Heavy Chain Variable Gene Research Articles

Chronic Lymphocytic Leukemia Subset Expressing Mutated IGHV3-23 Has Peculiar Clinical and Biological Features.

Abstract 1256Poster Board I-278 IntroductionIn the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes. Patients and MethodsA HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells. ResultsIGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p<0.0001), compared with the distribution of IGHV genes belonging to stereotyped BCR clusters observed in our series. Although 109/134 IGHV3-23 were mutated (M), alignment of IGHV sequences revealed a high degree of conservation in the context of the 13 AA positions involved in superantigen binding by IGHV3 subgroup genes, suggesting that the majority of M IGHV3-23 cases maintained the capacity to mediate superantigen recognition and binding. Median TTT (73 months) of 43 M IGHV3-23 CLL was significantly shorter than median TTT (253 months, p=0.0153) of 333 M CLL, as well as of 326 M CLL in which 7 cases belonging to the bad prognosis IGHV3-21/IGLV3-21 cluster were excluded (253 months, p=0.0082). Multivariate Cox proportional hazard analyses selected IGHV3-23 usage (p=0.029), Rai stage (p<0.0001) and FISH group (p<0.0001) as independent markers of disease progression for 376 M CLL, and for the cohort in which 7 M CLL from the IGHV3-21/IGLV3-21 cluster were excluded. Comparing 5 M IGHV3-23 and 22 M non-IGHV3-23 CLL for their differential GEP, 212 genes were selected, 108 up-regulated and 104 down-regulated in M IGHV3-23 CLL. Using the “Gene-Ontology Tree Machine” platform, a set of growth/tumor suppressor genes (PDCD4, TIA1, RASSF5), all down-regulated in M IGHV3-23 CLL, was constantly found in several gene-ontology categories related to apoptosis. QRT-PCR confirmed a significant down-regulation of these genes in 15 M IGHV3-23 compared to 35 M non-IGHV3-23 CLL. Given the notion that PDCD4 and TIA1 are among the genes under control of miR-15a and miR-16-1 a “Gene Set Enrichment Analysis” carried out on the 212 differentially expressed genes, confirmed that M IGHV3-23 samples were significantly deprived in genes whose expression is under control of miR-15a and miR-16-1. Accordingly, QRT-PCR experiments performed on 15 M IGHV3-23 and 35 M non-IGHV3-23 CLL revealed significant higher levels of both miR-15a (p=0.0007) and miR-16-1 (p=0.0031) in M IGHV3-23 cases. No difference was found in the distribution of patients with 13q14 deletion between M IGHV3-23 CLL and M non-IGHV3-23 CLL (p=0.19). Considering the cases used for microRNA expression experiments (data available in 47/50 cases), 8/15 M IGHV3-23 CLL bore the 13q14 deletion in more than 20% of nuclei, against 19/32 cases in the group of M non-IGHV3-23 CLL (p=0.94). ConclusionExpression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1. DisclosuresNo relevant conflicts of interest to declare.

CD38 Induces Homing of Chronic Lymphocytic Leukemia Cells to the Lymphoid Organs through a Functional Interplay with CXCR4.

Abstract 2328Poster Board II-305Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature B lymphocytes expressing surface CD5, CD23 and low levels of immunoglobulin (Ig). Originating in the bone marrow (BM), neoplastic cells accumulate in blood (the circulating pool) and in lymphoid organs (the residential pool). Data obtained ex vivo and in patients suggest that the proliferative core of the disease dwells within the residential pool, while little or no proliferation is seen among circulating CLL cells. Threrefore, homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth is a critical step in disease progression.Human CD38 is a pleiotropic glycoprotein belonging to a complex family of genes coding for enzymes of the cell surface and involved in the catabolism of extracellular nucleotides. Along with the absence of mutations in the immunoglobulin variable region heavy chain gene (IgVH) and expression of the cytoplasmic kinase ZAP-70, CD38 expression above a critical threshold represents a reliable negative prognostic marker for CLL patients. The working hypothesis of our group is that CD38 is not merely a marker in CLL, but a cell surface receptor and adhesion molecule directly involved in the delivery of growth signals. Prolonged interactions between CD38 and its non-substrate ligand CD31 under static conditions increase survival of the CLL clone and sustain its proliferation in vitro. Here we show that CD38, is directly involved in chemokine-mediated homing of CLL cells. CD38 signals facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by the CXCL12 chemokine. This was first shown by testing functional responses to CXCL12 in a large cohort of clinically and molecularly characterized patients. The superior chemotactic performance of CD38+ CLL cells was confirmed by comparing the CD38+ and CD38- leukemic components within the same patient presenting with a bimodal clone. Assays using i) a specific inhibitor of CD38 enzymatic activities, ii) a panel of agonistic and blocking monoclonal antibodies (mAbs) and iii) microarray technology indicate that increased homing depends on the receptor functions of CD38. Indeed, interactions between CD38 and its non-substrate ligand CD31 define a genetic signature characterized by modulation of pathways involved in proliferation and migration of CLL cells. Conversely, blocking CD38 with domain-specific mAbs significantly weakens CXCL12 responses. This is attributed to physical proximity on the cell membrane between CD38 and CXCR4 (the CXCL12 receptor). The existence of a CD38/CXCR4 complex was demonstrated by i) co-immunoprecipitation, ii) confocal microscopy and iii) direct measurement of CXCL12 binding to its receptor. The relevance of CD38 in homing is substantiated by results from an in vivo mouse model, where CLL cells are allowed to home to lymphoid organs for a short period of time. The compromised homing ability of CLL cells to spleen and BM in the presence of blocking anti-CD38 mAbs confirms in vivo the in vitro evidence. Furthermore, the lack of effects induced by an isotype-matched mAb with the same anti-CD38 specificity convincingly rules out the possibility that the effects observed are secondary to sequestration of mAb-coated cells. Future studies will address the issue of whether CD38 may become a therapeutic target for selected CLL patients. Disclosures:No relevant conflicts of interest to declare.

Rituximab Combining with Fresh Frozen Plasma for the Treatment of Patients with Refractory Advanced CLL.

Abstract 3450Poster Board III-338Chronic lymphocytic leukaemia (CLL) is characterized by a progressive accumulation of monoclonal B lymphocytes in the bone marrow, lymphatic organs and blood. Even with the use of newer therapies, many patients with CLL develop progressive, treatment-resistant disease. Rituximab (RTX), a monoclonal antibody, targets CD20 on B lymphocytes and widely used in indolent B cell neoplasms. The lower density of CD20 on CLL cells may play a role, however, the causes of the lesser susceptibility of CLL to RTX remain poorly understood. Complement deficiencies have been identified in many CLL patients and appear to be more pronounced in patients with more advanced disease. Since complement mediated cell lysis is one of the major mechanisms of RTX action, we hypothesized that the therapeutic effect of RTX in CLL may be enhanced by the provision of complement through concurrent administration of fresh frozen plasma (FFP). Between August 2008 and April 2009, twelve patients with refractory advanced CLL were enrolled in this open clinical trial. All had been previously treated with fludarabine and two also failed treatment with RTX. The median age patients was 57 years (range, 48-82 years). According to the Binet staging system, 2 patients were in Binet B, 10 patients in Binet C. Florescence in situ hybridization (FISH) studies identified del(13q14) in 8 patients, and 4 patients with del(13q14) as the sole abnormality. Trisomy 12 was found in 3 patients, del(11q22.3) in 2 paitents, del(17p13) in 2 patients, del(6q23) in 1 patient, and IgH translocation in 1 patient, respectively. By flow cytometry, CD38 was positive in 9 patients, and ZAP-70 positive in 8 patients. Immunoglobulin heavy chain variable gene (IgVH) mutational status was detected by multiplex PCR, and six patients had unmutated VH. Two units of FFP followed with RTX 375 mg/m2 as a single agent, repeated every 1-2 weeks up to a total of 2-5 cycles. Complete response (CR) was achieved in 4 patients (33.3%), partial remission (PR) in 6 patients (50.0%). A major improvement of clinical signs and symptoms was achieved in all patients. Lymphocyte counts dropped markedly followed by shrinkage of lymph nodes and spleen and improvement of the anaemia and thrombocytopenia. This could be maintained over 9 months (range, 3-11 months) without progression. Toxicity was minimal and the treatment was well tolerated in all cases. It was show that RTX combining with FFP may provide a useful therapeutic option in patients with refractory advanced CLL. DisclosuresNo relevant conflicts of interest to declare.

Immunoglobulin heavy chain variable region gene usage and mutational status of the leukemic B cells in Iranian patients with chronic lymphocytic leukemia

The mutational status of the immunoglobulin variable region heavy chain genes (IGHV) is an important prognostic marker in chronic lymphocytic leukemia (CLL). The data accumulated in the literature has largely been derived from studies conducted on Caucasian Western populations. Little is known about Asian CLL patients. In this study the IGHV genes usage and somatic hypermutation status have been investigated in 87 Iranian CLL patients. Based on a cut-off of 98% nucleotide sequence homology, 64.4% and 35.6% of the patients expressed mutated and unmutated IGHV genes, respectively, with most non-progressive patients being in the mutated group (35/44 vs 19/40; P = 0.009). Progression-free survival (PFS) and time to first treatment (TTFT) were significantly higher in our mutated and non-progressive patients compared to unmutated and progressive subtypes, respectively. The most frequently used IGHV gene was IGHV3-7 (12.6%) followed by IGHV3-30 (11.4%), IGHV3-48 (9.2%), IGHV4-39 (6.9%), and IGHV1-8 (6.9%) genes, which taken together comprised nearly half of the IGHV genes expressed in the Iranian CLL patients. Of the IGHV genes, IGHV3-7 was significantly over-represented in non-progressive compared to progressive CLL patients (P = 0.036), whereas IGHV1-69 and IGHV1-2 were expressed at a higher frequency in unmutated compared to mutated CLL patients (P < 0.03). Comparison of IGHV gene usage in our patients with that of Western CLL patients revealed significant differences in expression of IGHV1-69, IGHV3-7, IGHV3-21, and IGHV4-34 genes. Analysis of the IGHV third complementary determining region (HCDR3) sequences revealed a high frequency use of certain HCDR3 motifs, such as YYYGMDV, in our samples. These findings imply contribution of antigen selection and regional (ethnic/geographic) parameters in the leukomogenesis of CLL.

Variation in WNT genes expression in different subtypes of chronic lymphocytic leukemia

The Wnt molecules are a family of secretory glycoproteins implicated in proliferation and differentiation of both normal and malignant cells. Despite extensive investigation of the WNT genes expression profile in various tumors, little is known about their expression in chronic lymphocytic leukemia (CLL). In this study, the expression profile of 14 WNT genes was investigated in a large number of Iranian patients with CLL. Semi-quantitative RT-PCR was performed on peripheral blood leukemic cells obtained from 62 patients with CLL. Peripheral blood mononuclear cells isolated from 11 age matched normal subjects served as control to determine baseline expression level of these genes. Our results have demonstrated significant up-regulation of WNT-3, WNT-4, WNT-5B, WNT-7B, WNT-9A, WNT-10A, and WNT-16B in patients with CLL compared to normal subjects (p < 0.05 to p < 0.0001). WNT gene expression analysis in different CLL subtypes showed a similar pattern of expression in progressive and indolent clinical subtypes. Over-expression of WNT-5A and WNT-9A genes was observed in patients with no mutation in their immunoglobulin (Ig) variable region heavy chain (Ig VH) genes compared to those with mutated Ig VH genes. Comparison between patients expressing VH1 (n = 9), VH3 (n = 40) and VH4 (n = 12) gene families, revealed down-regulation of WNT-3 and WNT-9A in VH3 positive patients. Our results indicate up-regulation of many members of the WNT gene family in CLL suggesting involvement of the Wnt canonical and/or noncanonical signaling pathways in CLL tumorigenesis.

IGHV Unmutated Status Influences Outcome More Than IGHV1-69 Gene Usage Per Se in Patients With Chronic Lymphocytic Leukemia

In this study, IGHV1-69 gene usage was detected in 46 out of 379 cases (12%) of chronic lymphocytic leukemia (CLL). In comparison with patients using alternative immunoglobulin heavy-chain variable (IGHV) genes, patients with IgHV1-69 CLLs more often presented at advanced stage, lacked somatic hypermutation (unmutated cases, 87% vs. 35%; P = .00001), and expressed unfavorable biologic characteristics. In 12 patients (26%), common amino acid motifs within the heavy-chain third complementarity-determining region were identified, allowing assignment to previously reported stereotyped subsets. In our study, treatment-free survival of patients with unmutated IGVH1-69 did not differ significantly from that of patients expressing unmutated alternative IGHV genes. As such, IGHV1-69 gene usage per se did not seem to be predictive of progressive disease, progression being primarily related to the unmutated IGHV profile.

In vitro activity of 20 agents in different prognostic subgroups of chronic lymphocytic leukemia – rolipram and prednisolone active in cells from patients with poor prognosis

There is a need for development of new drugs for treatment of B-cell chronic lymphocytic leukemia (CLL), especially for poor-prognostic subgroups resistant to conventional therapy. The in vitro antileukemic activity of 20 different anticancer agents was characterized in tumor cells from CLL, aiming at identifying agents active in poor-prognostic subgroups. In tumor cells from 40 CLL patients and in peripheral blood mononuclear cells (PBMC) from three healthy controls, the activity of 20 substances was assessed using a non-clonogenic assay. The CLL samples were characterized regarding genomic aberrations by interphase fluorescence in situ hybridization and immunoglobulin heavy-chain variable (IGHV) gene mutational status. In line with clinical experience, cells from patients with unfavourable genomic aberrations [del(11q)/del(17p)] showed lower drug sensitivity to fludarabine and chlorambucil than cells from patients with favourable cytogenetics [del(13q)/no aberration]. Most investigated drugs demonstrated similar activity in CLL cells from patients with unmutated and mutated IGHV genes as well as in CLL cells vs. PBMC. Interestingly, prednisolone and rolipram displayed high CLL specificity, high activity in CLL cells with unmutated IGHV genes and retained the effect in several cases with 11q/17p deletion. Further studies on prednisolone and rolipram revealed a synergy when these agents were combined in CLL cells, and suggested correlation between drug sensitivity and difference in downstream signaling. Prednisolone and rolipram are interesting for further studies in CLL with inferior prognosis. The study can also be considered a basis for future efforts to find drugs active in subsets of CLL patients that are resistant to conventional therapy.

Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1

Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.

Relative seroprevalence of human herpes viruses in patients with chronic lymphocytic leukaemia

Herpes virus infections may have a significant role in chronic lymphocytic leukaemia (CLL) due to their ability to modulate the host's immune system. We examined the seroprevalence of four herpes viruses [Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), human herpes virus (HHV)-6 and -7] in a cohort of European CLL patients (cohort 1, n = 100) in relation to the immunoglobulin variable heavy (IGHV) chain gene use and compared serological results with those obtained from age- and gender-matched healthy adults (n = 100). CMV-seroprevalence was significantly higher in CLL cohort 1 (79%) than in the control cohort (57%, P = 0.001); the seroprevalence of EBV (89% vs. 94%), HHV-6 (73% vs. 60%), or HHV-7 (35% vs. 35%) was not. In CLL cohort 1, use of IGHV3-30 was more prevalent among CMV-seropositive and of IGHV3-21 among HHV-7-seronegative cases. To investigate the generalizability of these findings, we investigated the herpes virus seroprevalence in a second cohort of age-matched CLL patients from a different geographical area (USA, n = 100, cohort 2). In cohort 2, CMV-seroprevalence was comparable with that of the control cohort (53%). Seroprevalence of EBV, HHV-6 and HHV-7 were 85%, 88% and 73% respectively. In CLL cohort 2, use of IGHV3-30 or IGHV3-21 was not associated with any of the herpes viruses investigated. CMV-seropositivity is associated with CLL in selected patient cohorts. However, the considerable variation in herpes virus-specific seropositivity between geographically distinct CLL cohorts indicates that seropositivity for any of the four human herpes viruses investigated is not generally associated with CLL.

Difference in the Relative Distribution of CD4+ T-cell Subsets in B-CLL With Mutated and Unmutated Immunoglobulin (Ig) VH Genes

B-cell chronic lymphocytic leukemia (B-CLL) is a clinically heterogeneous disease in which the clinical course is influenced by the presence or absence of immunoglobulin (Ig) variable heavy chain (VH) gene mutations. The poor clinical outcome of the subgroup with unmutated Ig VH genes has been linked to the persistent ability of the B-cell receptor in tumor cells from these cases to respond to antigen. As B-cell receptor signaling generally relies on T-cell help, we hypothesized that the course of B-CLL might not only be influenced by the Ig VH mutational status but also by the activation/differentiation status of T cells. We assessed the relative distribution of naive and memory T-cell subsets in peripheral blood from patients with mutated (M-CLL, n=71) and unmutated Ig VH genes (UM-CLL, n=42) and correlated it with the course of disease. We also compared the prosurvival potential of naive and memory T cells cocultured with B-CLL cells in vitro. A significant increase in relative numbers of central and effector memory T cells was observed in the CD4 T-cell pool from UM-CLL as compared with M-CLL cases and was associated with high Rai stage, progressive disease and shorter treatment-free survival (TFS). In a multivariate analysis, the relative number of CD4 central and effector memory T cells remained a significant prognostic parameter for TFS after correction for CD38 expression, Ig VH status, genomic aberrations, and Rai stage. The inverse correlation of memory CD4 T cells with TFS might be explained by their potential to support survival of B-CLL cells.

Clinical features and outcome of Chinese patients with monoclonal B-cell lymphocytosis

B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemias in the Western countries, however, infrequent in the Eastern. A diagnosis of CLL requires a count of B-lymphocytes ≥5.0×109/L. Asymptomatic person with <5.0×109/L B-lymphocytes is defined as monoclonal B-cell lymphocytosis (MBL). To compare the clinical characteristics, prognostic factors, and outcome of Chinese patients with MBL and CLL, we present a study from our single centre of 20 patients with MBL and 136 patients with CLL. The factors included: age at diagnosis, gender, direct antiglobulin test (DAT), immunoglobulin heavy chain variable gene (IgHV) mutational status, ZAP-70 protein, CD38 expression level, and molecular cytogenetic aberrations were analyzed in MBL and CLL subgroups. The Kaplan–Meier method was used to construct survival curves, and results were compared using the log-rank test. Patients in the MBL category were slightly older than in the CLL category. There was no significant difference of these clinical and biological characteristics between patients in MBL subgroup and early stage CLL (Binet A). The incidence of positive DAT was significantly increased in CLL patients at Binet B and C, compared with MBL (P=0.036). IgHV gene mutation in MBL is skewed, with more than 92.3% of subjects harbored mutated IgVH genes (P=0.025). The proportion of MBL patients with a 13q14 deletion or trisomy 12 was similar to that of CLL patients. Moreover, markers associated with poor prognosis (deletion of 11q22 or 17p13) in these MBL populations were less than those in Binet B and C CLL patients (P=0.025). No statistically significant differences in ZAP-70 and CD38 status were observed between the MBL and CLL subgroups. During a median follow-up period of 45.5 months, MBL patients had a low probability of progression, with no patients transformed to aggressive non-Hodgkin's lymphoma or dying of CLL-related causes. The overall survival of MBL was very similar to Binet A CLL, but longer than that of CLL patients at advanced stages (Binet B and C) (P=0.024). Our study demonstrated that a more indolent clinical course and superior clinical outcome for patients with MBL compared to CLL.

B-Cell Clones as Early Markers for Chronic Lymphocytic Leukemia

Otherwise healthy persons with a small number of B-cell clones circulating in the peripheral blood have been designated as having monoclonal B-cell lymphocytosis (MBL). Hospital-based series indicate an excess risk of progression from MBL to chronic lymphocytic leukemia (CLL). In this prospective cohort study, we tested the hypothesis that CLL is always preceded by MBL. Among 77,469 healthy adults who were enrolled in the nationwide, population-based Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial, we identified 45 subjects in whom CLL was subsequently diagnosed (up to 6.4 years later) through the collection of a peripheral-blood sample. Using six-color flow cytometry (with antibodies CD45, CD19, CD5, CD10, kappa, and lambda) and immunoglobulin heavy-chain gene rearrangement by reverse-transcriptase-polymerase-chain-reaction assay, we determined the association between MBL and subsequent CLL and characterized the immunoglobulin gene repertoire of the prediagnostic B-cell clones. On the basis of either flow-cytometric or molecular analysis, 44 of 45 patients with CLL (98%; 95% confidence interval [CI], 88 to 100) had a prediagnostic B-cell clone; in 41 patients (91%; 95% CI, 79 to 98), the presence of the B-cell clone was confirmed by both methods. The presence of immunoglobulin heavy-chain variable (IGHV) genes was determined in 35 of 45 prediagnostic clones (78%). Of these clones, 16 (46%) were IGHV3 subgroup genes (including 6 [17%] IGHV3-23 genes) and 9 (26%) were IGHV4 subgroup genes (including 4 [11%] IGHV4-34 genes). Furthermore, 27 of 35 of the IGHV sequences (77%) had mutations, with similar distributions after stratification either below or above the median time between the collection of the prediagnostic blood sample and the subsequent CLL diagnosis. In peripheral blood obtained up to 77 months before a CLL diagnosis, prediagnostic B-cell clones were present in 44 of 45 patients with CLL.

Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: results from an Italian multicentre study

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such 'stereotyped' BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22.4%) with stereotyped BCR were identified. Among 30 confirmed clusters (>or=3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these 'M clusters'. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.

BCL6: somatic mutations and expression in early-stage chronic lymphocytic leukemia

BCL6 somatic mutations affect normal and tumoral post germinal center B-lymphocytes. Our objective was to analyse expression, mutations and polymorphisms in the BCL6 gene and to correlate those variables with the clinical outcome in early-stage chronic lymphocytic leukemia (CLL). CLL samples were used for characterisation of the mutational status of BCL6/ immunoglobulin variable heavy chain (IGHV) genes, and expression of BCL6 was determined by real time PCR and immunoblot. Out of 68 cases, 29% show somatic mutations on BCL6 which occur exclusively in IGHV mutated cases. They are single nucleotide substitutions located mainly in two short mutational hot spots. CLL cells express different levels of BCL6 regardless of the mutational status, the number of mutations and polymorphisms. CLL cases expressing high levels of BCL6 have significantly shorter treatment-free interval. In conclusion, in early-stage patients with CLL, we found no correlation between expression and the mutations or polymorphism in BCL6, but high levels of BCL6 can discriminate patients with a worse prognosis.

The negative prognostic significance of positive direct antiglobulin test in Chinese patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western countries, however, it is infrequent in the Eastern countries. The direct antiglobulin test (DAT) may be positive at some time during the disease course of CLL. The aim of this study was to explore the prognostic impact of positive DAT at diagnosis in Chinese patients with CLL. In 123 Chinese patients with CLL, 34 (27.6%) patients presented with a positive DAT at diagnosis. However only 12 patients (9.8%) with a positive DAT developed autoimmune hemolytic anemia (AHA). According to the correlation analysis, advanced Binet stage (p < 0.001), high level of serum lactate dehydrogenase (LDH) (p = 0.003) and β2-microglobulin (β2-M) (p = 0.011), unmutated immunoglobulin heavy chain variable gene status (p < 0.001), positive ZAP-70 (p = 0.012), and trisomy 12 cytogenetic aberration (p = 0.004) emerged as factors significantly related to the occurrence of DAT-positive. Patients with positive DAT had significantly shorter survival times than patients with negative DAT (p = 0.009). Five-year OS percentages of DAT-positivity and DAT-negative patients were 72.8% and 97.5%, respectively. It was showed that DAT status might be applied for the assessment of prognosis in patients with CLL.

Errata

Microbiology and ImmunologyVolume 53, Issue 1 p. 58-58 Free Access Errata This article corrects the following: Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains Terri K. Wade, William F. Wade, Volume 52Issue 12Microbiology and Immunology pages: 611-620 First Published online: December 3, 2008 First published: 15 January 2009 https://doi.org/10.1111/j.1348-0421.2008.00110.xCitations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Microbiology and Immunology, the footnotes for Tables 2, 3 and 4 were incorrect.1 The correct table legends and their footnotes should be as follows: Table 2 Hybridomas investigated in the present study No footnote. Table 3 O1 LPS binding and vibriocidal capacity of the anti-O1 LPS mAbs ELISA (OD) and vibriocidal data were generated using a standard volume from supernatants of individual hybridomas that contained different concentrations of mAb. The effective concentration of mAb was calculated by dividing the concentration of mAb (μg/mL) by the reciprocal of the end-point dilution. The lack of vibriocidal activity was because IgA does not fix complement. Other isotypes (e.g. IgM, IgG1) of mAbs that fix complement that were not vibriocidal were also denoted as negative (neg). Table 4 Variable heavy chain gene usage (family member) of the anti-O1 LPS mAbs The V family usage was determined from the DNA sequences of the mAbs using the Ig BLAST program from NCBI (http://www.ncbi.nlm.nih.gov/igblast). The cDNA sequences for all the mAbs are available upon request. The specific family member for individual mAb that was assigned and the percent identity to that member are: mAb 22 (Vh9.1, 99.0%), mAb 23 (Vh5, 7183.a15.24, 99.3%), mAb 24 (Vh1, J558.8, 99.7%), mAb 26 (Vh5, 7183.a47.7, 96.2%), mAb 35 (Vh1, J558.47, 100%), mAb 36 (Vh1, J558.45, 99.7%), mAb 40 (Vh6, J606.4.82, 99.7%), mAb 47 (Vh5, 7183.a47.76, 99.0%), mAb 55 (Vh3, 36–60, 96.5%), mAb 58 (Vh1, J558.8, 99.7%), mAb 1 (Vh1, J558.29, 95.2%), mAb 6 (Vh11, 100%), mAb 9 (Vh5, 7183.a33.55, 95.6%), mAb 11 (Vh5, 7183.a37.59, 96.6%), mAb 16 (Vh9.1, 100%), mAb 18 (Vh3, 36–60, 92.5%) and mAb 19 (Vh5, 7183.a47.24, 96%). Reference 1 Wade T.K., Wade W.F. (2008) Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains. Microbiol Immunol 52: 611– 20. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume53, Issue1January 2009Pages 58-58 ReferencesRelatedInformation

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

BackgroundThe hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.Methods and FindingsHere we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.ConclusionsThe human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.

Co‐ordinated expression of lymphoid and myeloid specific transcription factors during B‐1b cell differentiation into mononuclear phagocytes in vitro

We previously demonstrated that B-1b cells can undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to substrate in vitro. Here we followed the expression of surface markers and transcription factors during this differentiation. B-1b cells spontaneously express both myeloid and lymphoid restricted transcription factors. When induced to differentiate into a phagocyte, the lymphoid genes E box protein (E2A), early B-cell factor (EBF), paired box 5 (Pax5) are down-modulated, while expression of genes related to myeloid commitment is sustained. Furthermore, B-1b cell-derived phagocytes (B-1CDPs) decrease immunoglobulin M (IgM) expression but retain the expression of the heavy chain variable gene VH11 or VH12, an immunoglobulin gene rearrangement predominantly expressed by B-1 cells. The maintenance of lymphoid characteristics in B-1CDPs characterizes a unique type of phagocyte, not related to monocyte-derived macrophages.

A Different Ontogenesis for CLL Cases Carrying Stereotyped Antigen Receptors: Molecular and Computational Evidence

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; andHCDR3 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.

ATM Gene Alterations in CLL Patients Induce Gene Profile Clusters and Predict Disease Progression.

Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients surviving for many years without requiring treatment, while others have rapidly progressing disease, require aggressive treatment and show a short life expectancy. Several biological features of the leukemic cells - the mutational status of the immunoglobulin (Ig) heavy (H) chain variable (V) genes, cytogenetic alterations, ZAP-70 and CD38 antigen expression, p53 dysfunctions – bear prognostic value and allow to stratify patients into risk categories. The deletion of the chromosome region 11q22–q23 that occurs in 10–20% of cases represents the second most common genetic abnormality in CLL and defines a subgroup of patients characterized by advanced progressive disease and poor survival. The ATM (ataxia telangiectasia-mutated) gene is located in this region and several studies suggest that the 11q deletion results in ATM gene inactivation. The ATM gene consists of 66 exons, 62 (5–66) of which are coding exons. The ATM protein is a nuclear serine/threonine kinase of 350 kDa induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and drugs. It protects the integrity of the genome in several ways: it regulates the arrest of the cell cycle at the G1/S and G2/M phases to prevent the processing of damaged DNA; it activates DNA repair pathways and induces apoptosis if the DNA damage cannot be repaired. In the present study we examined the status of the ATM gene by DHPLC (Denaturing High Performance Liquid Cromatography) in a series of untreated CLL patients studied at the Division of Hematology, “Sapienza” University of Rome. In addition, a multiplex gene dosage analysis of the ATM gene was performed by MLPA (Multiplex Ligation Probe Amplification) in all patients. The results obtained have been correlated with the molecular status of the IgVH genes, the presence of cytogenetic abnormalities, ZAP-70 and CD38 expression, p53 mutations, and with the gene expression profile. Mutational screening of the ATM gene identified mutations in 8 of the 57 CLL patients studied (14%): 6 missense, 8095C>T (Pro2699Ser), 8071C>T (Arg2691Cys), 2476A>C (Ile826Leu) and, in 3 patients, 1435G>T (Asp479Tyr); 1 frameshift: 2502insA; 1 splicing mutation: IVS29+5G>A. In addition, 14 different variants and polymorphisms of unknown functional significance were found. The 8 ATM mutated cases showed the following features: 6 were IgVH unmutated, 4 were ZAP70+, 5 were CD38+, 4 harbored a 13q14 deletion, only 1 patient showed a 17p13 deletion and 2 a 14q32 deletion; furthermore, 2/8 patients showed a p53 gene mutation; none showed the 11q deletion. MLPA analysis identified 6 patients with 11q deletion and all of them showed at least 20% of cells positive for the 11q23 deletion evaluated by FISH; only 1 case showed a 17p13 deletion. In these cases, the IgVH gene was always unmutated and ZAP70 was positive; 4 cases were also CD38+. ATM gene mutated and deleted CLL cases showed a peculiar gene profile characterized by the up-regulation and down-regulation of genes involved in apoptosis and DNA repair function. On the contrary, no gene profile difference was found in patients carrying ATM gene polymorphisms. Patients with ATM gene mutated and deleted showed a short time to treatment, 23 and 19 months respectively. The results of this study indicate that ATM gene alterations - mutations or deletions - account for 24% of untreated CLL patients, are associated with a defined gene profile and are characterized by a poor prognosis. It is important to underline that these patients have been evaluated before any treatment and that ATM mutations have been found in the absence of 11q23 deletions. ATM alterations appear to represent the most frequent genetic abnormality with unfavorable prognostic implications in CLL and should be investigated prior to initiating therapy in order to plan an appropriate treatment algorithm.