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Approximately 70% of chronic lymphocytic leukaemia (CLL) patients present with early stage disease, therefore defining which patients will progress and require treatment is a major clinical challenge. Here, we present the largest study of prognostic markers ever carried out in Binet stage A patients (n = 1154) with a median follow-up of 8 years. We assessed the prognostic impact of lymphocyte doubling time (LDT), immunoglobulin gene (IGHV) mutation status, CD38 expression, ZAP-70 expression and fluorescence in situ hybridization (FISH) cytogenetics with regards to time to first treatment (TTFT) and overall survival (OS). Univariate analysis revealed LDT as the most prognostic parameter for TTFT, with IGHV mutation status most prognostic for OS. CD38 expression, ZAP-70 expression and FISH were also prognostic variables; combinations of these markers increased prognostic power in concordant cases. Multivariate analysis revealed that only LDT, IGHV mutation status, CD38 and age at diagnosis were independent prognostic variables for TTFT and OS. Therefore, IGHV mutation status and CD38 expression have independent prognostic value in early stage CLL and should be performed as part of the routine diagnostic workup. ZAP-70 expression and FISH were not independent prognostic markers in early stage disease and can be omitted at diagnosis but FISH analysis should be undertaken at disease progression to direct treatment strategy.

Immunophenotypic studies, fluorescence in situ hybridization (FISH) and conventional karyotyping were used to define the clinicobiological significance of 14q32 translocations involving the immunoglobulin gene locus (14q32/IGH) in 252 chronic lymphocytic leukaemia (CLL) patients. The following regions were studied: 13q14, centromere 12, 6q21; 11q22/ATM; 17p13/TP53, 14q32/IGH. Patients were classified as group 1 (favourable, i.e. 13q-single or normal), group 2 (intermediate risk, i.e. +12, 6q-, 1-2 anomalies), group 3 (unfavourable, i.e. 17p-, 11q-, complex karyotype), or group 4 (14q32/IGH translocation). Endpoints were treatment-free survival (TFS) and overall survival (OS). One hundred and ten patients were included in group 1, 99 in group 2, 25 in group 3 and 18 in group 4. 14q32/IGH translocation partners were identified in eight cases (BCL2 in five cases, BCL11A, CCND3 and CDK6 in one case each). group 4 showed shorter TFS versus groups 2 and 1 (25% patients treated at 2 months vs. 12 (P = 0.02) and 20 months (P = 0.002), respectively) and shorter OS (25% patients dead at 18 months versus 50 (P = 0.0003) and >60 months (P < 0.0001) respectively. The 14q32/IGH translocation maintained prognostic significance at multivariate analysis on TFS (P = 0.025) and OS (P < 0.001), along with advanced stage and CD38+. These findings show that the 14q32/IGH translocation predicts for an unfavourable outcome in CLL and that this cytogenetic subset might be included as a separate entity in a hierarchical cytogenetic classification of CLL.

Summary. We report a case of acute leukaemia with the t(4;l 1) chromosomal translocation which, at initial diagnosis, had L-l lymphoblasts that were positive for terminal deoxynucleotidyl transferase (TdT) and HLA-DR but negative for myeloid cytochemical markers. At last relapse the patient had mostly monocytoid blasts which were now TdT negative but were positive for HLA-DR, weakly positive for Sudan Black B (SB), periodic acid Schiffs (PAS), naphthol AS-D acetate esterase (NSE), chloroacetate esterase (CAE) and negative for acid phosphatase (AP) and nitroblue tetrazolium (NBT) reduction. Treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA) in vitro induced differentiation to macrophage-Iike cells that were strongly positive for SB, PAS, NSE, AP, CAE and NBT reduction, indicating a latent monocyte-like phenotype. Thus the leukaemic cell clone or a precurser clone with the t(4;l 1) translocation manifested a lymphoid phenotype at initial diagnosis and a monocytoid phenotype at relapse. Immunoglobulin gene analysis of the monocytoid relapse blasts revealed rearrangements of the heavy chain gene alleles and germline light chain genes. Thus, the leukaemia clone with the t(4;ll) chromosomal translocation could be a bipotential cell with heavy chain gene rearrangements occurring in a primitive cell which may retain the ability to differentiate along the myeloid-monocytoid lineage in response to the appropriate stimulus. Alternatively, these characteristics may result from a transformation associated event.

The mutational status of the variable region of the immunoglobulin heavy chain gene (IgV(H)) is an important prognostic marker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutated patients having improved outcome. To examine the impact of mutational status on V(H), D(H), and J(H) gene segment location and immunogenicity, we analysed 375 IgH sequences from 356 patients with B-CLL. Although V(H) and D(H) gene usage was different in mutated compared to unmutated patients, there was no impact of gene location on frequency of use or clinical outcome. Surprisingly, somatic mutations did not increase the immunogenicity of the Ig, as assessed by predicted binding affinity of Ig-derived peptides to major histocompatibility Class I and Class II molecules. Even excluding patients using V(H)1-69, cases using the V(H)1 gene family had a poor outcome. Both mutated and unmutated CLL patients demonstrated evidence of antigen selection. The worst outcome was seen in the subset of 14 unmutated patients with similar HCDR3 amino acid sequence using V(H)1-69, D(H)3-3 and J(H)6, suggesting an antigen-driven process modulating the clinical course.

We report on the different expression of CD56 and CD44 in plasma cells (PCs) simultaneously collected from bone marrow, extramedullary locations and peripheral blood in seven patients with multiple myeloma. Extramedullary PCs showed absence of CD56. In the bone marrow, however, subsets with varying CD56 expression were found in five out of seven patients, with one subset corresponding to that of extramedullar PCs. This differs from the de novo downregulation of CD56 in PC leukaemia, and suggests different mechanisms of spread of myeloma cells. CD44 expression was generally upregulated on extramedullary PCs. In three of the patients we investigated the clonal origin of extramedullary myeloma cells by sequencing the variable portion of the heavy chain immunoglobulin gene in phenotypically defined PCs isolated from different locations. In each patient we found malignant PCs with different homing behaviour originating from a common precursor cell.

In T cell-dependent immune responses, class switch recombination occurs in germinal centres. There is now evidence that Hodgkin/Reed–Sternberg cells are derived from germinal centre B cells. Cytokines specifically determine the direction of class switching, i.e the isotype of the new antibodies. We performed restriction analyses and polymerase chain reaction on the immunoglobulin heavy chain loci for five Hodgkin's disease-derived B-cell lines and one Hodgkin's disease-derived T-cell line in order to analyse class switch recombination. In all the B-cell lines, class switch recombination had been targeted to Cα4 or Cα1/2. This showed that cell-line precursors had undergone class switching, probably under the influence of TH2 or TH3 cell-derived cytokines. Deletions comprising several constant region genes were observed in cell lines L428, L1236, L591 and KMH2. Karyotype analyses of two of these revealed translocational breakpoints within the immunoglobulin heavy chain gene locus. Our data support the view that a chromosomal instability may occur during class switch recombination in Hodgkin/Reed–Sternberg cells causing chromosomal breaks. Thus, as in other germinal centre B cell-derived lymphomas, the immunoglobulin gene locus may be frequently involved in structural chromosomal aberrations in Hodgkin's disease.

Autoimmune thrombocytopenic purpura (AITP) is a severe disease in children with a still unknown aetiology. It is not known why AITP can either be transient and self limiting or become chronic. The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of AITP patients has been proven. It is, however, not clear how IVIG functions. To analyse patient-derived monoclonal IgG platelet autoantibodies that interact with IVIG in an anti-idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients a large number of clones specifically reacting with IVIG molecules were derived. Many of these IVIG binders also reacted strongly with platelets in ELISA and FACS, in contrast to IVIG binders derived from a healthy individual. The heavy and light chain variable regions were sequenced and compared with each other and with databases. In all three AITP patients clones with a striking complementarity-determining region (CDR) sequence homology to each other and to many of the known anti-platelet antibodies were observed. Selected Fab-phages representing the characteristic variable regions that occurred in the investigated patients with AITP may now be used to clone potentially regulatory anti-idiotypes from healthy donors by phage display.

Waldenstrom macroglobulinaemia (WM) malignant cells have been considered incapable of undergoing class switch recombination (CSR). However, we report a WM patient who developed an IgG M-component 4 years after diagnosis. When the second monoclonal component appeared, reverse transcription-polymerase chain reaction showed the presence of pre (Cmu) and postswitch (Cgamma) clonotypic isotypes; sequencing of these isotypes demonstrated that both corresponded to the single clone amplified at diagnosis, including the same complementarity-determining region 3 and somatic mutation pattern. This proves that WM cells can undergo a functional in vivo CSR.

Hodgkin-Reed Sternberg (H-RS) cells harbour clonal immunoglobulin gene (Ig) rearrangements in almost all cases of classical Hodgkin's disease but lack Ig gene expression. In the H-RS cell line L1236, a somatic mutation of the Ig heavy-chain gene promoter octamer motif has been described as a putative reason for absence of Ig gene expression. We addressed transcriptional activity of this mutated promoter by performing reporter gene studies and gel retardation assays. The results showed that the mutation outside the coding region of the rearranged Ig gene in L1236 cells leads to downregulation of Ig gene expression in H-RS cells.

Rearrangements of the heavy chain immunoglobulin gene and T cell receptor beta gene were investigated in 25 patients suffering from precursor B cell acute leukaemia and six patients suffering from T cell acute leukaemia using biotinylated DNA probes. All precursor B acute leukaemia patients had IgH gene rearrangements and 63% of those studied also had TCR beta gene rearrangements. All T cell acute leukaemia patients had TCR beta gene rearrangements and germline IgH configuration. Dilution experiments indicated that DNA from leukaemic cells representing 1-2% of a tested sample could be detected using this technique which compares favourably to radioactive DNA probes.

Summary. We report three family members, including a set of identical twins, who developed CD5 positive B-CLL. The patients are female Ashkenazi Jews sharing an identical HLA phenotype. Two of the HLA loci (B35 and Cw4) were common with those already described as being shared by Ashkenazi Jews with an increased incidence of CLL. The rearranged immunoglobulin genes of the malignant cells of all three patients were found to express genetically related VH regions belonging to the VH3 subgroup, and chromosomal studies suggest a process of clonal evolution in one of the twins.

Mantle cell lymphomas (MCL) frequently show a vaguely follicular growth pattern. This phenomenon is thought to result from the colonization of reactive B-cell follicles by tumour cells. In view of the unique property of the germinal centre environment, antigen stimulation may play a role in the expansion of the tumour. To assess this, we have examined ongoing Ig mutations, which are genetic markers of B cells in persistent response to antigen stimulation, in five MCLs including two cases derived from the gastrointestinal tract known as lymphomatous polyposis (LP). We have specifically analysed Ig ongoing mutations in tumour cells from multiple lesions in one case and in tumour cells microdissected from colonized follicles in two cases. The consensus Ig V H sequences in four MCLs were identical, or almost identical (three cases 100%, one case 99% homology) to the published germlines. which in each case were those frequently employed by autoantibodies. The consensus Ig V H sequence in the remaining case displayed 95.5% homology to the closest published germline. This may represent derivation from an unknown V u germline or a rare instance of somatic mutations. Extensive sequencing of the rearranged Ig genes revealed ongoing mutations within the tumour clone in two cases: one was a LP with multiple lesions of the gastrointestinal tract and the other was a nodal MCL in which tumour cells from colonized follicles were analysed. Our results indicate that MCLs are derived from pre-germinal centre B cells, possibly autoreactive B-cell clones. The ongoing mutations identified suggest a possible involvement of antigen stimulation in the clonal expansion of a proportion of MCLs.

Techniques currently available enable the detection of clonal rearrangements of the immunoglobulin heavy chain gene using fluorescent PCR technology. It is possible to use this technique to analyse minimal residual disease throughout patient treatment ; however, without the development of a quantitative assay, only the presence or absence of a clonal population can be determined. We describe here the development of a quantitative competitive PCR technique using genomic DNA which enables the rate of clearance of disease to be measured. In future, the ability to detect and also quantitate minimal residual disease may enhance the application of molecular investigations in the clinical management of patients.

Malignant lymphomas (ML) with t(3;14) or variant t(2;3) and t(3;22) have recently been recognized. These translocations have been shown to associate predominantly with B-cell diffuse large cell lymphoma (DLCL) and less frequently with follicular lymphoma (FL). The molecular alterations associated with these translocations involve one of the immunoglobulin gene (Ig) loci and a recently cloned gene, bcl-6 located at 3q27 which codes for a zinc-finger protein that may function as a transcription factor. We have identified by cytogenetic analysis 22 cases of ML with a 3q27/Ig translocation. The pathologic diagnoses of these cases include DLCL, FL, small non-cleaved non-Burkitt lymphoma and chronic lymphocytic leukaemia. Molecular analysis confirmed a bcl-6 rearrangement in 10/12 cases tested. The karyotype in 5/22 cases revealed the t(3;14) or variant in association with another lymphoma-specific translocation, t(14;18) in three cases and t(8;14) in two cases. ML with dual translocations that implicate Ig genes in the deregulation of proto-oncogenes are being increasingly recognized and may represent distinct subtypes or ‘hybrid’forms of malignant lymphoma.

Summary Rearrangement of the immunoglobulin heavy chain (IgH) gene is widely exploited as a marker of B cell lineage and clonality in the pathology of lymphoproliferative disorders. We have developed a simple, polymerase chain reaction (PCR) based method for detecting IgH gene rearrangement which relies on the observation that by using a panel of PCR amplimers specific for each of the six heavy chain variable region families in conjunction with a common joining region amplimer, clonal rearrangement can be detected in over 90% of cases of B lymphoid malignancy. By using radiolabelled amplimers and exploiting the size heterogeneity resulting from independent IgH rearrangement events. we show that high resolution gel electrophoresis can be used to generate a‘fingerprint’representing the spectrum of B cell clonality in complex populations of B lymphocytes. The method effectively scans the entire IgH gene rearrangement repertoire and is capable of detecting rare clonal or oligoclonal B lymphoid cell populations. In normal bone marrow mononuclear cells. clonal IgH rearrangement could be readily detected at a sensitivity of 10−3. We illustrate the application of the method in assessing the spectrum of B cell clonality occurring in an autoimmune condition, Hashimoto's thyroiditis, and in a malignant B cell disorder, chronic lymphocytic leukaemia. In addition, we explore the potential application of the technique in tracking minimal residual disease and for monitoring clonal evolution in acute lymphoblastic leukaemia.

Peripheral blood mononuclear cells from 28 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of unknown significance (MGUS) were studied by immunoglobulin gene analysis. Clonal immunoglobulin gene rearrangements in peripheral blood mononuclear cells (PBIGRA) were demonstrated in 10 of the 28 MM patients (36%). Bone marrow and peripheral blood mononuclear cells were studied simultaneously in five of these 10 patients, and identical gene rearrangements were demonstrated in both. The incidence of such gene arrangements was higher in patients with active disease (cases at presentation or relapsed = 10/19 [47%]) compared to remission status (0/9) and higher in untreated (47%) compared to treated patients (11%) (P less than 0.05). Patients with this phenomenon had higher serum calcium levels (P less than 0.001), and higher bone marrow plasma cell counts (P less than 0.05). Serum creatinine and beta 2-microglobulin were also higher but did not reach statistical significance. None of the patients with monoclonal gammopathy of uncertain significance had gene arrangements. Our findings confirm that circulating B lymphocytes are part of the malignant clone in MM and their presence correlates with high tumour volume.