5 yrs (1990–1999) and not reached (2000 onwards), and the proportion of patients alive at 5 years in the same periods of time are 43%, 63% and 72% (figure). Unfortunately survival of patients > 65 yrs has not improved (median survival around 5.5 years across the three groups). Also, survival of patients in early stage (Binet A) has remained basically unchanged. From this large, single-institution study it can be concluded that the outcome of patients with CLL has been steadily improving since 1980. Nevertheless, the most significant progress has been made in younger patients with advanced disease, which constitute a small proportion of subjects with CLL. Besides to improving the outcome of these patients, future efforts should be aimed at prolonging survival of all patients, irrespectively of their age, and seeking the cure for the disease. 1980–1989 1990–1999 > 2000 P Number 254 388 258 Age (yrs). 67 (24–88) 66 (28–96) 67 (34–94) NS < 65 yrs (%) 43 46 45 > 65 yrs (%) 57 54 55 Males (%) 55 55 60 NS Binet A (%) 65 75 85 2 (%) 1.2 1.9 0 NS Treatment No treated (%) 59 37 60 8 NS PA = Purine analogs; Rit. = Rituximab

We examined SHM features in 1967 IGH rearrangements from 1939 patients with CLL. The sequences were divided into four “identity groups”; “truly unmutated” (100% identity to germline; 677 sequences), “minimally mutated” (99–99.9% identity; 133 sequences), “borderline mutated” (98–98.9% identity; 93 sequences) and “mutated” (

B-cell chronic lymphocytic leukemia (CLL), the most common adult leukemia, is a clonal disease manifested by an absolute lymphocytosis. Previously we identified genes capable of sorting and predicting the B-cells of patients with CLL or monoclonal B-cell Lymphocytosis (MBL) from aged matched controls. Using gene expression profiling, we and others have identified genes transcribed at significantly different levels between CLL patients and normal subjects. From these genes RT-QPCR verification was performed, in triplicate or more, on 22 gene candidates. A gene panel of seven genes, significantly different between CLL and normal individuals was chosen: FMOD, CKAP4, PI3Kc2b, LEF1, PFTK1, Bcl2 and GPM6a (in order of Receiver Operating Characteristic [ROC] concordance). This RT-QPCR verification step was based upon the comparison of freshly isolated PBMCs from CLL patients and age matched normal subjects after enriching for B cells with negative selection. The panel was then used to blindly categorize and predict the relationship of RNA from apparently normal subjects, some with and some without MBL, to the mRNA of patients with CLL as well as normal subjects, presumably without MBL. Since the numbers of cells within the expanded clones from individuals with MBL, after positive selection was limiting, RNA was amplified utilizing an RNA whole genome amplification approach and compared to similarly amplified RNA after negative selection; CLL cells, B-cells from normal age matched controls, and cord blood-derived B1 cells, enriched further by CD5 expression with positive selection. Using the Bayesian relevance determination method, a novel computer-learning tool to provide a probabilistic model, 56 samples were identified correctly at the rate of 100%. FMOD and PI3Kc2b were determined to be optimal at predicting blinded samples. In order to make the panel more clinically feasible, a universal reference RNA standard was used as a control, eliminating the need for pairing samples with age-matched controls. Analysis of this new model led to the discovery that a single gene; lymphoid enhancer binding factor 1 (LEF1), was not only capable of sorting CLL B cells from age-matched controls at a 100% rate (n=40) but it was also capable of predicting CLL B cells from other B cell malignancies (n=15). Verification of the protein expression levels for these genes is ongoing. The RNA expression levels of the genes in this panel provides a novel way to identify cell populations and gene expression patterns that change during the transition from B-cell clonal expansions that occur physiologically from those that occur among pre-leukemic and leukemic B-cell populations. The addition of a standard reference RNA enhances the clinical application of this gene panel. These data strongly suggest a unique role for LEF1 in CLL.

The absence of mutations in the VH gene (UM-VH) and the expression of ZAP-70 by leukemic cells are closely related adverse prognostic factors in CLL. Whether ZAP-70 should substitute VH gene analysis as prognostic predictor in patients with CLL is a matter of debate. We and others have shown that allogeneic but not autologous stem-cell transplantation may overcome the negative impact of the UM-VH on the outcome of patients with CLL. Herein we report on transplantation results based on ZAP-70 expression in leukemic cells. Thirty-seven patients (24 M/13 F; median age: 48 years, range: 29-63) received either an autologous (auto-SCT) (n=22) or an allogeneic (allo-SCT) (n=15) transplant. ZAP-70 was assessed by flow cytometry (19 cases), lymph node immunohistochemistry (10 cases) or both (8 cases). No discrepancies were observed in those cases in which both peripheral blood and lymph nodes were analyzed. Thirty-one patients were ZAP-70 positive and 6 ZAP-70 negative. ZAP-70 was positive in all UM-VH (n=24) and in 4 of 8 mutated VH patients. After a median follow-up of 62 months (range: 4 – 142), 5-year survival is 86% (66% to 100%) for allo-SCT and 50% (28% to 72%) for auto-SCT (p=NS), whereas 5-year disease-free survival (DFS) is 80% (54% to 100%) for allo-SCT and 37% (15% to 59%) for auto-SCT (p=0.03). Seventeen patients have relapsed: 16 of 31 of those being ZAP-70 positive and 1 of 6 of those ZAP-70 negative. In ZAP-70 positive patients the cumulative relapse rate at 5 years is 10% (2% to 65%) in the allo-SCT group and 53% (35% to 81%) in the auto-SCT group (p=0.04) (figure 1a). In addition, ZAP-70 positive patients submitted to allo-SCT have a trend for a longer DFS (76% at 5 years; 46% to 100%) than those receiving an auto-SCT (39% at 5 years; 15% to 63%) (p=0.07) (figure 1b). In summary, among transplanted patients those with ZAP-70 positive CLL have a better relapse rate and DFS if submitted to allo-SCT. Figure Figure

Insertions/duplications and deletions (I/D/Ds) in IG variable region genes are infrequent sequelae of the somatic hypermutation process. In the present study, we document the occurrence of nucleotide I/D/Ds in rearranged IGHV genes in CLL patients. Among 809 IGHV-D-J sequences amplified in 760 CLL cases, 9 (1.11%) and 15 (1.85%) sequences exhibited IGHV sequence changes consistent with nucleotide insertions/duplications or deletions, respectively. I/D/Ds were found in genes of the IGHV1, IGHV3 and IGHV4 subgroups, always within mutated IGHV-D-J rearrangements. In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. I/D/Ds were located completely or in part within CDRs in 21/24 cases; sequence motifs (AGY, AGA, AAC trinucleotides) that resemble intrinsic hotspots for somatic hypermutation were identified in 21/24 sequences. Short stretches with high homology (misalignment feet) that would offer the DNA polymerase an alternative template for re-annealing in cases of replication slippage were identified in all CLL sequences carrying I/D/Ds. I/D/Ds were generated somatically since (i) they always occurred in cases with somatic mutations, (ii) they could not be found on sequencing analysis of the corresponding germline IGHV genes amplified on DNA isolated from selected patients’ neutrophils, thus excluding a possible genetic polymorphism. The incidence of I/D/Ds in CLL is consistent with previous reports in normal, autoreactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease-specific associations. A striking exception to this rule was identified in the case of CLL IGHV3-21 expressing cases: one aminoacid was deleted from the CDR2 region in 16/74 (21.6%) IGHV3-21 CLL sequences (database-derived IGHV3-21 CLL cases + present series) vs. only 2/340 (0.59%) non-CLL IGHV3-21 sequences; 15/16 CLL IGHV3-21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3s and light chain CDR3 motifs. The effect of the CDR2 deletion on the structure of the IG molecules in this subset of CLL patients was studied with molecular modelling and dynamics simulations. The models suggested that the deletion could be accommodated without significantly affecting the local structure. The close association of deletions in CDR2 with the homologous subset of IGHV3-21 expressing CLL cases provides further evidence for the importance of an antigenic drive in malignant transformation and/or maintenance of the neoplastic clone in at least some CLL cases.

MRD detection has been associated with a higher relapse risk in patients with CLL having achieved complete response (CR) by conventional NCI-WG criteria. Although there are a number of techniques to evaluate MRD, their respective clinical value is still controversial. Against this background, we analyzed MRD, as assessed by consensus PCR, real time quantitative PCR (qPCR), and flow cytometry, in peripheral blood (PB) and/or bone marrow (BM) in 40 patients with CLL submitted to SCT (25 autologous and 15 allogeneic). Patients were considered MRD positive if a monoclonal pattern was obtained by consensus PCR, qPCR MRD levels were ≥ 10−5 or flow cytometry MRD levels were ≥ 10−4. 97.4%, 89% and 100% of the patients could be studied by consensus PCR, qPCR, and flow cytometry, respectively. One hundred sixty-four of 248 samples were MRD negative by consensus PCR. Among those, CLL cells were detected by qPCR and by flow cytometry in 47% (77/164) and 23% (39/164) of the cases, respectively, whereas all 84 PCR positive samples had detectable CLL cells by qPCR and flow cytometry. A good correlation was seen between MRD levels detected by flow cytometry and by qPCR (n: 254, r: 0.826; p