Immunoglobulin Heavy Chain Variable Gene Research Articles

Facteurs pronostiques utiles dans l’évaluation personnalisée d’un patient avec une leucémie lymphoïde chronique (LLC)

B cell chronic lymphocytic leukaemia (B-CLL) is the most frequent leukaemia, particularly in older patients. B-CLL is defined by the presence of an absolute lymphocytosis with a RMH scoring system above or equal to 4. Clinical staging of the disease has assisted in deciding treatment options and more recently the cytogenetic (del 17p13, del 11q22-q23), molecular expression of unmutated immunoglobulin heavy chain variable region genes (IGHV), proliferation markers (thymidine kinase or CD23s) and surrogate markers (CD38, ZAP-70) have assisted in further risk stratification. A high number of other prognostic factors (LPL, ADAM) have been recently described but they are not useful in routine. The improvement of the knowledge of B-CLL is the basis of an optimal management of patients with B-CLL, including individualized treatment decisions. La leucémie lymphoïde chronique (LLC) est une hémopathie fréquente, en particulier chez le sujet âgé. Elle se définit par la présence d’une lymphocytose sanguine absolue supérieure à 4 × 109/l avec à l’examen par cytométrie en flux une prolifération monotypique et un score RMH supérieur ou égal à 4. Quatre facteurs avec un impact fort sur le pronostic ont été identifiés : temps de doublement des lymphocytes (TDL) supérieur à 12 mois, profil non muté des gènes des chaînes lourdes des immunoglobulines (IGHV), thymidine kinase ou CD23 soluble sériques élevés, présence d’une del 17p13 ou del 11q22-q23. De nombreux autres facteurs ont été plus récemment décrits mais ne sont pas utilisés en pratique quotidienne comme facteur de substitution de l’étude du profil IGHV. L’amélioration des connaissances permet une meilleure prise en charge des patients avec une LLC, prise en charge individualisée dépendante du parcours du patient.

Shorter Telomeres Are Associated with the Unfavourable Chromosomal Aberrations Del 17p and Del 11q in Chronic Lymphocytic Leukemia (CLL)

BACKGROUND: In chronic lymphocytic leukemia (CLL) short telomeres were shown to be associated with mutational status, progression free survival (PFS) and overall survival (Damle et al., 2004). Chromosomal instability increases with shortening of telomeres. Recently, a relationship between telomere length and number of chromosomal aberrations has been shown if telomere length was investigated by quantitative real-time polymerase chain reaction (Tel-PCR) (Roos et al., 2008). The aim of the present study was to correlate average telomere length of individual cells measured by multicolor flow-FISH to established prognostic factors and genomic aberrations.PATIENTS and METHODS: Blood samples from 64 patients with CLL were analyzed. Flow cytometry was performed for quantification of ZAP-70 and CD 38 expression with a cut off at 20%. Immunoglobulin variable heavy chain (IGVH) genes were sequenced. An IGVH gene sequence with less than 98% homology with the corresponding germ-line sequence was considered to be mutated. Chromosomal alterations were investigated by fluorescence in situ hybridization (FISH) with the following gene probes: LSI 13q14, LSI 13q34, CEP 12, LSI 17p13, LSI 11q22–23. Copy number changes were also detected in 18 samples by SNP-chip analysis. The average length of telomere repeats at chromosome ends was measured by multicolor flow-FISH. Values of telomere length from CLL cells were correlated to values of telomere lengths of B lymphocytes from healthy age matched individuals (delta telomere length=Δtel).RESULTS: The average telomere length of the clonal B-cells was short. Patient samples from advanced Binet stages (B/C) had significantly shorter telomeres (Δtel −4.8 ± 1.0 kb) than patients samples from Binet A (Δtel −3.4 ± 1.2 kb, p=0.03). The average telomere length was significantly shorter for ZAP-70+ (Δtel −5.0 ± 0.5 kb) and CD38+ (Δtel −4.9 ± 0.7 kb) patient samples than for ZAP-70− (Δtel −2.4 ± 0.8 kb) and CD38− (Δtel −3.0 ± 1.0 kb) patient samples, respectively (p<0.005, p<0.005). IGVH unmutated CLL samples exhibited significant shorter telomere lengths (Δtel −4.8 ± 0.4 kb) than mutated samples (Δtel −2.8 ± 0.9 kb, p<0.005). Interestingly CLL samples harbouring del 17p and del 11q had significantly shorter average telomere length (Δtel −5.3 ± 0.2 kb, n= 8) than samples without these aberrations (Δtel −4.0 ± 1.2 kb; n= 56, p<0.005). Furthermore we found a tendency of an increase in the number of chromosomal aberrations detected by SNP-chip with shorter telomeres.DISCUSSION: We are able to confirm significant shorter telomeres in CLL samples with unfavourable prognostic factors like advanced Binet stage, positivity for ZAP-70 or CD38 and unmutated IGVH genes compared to their favourable counterparts. CLL samples with the chromosomal aberrations del 17p and del 11q are associated with bad clinical outcome. CLL with these aberrations of the present study demonstrated significantly shorter telomeres compared to cases without these abnormalities. Additional studies relating impact of telomere length on genomic instability detected by SNP-chip are ongoing.

Effects of Anti-Retroviral Drugs to Expression of Cell Surface Adhesion Molecules and Hypermutation of Immunoglobulin Heavy Chain Variable Gene of HIV-Related Lymphoma

It has been known that non-Hodgkin's lymphomas (NHL) are a major complication in human immunodeficiency virus (HIV) infection, and a major cause of death in HIV infected patients. Recently, it has been reported that highly active anti-retroviral therapy (HAART) declines the incidence of HIV-related lymphoma. In this study, we tried to make in vitro model of HIV-related lymphoma by Epstein-Barr virus transformation, and to examine the cellular and molecular characteristics of the HIV-related lymphoma cells derived from patients with HIV infection. PBMCs from 10 patients HIV infection and 10 HIV-negative normal individuals were obtained by Ficoll-Paque (Amersham Biosciences Corp. NJ) density gradient centrifugation. Lymphoblastoid cell lines (LCL) were obtained from each patient through transformation with Epstein-Barr virus (B95-8). Cells were cultured with RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (JRH Bioscences, Inc. Lenexa, Kansas), antibiotics, and L-glutamine and maintained at 37°C in an atmosphere containing 5% CO2. FACS analysis revealed the cells to be CD3−, CD4−, CD8−, CD5−, CD19+, CD20+, HLA-class I+, -class II+. After the establishment of LCLs, to obtain a highly purified population, CD19+LCLs were sorted on a FACS Vantage SE (Becton Dickinson, San Jose, CA). The purity of each CD19+LCLs population evaluated by FACS analysis was always higher than 98%. After growth advantage of LCLs, growth ability of the LCLs from HIV patients was examined by MTT assay. In results, there is no significant difference of growth ability between LCLs derived from HIV patients and those derived from normal individuals. Furthermore, the expression of cell adhesion molecules (CD11a, CD11b, CD18, CD50, and CD54) of LCLs was observed by FACS. The results showed that the expression of CD18 on LCLs derived from HIV patients was enhanced significantly in compared to those of normal LCLs. This phenomenon seems to be consistent with development of primary central nervous system (CNS) lymphoma. Furthermore, effects of anti-retroviral drugs to the expression of the adhesion molecules are examined. Somatic hypermutation of immunoglobulin heavy chain variable gene (IGHV) has been detected over 90% of HIV-related lymphoma. The effects of HAART to hypermuation of IGHV of LCL derived from HIV patients will be addressed.

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.

Immunoglobulin Gene Repertoire in Ocular Adnexa Lymphomas (OAL): Hints on the Nature of the Antigenic Stimulation

OALs arising in the ocular adnexae account for 15% of all extranodal lymphomas and are mostly represented by mucosa-associated lymphoid tissue (MALT)-type lymphomas (OAML). OAML have been described to be significantly associated, although with a variable geographic distribution, with Chlamydiophila psittaci (CP) infection, the etiologic agent of human psittacosis. Though the role played by Chlamydiae in OAML pathogenesis is also strongly supported by the observation of clinical remissions after bacterial eradication alone, the actual mechanisms have not been yet elucidated.Previous analysis of the Immunoglobulin Heavy Chain Variable (IGHV) genes in OAML showed a restricted repertoire with mutated genes and ongoing somatic mutations, thereby alluding to an origin from an antigen–experienced B cell, which has undergone antigen-selection; however, data on the potential antigenic elements stimulating OAML B lymphocytes are not available.In order to get hints on the potential ligands, we aimed at investigating the Immunoglobulin structure and, in particular, the molecular features of the antigen binding site (i.e. the HCDR3) of IG genes expressed by OAML. We analyzed the monoclonal IGHV-D-J rearrangements in a series of 30 OAML cases. IGHV3 subgroup genes predominated (24/30, 80%), followed by IGHV4 (4/30, 13.3%) and IGHV1 (2/30, 6,7%) subgroup genes, in keeping with the normal B cell IG gene repertoire. According to the 98% identity cut-off value, 23/30 sequences (76,7%) were defined as “mutated”, with a percentage of identity ranging between 89.3% and 97.7%, whereas the remainder (7/30 sequences; 23.3%) had “unmutated” IGHV genes. At individual gene level, IGHV3-23 and IGHV3-43 were the most frequently used genes (6/30 sequences, 20%, and 4/30 sequences, 13,3%, respectively); interestingly, the latter is rarely used in the normal repertoire. No significant correlations were found between IGHV gene usage and CP infection. HCDR3 length ranged from 9 to 30 amino acids (median 16) and 20/28 HCDR3 sequences showed an isoelectric point (pI) value >6.0, (in 10/28 pI>8.0), a feature shared with antibodies with anti–DNA reactivity. Shared epitope recognition was excluded by cluster analysis of HCDR3 sequences, also among OAML cases using the same IGHV genes. When we aligned HCDR3 sequences to a panel of 8214 unique IGHVD- J sequences from various types of normal, autoreactive or malignant B cell clones, we observed significant similarity between 3 OAML cases and 2 auto-reactive antibodies, present in rheumatoid arthritis and Sjoegren's syndrome, respectively, and 1 antibody with anti-thyroid peroxidase (TPO) reactivity. The latter case showed a HCDR3 similarity also with the IG gene of a Chronic Lymphocytic Leukemia case, a disease characterized by frequent expression of auto-reactive Igs. Interestingly, the same search performed using anti-Chlamydia antibodies did not show any significant similarity.In conclusion, these results suggest that OAML express a distinctive IG gene repertoire and may originate from B cells selected for their capacity to bind to auto-antigens in a similar way to what reported for gastric MALT lymphomas, where malignant B cells, instead of directly reacting against Helicobacter Pylori, are actually stimulated by tissue auto-antigens exposed during the inflammatory reaction, as confirmed by molecular analyses of antigen binding sites of the monoclonal IG. Our findings support the hypothesis that stimulation by auto-antigens, likely generated within an inflammatory background promoted by Chlamydial infection, may be the driving force also in OAML pathogenesis, rather than a direct action of the bacteria on B 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.

Indolent Mantle Cell Lymphoma (MCL): A Retrospective Detailed Clinical and Morphological Analysis of 21 Patients, with Histological, Cytological, Cytogenetic, Interphase Genetic, Immunoglobulin Gene, and Gene Expression Profiling Analysis.

MCL is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes expressing CD5 and with a genetic hallmark, the t(11;14) (q13;q32) translocation leading to the overexpression of cyclin D1, considered as the initial oncogenic event. However this entity may show a spectrum of morphological features broader than initially described. Clinically most of the patients have a poor prognosis with a rapid acquisition of chemoresistance. It has been recently described that some patients may follow an indolent clinical evolution, with the possibility of confusion in diagnosis because of overlapping morphological features with other small B cell lymphomas such as marginal zone lymphoma (MZL) and small lymphocytic lymphoma (SLL). The aim of our study was to analyze patients treated for a B cell lymphoma bearing the t(11;14)(q13;q32) who had a good outcome with a long survival. We retrospectively selected 21 patients (pts) with a t(11;14) B cell lymphoma and who followed an indolent outcome defined by an overall survival of more than 5 years after no more than 1 therapeutic regimen. Feature review included morphologic aspect, immunophenotype, cytogenetics, immunoglobulin (Ig) variable heavy chain (VH) gene usage and mutation patterns. Expression of CD5, CD10, CD23, and CD43, were evaluated on CD19+ cells by FCM. Cytogenetic analysis consisted in conventional cytogenetics on fresh tissue samples completed by a FISH analysis against 11q, 12p, 17p, and 18p realized on fresh frozen material. We further analysed the gene expression profiling of available samples using quantitative real-time PCR using microfluidic cards designed with selected genes of 3 independent signatures (Rosenwald A, et al. Cancer Cell 2003 - Ruiz-Ballesteros E, et al. Blood 2005 - Thieblemont C, et al. Blood 2004). Clinical characteristics showed that median age was 61 (range 45–81). All pts had a good PS and presented with a disseminated disease: bone marrow and blood involvement in 19 (90%) and 11/17 (65%) pts, respectively; peripheral and profound nodes involvement in 15 (71%); and splenomegaly in 13 (62%). LDH and b2-microglobulin levels were elevated in 4 pts. None of the pts had a monoclonal component. IPI score was low in 18 (86%) pts. Splenectomy alone or with chemotherapy (chlorambucil or fludarabine in 4; CHOP in 1) was proposed in 7 pts; monochemotherapy (chlorambucil or fludarabine) in 4 pts, CHOP +/− rituximab (R) in 2 pts, CHOP +/− R followed by autotransplant in 8. One pt did not receive any treatment. With a median follow-up at 6.44 years, median overall survival was 9.18 years. Morphologic review distinguished 2 groups of cases: true MCL cases (MCL, n = 8) and BorderLine Cases (BLC, n=13). Among the 8 MCL and 13 BCL, 7 and 12 were CD5+, 1 and 4 CD5/CD23+, 4 and 5 CD43+, respectively. All cases exhibited a t(11;14), associated with a complex caryotype in 5/8 (62%) MCL and in 9/13 (69%) BLC. Chromosome 7q deletion (n=1) and trisomy chr3/3q (n=3) were present only in BLC cases. IgVH gene was unmutated in 10 cases (3 MCL, 7 BLC) and mutated in 4 (2 MCL, 2 BLC). The most common VH families were VH1 (n=4), VH 3 (n=8) and VH4 (n=3). From the gene expression profiling studies we selected 70 genes specific of the diagnosis of MCL, MZL and SLL. A set of 5 MCL, 5 SLL, and 5 MZL control samples allowed us to validate 25 out of these 70 genes. These 25 genes were further used to analyse MCL and BLC samples. All these samples exhibited a more heterogeneous profile than control samples. Three MCL were more similar to MCL samples, whereas two were closer to MZL samples. All the BLC exhibited a profile close to that of MZL. We further analysed these samples with the 18 gene-signature predicting MCL survival. This signature could not discriminate survival of these MCL and BCL pts. In conclusion, pts with B cell lymphoma bearing the translocation t(11;14)(q13;q32) and with an indolent outcome can be distinguished in at least 2 groups with different characteristics in terms of morphology, immunophenotype, caryotype, and molecular profiles. Using a 25 gene-signature, we found profiles similar to either MCL or MZL for MCL samples, and similar to MZL for BCL samples. The signature discriminating the survival of MCL could not discriminate these indolent MCL. These findings will be validated on a larger series.

Single cell analysis of B lymphocytes from Wegener's granulomatosis: B cell receptors display affinity maturation within the granulomatous lesions

Increased amounts of anti-neutrophil cytoplasm antibody (ANCA) directed against proteinase 3 (PR3) are a diagnostic and pathogenic hallmark of full-blown Wegener's granulomatosis (WG). Aggregates of B lymphocytes proximal to PR3+ cells as well as plasma cells have been described as substantial components of Wegener's granuloma and could participate in forming tertiary lymphoid structures, which might promote autoantibody formation. Our aim was a molecular analysis of single B cells in order to develop a methodological approach that allows examination of potential ANCA formation in the tissue. Single B cells from cryo-conserved endonasal biopsies of three WG patients were isolated, using laser-assisted microdissection. Subsequently, their immunoglobulin variable heavy (VH) and light (Vkappa, Vlambda) chain genes were analysed by single cell polymerase chain reaction and direct sequencing. Sixteen immunoglobulin VH-Vkappa or VH-Vlambda chain gene couples were characterized. Twelve of these immunoglobulin gene couples resembled memory B cells. Two offsprings of one B cell were detected, indicating clonal expansion. VH genes representing 39 single B cells of WG tissues displayed significantly more mutations when compared with VH genes from peripheral blood of a healthy donor. The findings confirm and extend our previous results, arguing for an initial selection and affinity maturation of B cells within Wegener's granuloma. Further, the methodology provides the initial basis for the recombinant generation of antibodies derived from tissue cells.

Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia

AbstractLeukemia-cell expression of ZAP-70, CD38, or unmutated immunoglobulin heavy chain variable region genes (U-IGHV) each is associated with aggressive disease in patients with chronic lymphocytic leukemia (CLL). To assess the relative strength of each marker, we defined thresholds for designating a case as positive for CD38 or ZAP-70 in a test cohort of 307 patients and used these data-defined criteria to stratify patients in an independent cohort of 705 patients. Multivariable analysis revealed that ZAP-70 was the strongest risk factor. Knowledge of the IGHV mutation status or CD38 did not improve our ability to predict the time to first treatment except for ZAP-70–negative cases, which could be segregated into 2 groups of intermediate-risk or low-risk disease based on whether they expressed unmutated or mutated IGHV. ZAP-70 maintained its high relative prognostic value for the subset of patients with early-stage, asymptomatic disease, including patients evaluated within 1 year of diagnosis. Although it is premature to recommend therapy based on these risk factors, patients with ZAP-70–positive CLL cells should be monitored closely for disease progression as they have a median time from diagnosis to requiring initial therapy by standard criteria of approximately 3 years.

Systematic genomic screen for tyrosine kinase mutations in CLL

Systematic genomic screen for tyrosine kinase mutations in CLL

The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes

AbstractSrc family kinases (SFKs) were described to be overexpressed in chronic lymphocytic leukemia (CLL). We wished to examine the effects of the Src and Abl kinase inhibitor dasatinib on the intracellular signaling and survival of CLL cells. Dasa-tinib showed a dose- and time-dependent reduction of global tyrosine phosphorylation and of activating phosphotyrosine levels of SFKs. Treatment with 100 nM dasatinib led to decreased levels of the activated, phosphorylated forms of Akt, Erk1/2, and p38, and induced PARP cleavage through caspase activity. In Mec1 and JVM-3 cell lines, dasatinib increased p53 protein levels and inhibited proliferation. In freshly isolated CLL cells, dasatinib reduced the expression of Mcl-1 and Bcl-xL. Combination of 5 μM dasatinib and fludarabine increased the apoptosis induction of each by approximately 50%. In 15 primary CLL samples, cells with unmutated immunoglobulin variable heavy chain (IgVH) genes were more sensitive to dasatinib than those with mutated IgVH genes (P = .002). In summary, dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro, most prominently in samples obtained from patients with unfavorable prognostic features.

Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.

Oral fludarabine and cyclophosphamide as front-line chemotherapy in patients with chronic lymphocytic leukemia. The impact of biological parameters in the response duration

We tested the efficacy and safety of oral fludarabine and cyclophosphamide as front-line therapy in chronic lymphocytic leukemia (CLL) and assessed the influence of immunoglobulin variable region heavy chain (IgVH) gene mutation status, interphase cytogenetic abnormalities, and expression of ZAP-70 and CD38 on clinical outcome. Thirty-seven patients with previously untreated CLL received oral fludarabine (30 mg m(2)) and oral cyclophosphamide (250 mg m(2)) for three consecutive days every 4 weeks for six cycles. Eighteen patients had unmutated and 15 had mutated IgVH genes. Nine patients had the 'high risk' cytogenetic abnormality del(11q22.3) or del(17p13.1). Fifteen patients were ZAP-70-positive and eight patients were CD38-positive. Among the 35 valuable patients, 14 patients (40%) obtained a complete response and 13 (37%) a partial response. The median progression-free survival (PFS) was 23 months and median time to re-treatment (TTR) was 38 months. A significantly lower overall response rate (43% vs. 85%, p = 0.011), a shorter PFS (22 vs. 27 months, p = 0.015), and a shorter TTR (22 vs. 40 months, p = 0.031) were noticed in the 'high risk' cytogenetic abnormalities group; TTR was also shorter in IgVH-unmutated than in IgVH-mutated patients (26 vs. 41 months, p = 0.035). Hematologic toxicity included grade IV neutropenia (ten patients) and grade III/IV anemia (three patients). Gastrointestinal toxicity was mild and no patient required hospitalization. The oral combination of fludarabine and cyclophosphamide is an effective, safe, and well-tolerated regimen that, if confirmed with larger series, will be appropriate especially in patients with low risk biological parameters.

Spotlight

Spotlight

PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts

Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments.

Distinctive IgVH gene segments usage and mutation status in Chinese patients with chronic lymphocytic leukemia

Background and objectives The incidence of chronic lymphocytic leukemia (CLL) in Asian countries is lower than that in the Western ones, where CLL is the most common leukemia. It is a clinically heterogeneous disease, with survival ranging from a few months to decades. The mutation status of the immunoglobulin variable heavy chain (IgVH) gene has significantly improved prediction of the risk for disease progression. We investigated the frequency and mutation status of IgVH gene expression in Chinese patients with CLL. Methods IgVH gene segments usage and mutation status were investigated by multiplex RT-PCR, and the relationship between IgVH somatic mutation status and the expression of CD38 and ZAP-70 was analyzed in 65 CLL patients. Results Forty-five (69.2%) patients had mutated IgVH, and 20 (30.8%) had unmutated IgVH. The most frequently expressed VH gene family was found to be VH3 (47.7%) followed by VH4 (40%), VH1 (6.2%), VH2 (4.6%) and VH7 (1.5%), with no expression of VH5 or VH6 gene families. VH1-69 and VH3-21 which commonly overused in Western CLL were very low in our cohort. IgVH gene mutation status was significantly associated with the expression of CD38. Conclusions The frequency of IgVH gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. The expression of them may be simple and reliable surrogates for the identification of IgVH mutations.

Nonstochastic pairing of immunoglobulin heavy and light chains expressed by chronic lymphocytic leukemia B cells is predicated on the heavy chain CDR3

AbstractWe analyzed the immunoglobulin (Ig) variable heavy (IGHV) and variable light chain genes used by leukemia cells of 258 unrelated patients with chronic lymphocytic leukemia (CLL) found to express unmutated Ig heavy chains (IgH) encoded by a 51p1 allele of IGHV1-69 among 1846 CLL patients examined. We found each had at least 98% homology to an identified germline IGKV or IGLV gene. Within the 258 IgH, we identified heavy chain CDR3 (HCDR3) motifs encoded by certain unmutated IGHD and IGHJ genes with restricted reading frames. Frequent and restricted use of particular IGKV and IGLV genes revealed nonstochastic pairing of disparate Ig light chains (IgL) with IgH that had restricted HCDR3 motifs designated CLL69A, -B, -C, and -D. Eighty-six percent (19/22) of CLL cases that expressed motif CLL69B encoded by IGHD2-2/IGHJ6 had distinctive IgL encoded by IGKV1-39. Similarly, 83% (5/6) of samples with motif CLL69D encoded by IGHD2-2/IGHJ6 expressed IGKV3-11, 100% (25/25) with motif CLL69A encoded by IGHD3-16/IGHJ3 used IGKV3-20, and 77% (10/13) with motif CLL69C encoded by IGHD3-3/IGHJ6 expressed IGLV3-9. This study reveals nonstochastic pairing of IgH with particular IgL that is predicated upon Ig HCDR3 structure, providing compelling evidence for selection of antibodies expressed in CLL by conventional antigens.

Short telomeres are associated with genetic complexity, high-risk genomic aberrations, and short survival in chronic lymphocytic leukemia

Telomere length is associated with mutation status of the immunoglobulin heavy chain variable (IGHV) gene and clinical course in B-cell chronic lymphocytic leukemia (B-CLL). In a B-CLL cohort of 152 patients, we analyzed telomere length, genomic aberrations, IGHV mutation status, CD38 and ZAP-70 expression to study the prognostic impact and associations among these factors. An inverse correlation existed between telomere length and IGHV homology (P < .001), CD38 (P < .001), and ZAP-70 expression (P = .01). Patients with telomere lengths below median (ie, "short telomeres") and above median (ie, "long telomeres") had similar incidences of genomic aberrations (74% vs 68%), 13q- (57% vs 49%), and +12q (5% vs 12%). In contrast, 13q- as a single aberration was more frequent in patients with long telomeres (51% vs 21%; P = .006), whereas 11q- (27% vs 9%; P = .014), 17p- (17% vs 0%; P < .001), and 2 or more genomic aberrations (39% vs 8%; P < .001) were more frequent in patients with short telomeres. Compared with patients with long telomeres, treatment-free survival (TFS) and overall survival (OS) was significantly shorter (P < .001 and P = .015, respectively) in the group with short telomeres, and telomere length was an independent prognostic indicator for TFS. These observations have biological and prognostic implications in B-CLL.

Enzymatic activity of circulating proteasomes correlates with clinical behavior in patients with chronic lymphocytic leukemia

The ubiquitin-proteasome pathway has been implicated in the pathogenesis of many hematologic malignancies. The authors measured proteasome peptidase activity levels in plasma samples from 225 patients with chronic lymphocytic leukemia (CLL) and correlated the results with clinical behavior. By using fluorogenic kinetic assays, the enzymatic activity levels of 3 proteasomes were measured: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L). All activity levels were significantly higher in patients who had CLL compared with the levels in a control group of healthy volunteers (P<.001). Rai stage was correlated with Ch-L activity (P<.001) but not with Cas-L or Tr-L activity. Levels of beta2 microglobulin (B2M) were correlated with Ch-L activity (correlation coefficient [R]=0.4; P<.001) and with Cas-L activity (R=0.25; P=.001) but not with Tr-L activity. Cas-L activity as a continuous variable was a strong predictor of survival. Ch-L and Cas-L activity levels as categorical variables both were strong predictors of survival; Cas-L activity was independent of B2M level but not of immunoglobulin variable heavy chain gene (IgVH) mutation status. However, the combination of elevated B2M levels (>3.2 mg/L) and Cas-L activity (>1.32 pmoL/second/mL plasma) was associated with significantly shorter survival independent of IgVH mutation status. The current results indicated that measuring plasma proteasome activity has prognostic value in CLL that, when combined with B2M, can be independent of IgVH mutation status.

Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia

CD49d/alpha4-integrin is variably expressed in chronic lymphocytic leukemia (CLL). We evaluated its relevance as independent prognosticator for overall survival and time to treatment (TTT) in a series of 303 (232 for TTT) CLLs, in comparison with other biologic or clinical prognosticators (CD38, ZAP-70, immunoglobulin variable heavy chain (IGHV) gene status, cytogenetic abnormalities, soluble CD23, beta2-microglobulin, Rai staging). Flow cytometric detection of CD49d was stable and reproducible, and the chosen cut-off (30% CLL cells) easily discriminated CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (P<.001) and ZAP-70 (P<.001), or with IGHV mutations (P<.001), was independent prognosticator for overall survival along with IGHV mutational status (CD49d hazard ratio, HRCD49d=3.52, P=.02; HRIGHV=6.53, P<.001) or, if this parameter was omitted, with ZAP-70 (HRCD49d=3.72, P=.002; HRZAP-70=3.32, P=.009). CD49d was also a prognosticator for TTT (HR=1.74, P=.007) and refined the impact of all the other factors. Notably, a CD49dhigh phenotype, although not changing the outcome of good prognosis (ZAP-70low, mutated IGHV) CLL, was necessary to correctly prognosticate the shorter TTT of ZAP-70high (HR=3.12; P=.023) or unmutated IGHV (HR=2.95; P=.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL.