Heavy Chain Variable Gene Research Articles

Chronic Lymphocytic Leukemia (CLL) with Discordance in ZAP-70 Expression and IgVH Mutation Status.

In CLL patients, ZAP-70 expression is associated with an unmutated status of the variable immunoglobulin heavy chain gene (IgVH) region. Both features bear an adverse prognostic value. However, in various published series there is a proportion of cases with discordance in the expression of these two markers (i.e. ZAP-70-/IgVH unmutated or ZAP-70+/IgVH mutated) ranging from 5 to 30%. In order to clarify the outcome of this subgroup of patients, information on the clinico-biological features of the discordant cases is becoming increasingly more relevant. From November 2002 to April 2006, we evaluated ZAP-70 and CD38 expression, IgVH mutation status and cytogenetic aberrations by FISH in 125 young and untreated patients: 69 males, 56 females, with a median age of 51 years (range 29–64). According to Binet's staging system, 81% were stage A, 15% stage B and 4% stage C. Eighty % of patients presented stable disease and 20% progressive disease. After a median follow-up of 40 months (range 3–191) from diagnosis, 32% of cases have required therapy. ZAP-70, evaluated by immunocytochemistry and flow-cytometry, was positive (≥7%) in 36% of cases, IgVH genes were unmutated (≥98% homology) in 25% and CD38 was positive (≥7%) in 18%. The correlations between ZAP-70/IgVH, CD38/IgVH and CD38/ZAP-70 were highly significant (p≤0.001 each) with a proportion of discordant cases of 21%, 16% and 24%, respectively. Focusing on 114 cases with available data for both ZAP-70 and IgVH mutation status, three groups were identified: 23 ZAP-70+/IgVH unmutated, 67 ZAP-70-/IgVH mutated, 24 discordant cases. Significant differences were found in terms of distribution of cytogenetic aberrations, CD38 expression and atypical lymphocyte morphology (Table I). Only 2 cases showed V3-21 usage: both were IgVH mutated, 1 was ZAP-70+ and the other ZAP-70-. Of the 24 discordant cases, 18 (75%) were ZAP-70+/IgVH mutated and 6 (25%) ZAP-70-/IgVH unmutated. No significant difference was shown between the two groups regarding the presence of poor risk genetic abnormalities del(17p), del(11q) and +12 (p=0.17), CD38 expression (p=0.2), atypical lymphocyte morphology (p=0.12). Although the follow-up is still short, ZAP-70 and IgVH status significantly predicted treatment-free interval (TFI), individually (p<0.01 and p<0.0001) and in combination (p<0.0001). Discordant cases showed a TFI similar to the better prognosis group, ZAP-70-/IgVH mutated (p<0.66). Thus, CLL with discordant features regarding ZAP-70 and IgVH status are mostly ZAP-70+/IgVH mutated, have a low incidence of poor risk genetic abnormalities del(17)p/del(11q) and a relatively long TFI, suggesting that the mutational status is more relevant than ZAP-70 in the clinical outcome of this category of patients.Table IBiological features of 90 concordant and 24 discordant cases for ZAP-70/IgVH mutation statusTotal (114)*ZAP–70+/IgVH unmutated (23)Discordant (23)ZAP–70–/IgVH mutated (67)pN (%)N (%)N (%)N (%)Del(17p)7 (6)5 (23)1 (4)1 (1.5)<0.01Del(11q)10 (9)7 (32)1 (4)2 (3)<0.001+1210 (9)1 (4)5 (21)4 (6)<0.1Norm28 (25)5 (23)7 (29)16 (25)<1Del(13q)56 (50)4 (18)10 (42)42 (64.5)<0.001CD38 pos (>7%)21 (18.4)12 (52)8 (33)1 (1.5)<0.001Atypical morphology23 (20)9 (39)6 (25)8 (12)<0.025* FISH results not available in 3 cases

Dipeptidyl Peptidase 2 - A Pro-Survival Molecule in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a unique malignancy characterized by persistent accumulation of quiescent B-cells. Disruption of survival pathways leads to apoptosis and renders CLL cells sensitive to chemotherapeutic agents. As clinical outcomes in CLL are heterogeneous, it is important to better predict prognosis in each individual case of the disease. In this respect, detection of somatic mutations in the immunoglobulin variable heavy chain (IgVH) genes has become the gold standard. Overexpression of ZAP-70 (Zeta-chain associated protein kinase - 70 kDa) by CLL cells correlates with unmutated IgVH genes and predicts poor outcome in B-CLL. Dipeptidyl Peptidase 2 (DPP2) is a newly discovered serine protease which maintains lymphocytes in a quiescent state (G0). In this study we investigated whether DPP2 is involved in cell cycle control in B-CLL. 38 patients with B-CLL and 20 healthy controls were included in the study. Median age of CLL patients was 67 years. Median time from diagnosis to enrollment in the study was 102 months. 21 patients (55.3%) received treatment before enrollment in the study. Standard Ficoll-Hypaque techniques were used to isolate peripheral blood mononuclear cells (PBMC) from healthy donors and CLL patients. CLL cells were isolated to >98% purity by means of a MoFlo using CD19−specific antibodies. Cells were treated with Val-boro-Pro, a pan-inhibitor of DPP, or with DPP2-specific inhibitor (DSI), incubated for 16 hours and stained with propidium iodide and Annexin V. Expression of CD38 was assessed by flow cytometry, DPP2 and ZAP-70 - by real-time reverse-transcription polymerase chain reaction, Bcl-2 and p27 - by western blot analysis.We report that CLL B-cells expressed higher levels of DPP2 mRNA than normal B-cells. Inhibition of DPP2 in PBMC with VbP or DSI resulted in caspase-dependent apoptosis. In individuals with CLL, death of B-lymphocytes (and rescue by caspase inhibitors) was observed in 22 cases (57.9%). In the remaining 16 cases (42.1%) malignant B-cells did not undergo apoptosis upon inhibition of DPP2 with either VbP or DSI. We found that CLL cells resistant to DPP2 inhibition-induced apoptosis expressed higher levels of ZAP-70. Meanwhile, protein levels of p27 (cell cycle inhibitor) were decreased in resistant CLL. These patients exhibited worse disease prognosis such as shorter treatment-free period (p<0.001), frequent episodes of treatment failure and faster disease progression. Between the two groups, we identified no differences in expression of Bcl-2. CLL B-cells express higher levels of DPP2, a serine protease involved in maintenance of G0 which may serve as a survival factor in CLL B-cells. Resistance vs. susceptibility to DPP2 inhibition-induced apoptosis can be employed as a prognostic factor in CLL.

Analysis of Immunoglobulin Gene Mutation Status in Lymphoplasmacytic and Splenic Marginal Zone Lymphomas with Bone Marrow Infiltration.

Splenic Marginal-Zone lymphomas (SMZL) and Lymphoplasmacytic lymphomas (LPL) correspond to entities that shared clinical, morphological and immunohistochemical features and are not always easy to discriminate. Immunoglobulin variable heavy chain gene (VH) usage and somatic mutation pattern were analyzed to clarify the relationship and the pathogenesis of these 2 entities.Bone marrow frozen samples from 27 patients, 16 LPL and 11 SMZL, were studied. B-cell clonality was first analysed using the standard PCR method with the Biomed 2 primers. Informative PCR products were then purified, sequenced and analyzed on IMGT and Ig-Blast websites.Overall, median rate of somatic mutations was 9% in LPL and 4% in SMZL (p=0.013).All cases of LPL were mutated. Mutation rate was always higher than 5%. A preferential usage of VH3 gene was observed in 13/16 cases (80%), with a predominance of VH3–23 and VH3–74, both representing 60% of cases. VH4 gene was used only in 2/16 cases (13%). Regarding JH usage, a predominance of JH4 gene rearrangement was observed in 11/16 cases (69%). JH5 and JH6 were used in 20% and 13% of cases respectively.In the group of SMZL, mutation rates and usage of VH and JH genes were more heterogeneous. Thirty six percent of SMZL were unmutated and 19% had a mutation rate lower than 5%. VH3 was used in 4/11 cases (36%), VH4 in 3/11 cases (27%) and VH1 in 4/11 cases (36%). JH4 and JH6 were used in 4/11 cases (36%) and the JH5 in 1/11 cases (9%).Therefore, the profile of somatic mutations and the VH and JH segment preferentially used are different in SMZL and LPL, suggesting a different process of antigen-driven selection and of transformation of the initial B-cell clone. These findings consolidate the existence of LPL as a genuine entity, different of SMZL despite their histopathological overlaps.

ZAP-70 expression, as detected by immunohistochemistry on bone marrow biopsies from early-phase CLL patients, is a strong adverse prognostic factor

Zeta-associated protein-70 (ZAP-70), mostly assessed by flow-cytometry (FC), recently emerged as reliable prognostic factor in chronic lymphocytic leukaemia (CLL) at presentation. We evaluated ZAP-70 expression in 156 CLL patients by immunohistochemistry (IHC) on formalin-fixed bone marrow (BM) biopsies at diagnosis. At presentation, 117 patients (75%) were with Binet stage A, 27 (17%) stage B and 12 (8%) stage C. Median follow-up was 61 months (range 6-242). ZAP-70 was expressed in neoplastic lymphocytes of 69 patients (44%). Concordance between ZAP-70 by IHC and ZAP-70 by FC, immunoglobulin heavy chain variable genes (IGHV) mutational status and CD38 expression was found in 41/46 (89%), 41/49 (80%) and in 60/88 (68%) tested cases, respectively. ZAP-70 expression significantly correlated with advanced Binet stage (B-C), diffuse BM infiltration, increased lactate dehydrogenase (LDH) and beta2-microglobulin serum levels and lymphocyte doubling time <12 months. ZAP-70 positivity was significantly related to poorer time to progression (median 16 months vs 158 of ZAP-70-negative cases) (P<0.0001) and overall survival (median 106 months vs not reached) (P=0.0002); this correlation was confirmed at multivariate analysis. ZAP-70 expression correlated with poorer outcome also when evaluated only in the 117 stage A patients. In conclusion, immunohistological detection of ZAP-70 on formalin-fixed BM biopsies at diagnosis appears a useful methodological approach to identify patients with poor prognosis in CLL.

Possible Role for Mutations in the Transactivation Domain of C-MYC in the Clinical Transformation of Follicular Lymphomas.

Introduction: Follicular lymphoma (FL) is the second most prevalent non-Hodgkins lymphoma (NHL) in the United States. Although the clinical course is usually indolent, about 50% of low-grade FL transform to an aggressive lymphoma within 15 years of diagnosis. Somatic hypermutation (SHM) is a normal process in B cells which targets the immunoglobulin locus and introduces point mutations and small insertions/deletions. In diffuse large B cell lymphoma, the SHM misfires and aberrantly targets multiple loci, including some proto-oncogenes (PIM-1, PAX-5, RhoH/TTF and C-MYC).Hypothesis: We hypothesized that acquired mutations in the same proto-oncogenes could be responsible for the clinical transformation of follicular lymphoma.Materials and methods: The clinical database of the Department of Pathology, Washington University School of Medicine, St. Louis was searched to find patients with serial biopsies and the diagnosis of either stable (sFL) or transformed follicular lymphoma (tFL). Thirty-five (35) formalin-fixed paraffin-embedded (FFPE) tissue samples from 17 patients were identified. Eleven of seventeen patients had transformed to large cell lymphoma and six had stable low-grade follicular lymphoma. To exclude the possibility of a second unrelated lymphoma the common clonal origin of the original and follow-up lymphoma was confirmed by comparing immunoglobulin heavy and/or light chain variable gene sequences. Previously identified mutational hot spots in four genes, BCL-6, PAX-5, RhoH/TTF and C-MYC, were amplified and sequenced directly.Results: Fifty-five mutations were found in the four genes sequenced; all patients had at least one mutation. The number of mutations was similar in both the low grade and the transformed specimens overall, and most paired specimens had identical mutations or a single mutation change over time. Of the mutations identified, only mutations in Myc exon 2 correlated with the transformation. Acquired missense mutations were detected in the trans-activation domain of Myc in two of the eleven transformed FL specimens but none of the 24 low-grade follicular lymphoma specimens. The first case had two mutations: Pro57 to Ser57 and Glu39 to Asp39. The second case had a single Glu39 to Asp39 change. One of the mutations (Pro57 to Ser57) is close to a functionally critical phosphorylation site, Thr58, and has been reported in both Diffuse large B cell and Burkitt's lymphomas. The other mutation (Glu39 to Asp39) has also been reported in both Burkitts and DLBCL cases, and is not reported as a normal variant.Conclusion: There is not an accumulation in the overall number of SHM with transformation as the frequency of SHM is similar in low-grade FL and transformed FL. However, mutations in the trans-activation domain in c-Myc, caused by an aberrant SHM, may play a role in the transformation of low-grade FL to a diffuse, large cell lymphoma in a subset of patients.Somatic Hypermutation Analysis of 17 serial Follicular Lymphoma cases (35 specimens)Gene Name% Specimens with SHMMutation frequency per 100 bpNumber of unique single base pair substitutionsNumber of unique deletions or insertionsNumber of substitutions targeting G+C vs A+TBCL-663%0.1425316 vs 9PAX-543%0.06727 vs 0Rho/TTF17%0.03821 vs 7C-MYC31%0.02616 vs 0All genes83%NA47831 vs 17NA = Not applicable

Analysis of immunoglobulin heavy and light chain variable genes in post‐transplant lymphoproliferative disorders

Post-transplant lymphoproliferative disorders (PTLD) derive from antigen-experienced B-cells and represent a major complication of solid organ transplantation. We characterized usage, mutation frequency and mutation pattern of immunoglobulin variable (IGV) gene rearrangements in 50 PTLD (polymorphic PTLD, n=10; diffuse large B-cell lymphoma, n=35; and Burkitt/Burkitt-like lymphoma, n=5). Among PTLD yielding clonal IGV amplimers, a functional IGV heavy chain (IGHV) rearrangement was found in 40/50 (80.0%) cases, whereas a potentially functional IGV light chain rearrangement was identified in 36/46 (78.3%) PTLD. By combining IGHV and IGV light chain rearrangements, 10/50 (20.0%) PTLD carried crippling mutations, precluding expression of a functional B-cell receptor (BCR). Immunohistochemistry showed detectable expression of IG light chains in only 18/43 (41.9%) PTLD. Failure to detect a functional IGV rearrangement associated with lack of IGV expression. Our data suggest that a large fraction of PTLD arise from germinal centre (GC)-experienced B-cells that display impaired BCR. Since a functional BCR is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC reaction. The high frequency of IGV loci inactivation appears to be a peculiar feature of PTLD among immunodeficiency-associated lymphoproliferations.

Comparison of bone marrow and peripheral blood ZAP‐70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia

: The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. : We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19(+) B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t-test. : A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. : These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. (c) 2006 International Society for Analytical Cytology.

Analysis of the young and elderly variable gene repertoire in response to pneumococcal polysaccharides using a reconstituted SCID mouse model

Significant changes in anti-pneumococcal polysaccharide (PPS) variable gene usage occur with aging and may be influenced by changes in cytokine environment. Severe combined immunodeficient (SCID) mice were engrafted with B cells obtained from young and elderly donors, supplemented with human cytokines and immunized with the pneumococcal polysaccharide vaccine. B cells specific for PPS serotypes 4 and 14 were isolated from mice and immunized donors, and variable region sequences analyzed. Significant differences in variable heavy and light chain gene usage were observed between young and elderly adults despite a more constant cytokine environment. Due to the limitations of the hu-PBL-SCID model, the use of alternative systems would be beneficial in the elucidation of mechanisms underlying the reduced vaccine efficacy in the elderly.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations inNicotiana tabacumL.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

Detection of weakly interacting anti‐carbohydrate scFv phages using surface plasmon resonance

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.

Amplification and Analysis of cDNA Generated from a Single Cell by 5′-RACE: Application to Isolation of Antibody Heavy and Light Chain Variable Gene Sequences from Single B Cells

The technique of 5'-rapid amplification of cDNA ends (5'-RACE) is widely used to amplify unknown sequences at the 5' end of a messenger RNA (mRNA). However, conventional 5'-RACE is inappropriate for producing cDNAs from a single cell due to the small quantity of mRNA present in one cell. In this study, we report an improved 5'-RACE method that is suitable for generating cDNA from a single cell. In this method, the first-strand cDNA was directly synthesized from a single cell, and both the tailing reaction and second-strand cDNA synthesis were performed in the same tube without purifying the cDNA sample. Using this method, we were able to amplify the cDNA of the immunoglobulin (Ig) variable region gene from more than 50% of single B cells. The amplified cDNA fragment contained a full-length Ig variable region including a 5'-untranslated region, a leader sequence, and an initiation codon. This method may thus be applicable for a comprehensive analysis of the Ig variable genes of the lymphocyte repertoire in humans and animals, thereby contributing to the development of antibody-based therapeutics for infectious diseases.

Disparate roles of ATR and ATM in immunoglobulin class switch recombination and somatic hypermutation

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal “blunt end-joining,” but impaired end joining with partially complementary (1–3 bp) DNA ends. There was also an increased usage of microhomology at the μ-α switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (≥10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia–mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8-associated polyclonal body cavity effusions that mimic primary effusion lymphomas

Department of Clinical and Experimental Medicine, Amedeo Avogadro University of Eastern Piedmont, NovaraDear Sir,Kaposi’s sarcoma-associated herpesvirus/human herpesvirus8 (KSHV/HHV-8) is involved in the pathogenesis of Kaposi’ssarcoma (KS) and 2 lymphoproliferative diseases, primaryeffusion lymphoma (PEL) and the plasmablastic variant ofmulticentric Castleman’s disease (MCD). PEL is defined as aliquid-phase lymphoma harboring monoclonal rearrangementsof immunoglobulin (Ig) variable genes or, more rarely, of T-cell receptor (TCR) genes. As a rule, these elements are usedfor PEL diagnosis together with the demonstration of HHV-8infection of the tumor clone. However, as pointed out by Bou-langer and colleagues in the 1 July 2005 issue of the Interna-tional Journal of Cancer,

A robust ratio metric method for analysis of Zap‐70 expression in chronic lymphocytic leukemia (CLL)

Since Zap-70 expression in chronic lymphocytic leukemia (CLL) cells correlates with a lack of somatic mutation of the immunoglobulin variable heavy chain (IgVH) genes, it has been proposed as a surrogate marker for disease prognosis. However, published studies of Zap-70 expression have used different commercial antibodies and analytic strategies. This study was undertaken to determine if any strategy was broadly applicable in a clinical flow cytometry laboratory. Expression of Zap-70 was determined in 37 CLL patients using four different commercial antibodies. T, NK, and CLL cells were identified by immunophenotyping along with Zap-70 expression. Data was analyzed in terms of both percent of CLL cells expressing Zap-70 and the ratio of Zap-70 expression in CLL cells compared to that in T + NK cells. Three Zap-70 antibodies showed wide ranges of Zap-70 expression as a percentage of tumor cells, while a fourth gave consistently elevated results. Comparing the percent Zap-70 expression with any two antibodies gave poor correlations (r(2) = 0.45-0.63). Our results indicated that the previous analytical strategies were not reproducible. A ratio metric is proposed, which gave better correlations (r(2) as high as 0.95) and would allow separation of CLL patients with elevated or decreased Zap-70 expression.

Silencing of APAF‐1 in B‐CLL results in poor prognosis in the case of concomitant p53 mutation

Apoptosis protease-activating factor 1 (APAF-1), a transcriptional target of p53, is a cytosolic adaptor protein that links the mitochondrial apoptosis pathway to the caspase cascade. Here, we aimed to study the impact of APAF-1 expression levels on cell death induced by anticancer drugs or ionizing irradiation (IR) and disease prognosis in B-type chronic lymphocytic leukemia (B-CLL) patients. Samples from 138 patients with B-CLL were investigated for APAF-1 expression and p53 mutations. The results were related to survival data, in vitro cytotoxicity of various cytotoxic drugs and IR and clinico-pathological data. Variable APAF-1 expression was observed in all investigated B-CLL samples. Reduction in APAF-1 expression was observed at both mRNA and protein level indicating transcriptional silencing whereas mutation of p53 or the immunoglobulin heavy chain variable genes (IgH(V)) had no impact on APAF-1 expression. Surprisingly, APAF-1 loss did not result in resistance to cytotoxic therapies. Likewise, APAF-1 downregulation on its own showed no impact on disease prognosis. Nevertheless, a poor prognosis was observed in patients with loss of APAF-1 expression and additional p53 mutation. Thus, loss of APAF-1 may become relevant when additional core apoptosis signaling components are disrupted.

Restricted Pairing of Immunoglobulin Heavy and Light Chains Expressed by Chronic Lymphocytic Leukemia B Cells Is Predicated on the Heavy Chain CDR3.

Analysis of the immunoglobulin (Ig) heavy chains expressed by the leukemic B cells of patients with chronic lymphocytic leukemia (CLL) has demonstrated that expression of Ig variable heavy chain (VH) genes in CLL is not random. Certain VH genes are more frequently expressed in CLL than in the normal adult B cell repertoire, and some, such as the 51p1 allele of VH1-69, also use of certain diversity (D) and junctional (JH) gene segments that encode third complementarity determining regions (CDR3) with conserved molecular structures. We identified 15 CLL cases among 1,220 examined that express nearly identical Ig heavy and light chains, encoded by 51p1/D3-16/JH3 and VKA27, respectively (Blood , 104:2499, 2004). The highly restricted and virtually identical structure of these B cell receptors strongly suggests selection for Ig in CLL that have a particular binding activity. However, little information is currently available about the light chains expressed by CLL B cells that have 51p1-encoded Ig heavy chains that use other D and JH segments encoding CDR3 that also are repeatedly observed in this disease. We analyzed the VL genes used by 235 CLL cases found to express 51p1-encoded Ig heavy chains among 1,605 CLL patients examined. First, we find restricted light chain isotype expression, as 72% of samples express kappa and 28% express lambda light chains, compared to 65% kappa and 35% lambda within the cohort of all 1,605 CRC CLL samples, and about 60% kappa and 40% lambda expression in normal blood B cells. Nucleotide sequence analysis of the Ig light chain V gene used by these 235 cases revealed that each had greater than 98% homology to an identified germline VK or Vl gene. Additionally, we identified non-stochastic pairing of particular VK and Vl genes with 51p1-encoded heavy chains that have highly-conserved CDR3. Twenty of the 235 cases (8.5%) were found to have Ig light chains encoded by VKO2. Seventeen (85%) of such cases had 51p1-encoded Ig heavy chains that used D2-2 and JH6, 15 of which had nearly identical CDR3 using the amino acid motif DIVVVPAAI. The VKO2-encoded light chains paired with these Ig heavy chains all had nearly identical CDR3 with the amino acid sequence motif QQSYSTPRT. Similarly, seven of the 235 cases (3%) were found to have Ig light chains encoded by Vl3-9. Six (86%) of such cases had 51p1-encoded Ig heavy chains that used D3-3 and JH6, and all had highly conserved heavy chain CDR3 with the amino acid motif YDFWSGYYPNYYYYGMDV. The Vl3-9-encoded light chains paired with these Ig heavy chains all had nearly identical CDR3 with the amino acid sequence motif QVWDSSTXV. Finally, we identified seven additional samples that express a heavy chain using D3-16 and JH3 that have nearly identical CDR3 amino acid sequences GGGYDYIWGSYRPNDAFDI, and also express light chains encoded by VKA27. These seven samples combined with the previous 15 represent all of the 51p1-encoded heavy chains that utilize D3-16 and JH3, as well as 52% (22 of 42) of all 51p1-encoded CLL samples that express VKA27-encoded light chains. These studies reveal for the first time that CLL cases using the same unmutated Ig heavy chain have non-stochastic pairing with disparate Ig light chains that is predicated upon the Ig heavy chain CDR3 structure. Because the CDR3 typically forms a major part of antibody binding site(s) for antigen, these data provide compelling evidence for antigen selection of the antibodies expressed in CLL.

B-Cell Scaffold Protein with Ankyrin Repeats (BANK1) Is Differentially Expressed in ZAP-70 Negative Compared to ZAP-70 Positive Chronic Lymphocytic Leukemia Cells.

The lack of somatic mutations of the immunoglobulin variable heavy chain (IgVH) gene has been established as poor prognostic marker for chronic lymphocytic leukemia (CLL) patients at early stage disease. Expression of the non receptor tyrosine kinase zeta chain associated protein (ZAP-70) was proposed as a surrogate marker for an unmutated IgVH, however, up to 30% discordant samples have been reported depending on the respective study. B cell receptor (BCR) mediated signaling is enhanced by ZAP-70 expression in CLL cells in vitro and ZAP-70 expression also tends to decrease the time from diagnosis to treatment irrespective of the IgVH status. Therefore, we wanted to identify differentially expressed genes between the ZAP-70 positive and negative CLLs by gene expression profiling of peripheral blood mononuclear cells (PBMCs) using Affymetrix microarrays (HG-U133 Plus 2.0). ZAP-70 expression was analyzed by quantitative real time PCR of CD19 purified (purity > 99%) PBMCs (n=62) using a LightCycler instrument. Expression of ZAP-70 mRNA was normalized against the housekeeping gene ABL and a relative quantitation against Jurkat T cells as a calibrator was performed. Results are expressed as normalized ratio and a cut-off of 0.5 normalized ratio gave the best correlation to the IgVH status with 77% concordant samples between ZAP-70 expression and the IgVH status. The discordant samples consisted of 5 unmutated IgVHs in the ZAP-70 negative group and 9 mutated in the ZAP-70 positive group. In a second step PBMCs of the same samples were analyzed by gene expression profiling and differentially expressed genes were identified by t-test. Among the two best genes that could be used in a classification algorithm (SVM) to distinguish between the 2 subsets with 92% accuracy were ZAP-70 and B cell scaffold protein with ankyrin repeats (BANK1). The expression of BANK1 was increased 3–4-fold in the ZAP-70 negative compared to the ZAP-70 positive CLL subset (P = 0,001). In the literature, BANK1 has been identified in human BCR expressing B cells and seems to be B cell restricted. In B cells the scaffolding protein BANK1 enhances BCR-mediated Ca2+-signaling, a signaling pathway that is also enhanced by ZAP-70 expression in CLL B cells. Based on these data we show that increased BANK1 expression correlates with a ZAP-70 negative status in CLL B cells. The functional consequences of BANK1 expression in the ZAP-70 negative subset of CLL B cells, which are usually associated with a more favorable prognosis, still need to be established further.

Short Telomeres Are Associated with Genetic Instability and the Occurrence of High Risk Genomic Aberrations in Chronic Lymphocytic Leukemia.

Telomere length has been associated with the mutation status of the immunoglobulin variable heavy chain (VH) gene and the clinical course in chronic lymphocytic leukemia (CLL). In an unicentric CLL cohort of 108 patients, we have analyzed the telomere length by quantitative real time PCR, genomic aberrations by FISH with a comprehensive set of DNA probes (11q, 12q, 13q, 14q, 17p), the VH mutation status by DNA sequencing, ZAP-70 expression (clone 2F3.2, Upstate, according to Crespo et al., NEJM 2003) and CD38 expression by flow cytometry, to further study the prognostic impact and associations among these factors. A relative telomere-single-copy-gene ratio (T/S) was calculated for each sample, where low and high T/S values correspond to short and long telomere lengths, respectively. The median T/S value was 0.33 (range 0.06–1.18). There was an inverse correlation between telomere length and the following parameters: 1. VH homology (r=−0.56, p<0.001), 2. CD38 expression (r=−0.44, p<0.001) and 3. ZAP-70 expression (r=−0.25, p=0.01). Cases with T/S values below the median of 0.33 (short telomeres) and cases with T/S values above the median (long telomeres) had similar incidences of genomic aberrations (76 vs. 67%), 13q- (54 vs. 52%) and +12q (9 vs. 9%). In contrast, 13q- as a single aberration was significantly more frequently observed in cases with long telomeres (43 vs. 17%, p=0.006), whereas 11q- (30 vs. 9%, p=0.014), 17p- (24 vs. 0%, p<0.001) and cases with two or more genomic aberrations (26 vs. 6%, p<0.001) were significantly more frequent in cases with short telomeres. Compared to cases with long telomeres the treatment free survival from diagnosis (TFS) and overall survival (OS) in the group with short telomeres were significantly shorter (TFS: 29 vs. 67 months, p=0.002; OS: last observed death at 100 months, survival probability 57% vs. last observed death at 141 months, survival probability 77%, p=0.02). In conclusion, telomere length was inversely correlated with the VH mutation status, CD38 expression and ZAP-70 expression. Short telomeres were associated with genomic instability indicated by a high number of aberrations and the occurrence of 11q- and 17p- in CLL. These observations have biological and prognostic implications in CLL.

Analysis with the LightCycler System® Identifies a Highly Significant Correlation between ZAP-70 mRNA Expression and Immunoglobulin Variable Heavy Chain Gene Mutational Status in Chronic Lymphocytic Leukemia.

The presence or absence of somatic mutations in the immunoglobulin variable heavy chain (IgVH) region of chronic lymphocytic leukemia (CLL) B cells has prognostic information already at the time of diagnosis. The lack of somatic mutations in about 50% of the CLL patients significantly separates those with poor prognosis from those with a more indolent course that carry somatic mutations in their IgVH gene. However, the analysis of this marker is time consuming. Global gene expression analysis of CLL cells identified the expression of the T cells marker zeta chain associated protein (ZAP-70) as a surrogate marker for an unmutated IgVH. In order to analyze ZAP-70 expression in CLL cells using a relative quantification approach, we developed an assay**For research use only. Not available in distribution. on the LightCycler® instrument. Here, we analyzed a cohort of 100 samples for ZAP-70 mRNA and compared the results with the IgVH mutational status. Mononuclear cells were isolated from blood (n=80) or from bone marrow (n=20). To exclude ZAP-70-expressing T and natural killer cells from analysis, cells were enriched for B cells to a purity of > 99% by CD19 magnetic beads positive selection and mRNA was isolated using a MagNA Pure LC instrument. cDNA was reverse transcribed from an equivalent of 5x 106 cells and used for the analysis of the IgVH status as described (Blood . 2003; 101:2049–2053) as well as for the analysis of ZAP-70 expression with a LightCycler® instrument. ZAP-70 expression levels as the target gene were normalized to the houskeeping gene ABL using relative quantitation. mRNA of the Jurkat cell line was used as a calibrator. In preliminary experiments, ABL was selected from a total of 4 different housekeeping genes based on its expression levels, which were in the range of ZAP-70 expression, and based on the resulting correlation to the IgVH status. Thus, results for ZAP-70 gene expression are given as normalized ratio. ZAP-70 mRNA expression (n=100) revealed a highly significant correlation to an unmutated IgVH and low ZAP-70 expression correlated to a mutated IgVH (r2=0,692, Spearman). A cut off of 0.5 ZAP-70 expression was optimal to distinguish samples with mutated from unmutated IgVH, resulting in 80 concordant samples (80%). Of the 11 mutated IgVHs with discordant ZAP-70 expression 7 (64%) had borderline mutation status and 4 (36%) expressed the poor prognosis VH3-21 gene. 7(78%) of the 9 unmutated IgVHs with low ZAP-70 expression were characterized by adverse cytogenetic features. In summary, the results of the present study show that the LightCycler® system based quantitative RT-PCR is suitable to achieve a highly significant correlation between the IgVH status and ZAP-70 expression in CLL samples. Unlike the flow cytometric detection of ZAP-70 protein expression, which uses ZAP-70 of endogenous T cells as an internal control, the assay* developed by us might be easier to standardize between laboratories and therefore, might be applicable for future routine applications.

Chronic Lymphocytic Leukemia Patients with a V1-69 Gene Rearrangement Do Not Have Inferior Survival with Respect to Patients That Express Other Unmutated VH Genes.

The mutational status of the immunoglobulin variable heavy chain genes (VH) is a major prognostic indicator in patients with CLL. More recently, additional prognostic categories have been identified by recognizing disease subsets which utilize unique VH genes. Examples include the V3-21 and V3-72 genes which are invariably associated with progressive and stable disease, respectively. The V1–69 gene is the most frequently rearranged VH gene in CLL and is almost always unmutated. We therefore investigated whether CLL patients that express this VH gene differ in clinical course and outcome with respect to CLL patients expressing other unmutated VH genes. The study group consisted of 106 consecutive CLLs that had been diagnosed at our institution prior to 2000. The 25 V1–69 cases (23.6%) were identified by allele-specific VH gene fingerprinting and all were found to be unmutated by nucleotide sequencing. Forty of the remaining cases were randomly selected for VH gene nucleotide sequence analysis; 19 cases expressed another unmutated VH gene, 14 cases expressed a mutated VH gene and the VH gene sequence could not be determined in 7 cases. The V1–69 cases did not differ significantly from cases with other unmutated VH genes with respect to age (median age 60 years for both subgroups), gender (male: female=21:4 for V1–69 cases and 13:6 for other VH unmutated cases, P=n.s.) and median follow-up (V1–69 cases: median 56 months, range 6 to 109; other VH unmutated cases: median 59 months, range 20 to 97, P=n.s.). The percentage of cases presenting at an advanced stage was higher in the V1–69 subgroup (36% of cases in Rai high risk category) than in the subgroup with other unmutated VH genes (5% of cases in Rai high risk category, P=0.027), whereas no significant difference was observed with respect to Ly counts and total tumor burden at diagnosis. Interestingly, 80% of the V1–69 cases compared to 42% of cases with other unmutated VH genes had received treatment immediately following diagnosis, but median time to treatment was not significantly different between the two subgroups (1 month vs. 9 months, respectively, P=0.085). To date 24 V1–69 cases (96%) and 15 cases with other unmutated VH genes (79%) have died, with median survival times of 56 and 60 months, respectively (P=0.9). For comparison, median survival of the 14 VH mutated cases from this series was 125 months (P<0.0001 with respect to both subgroups with unmutated VH genes). We therefore conclude that chronic lymphocytic leukemia patients with a V1–69 gene rearrangement constitute a uniform group with unfavorable prognosis, but their survival does not differ significantly from patients with other unmutated VH genes. [Display omitted]