Christian Magnac

Predictive value of serum thymidine kinase level for Ig-V mutational status in B-CLL.

In B-CLL IgVH genes mutational status is a major prognostic factor. Since sequencing of IgVH genes is not available in most laboratories, an easily performed surrogate assay is desirable. To identify the best surrogate assay, and to better discriminate prognostic subgroups we analyzed clinical and biological data from 58 typical CLL cases. A higher serum thymidine kinase level (>15 U/l) proved to be a strong predictor of mutational status, and the only independent one among the studied parameters. To further identify prognostic subgroups, cluster analysis was employed on 38 cases on which all data were available, which segregated two groups including 25 and 13 patients, respectively. These two clusters differed by their proliferative potential and appeared to discriminate patients with very different clinical course and outcome. s-TK was strikingly different among these two clusters, suggesting that s-TK level could be used routinely to identify patients at risk of progression.

VH gene usage by family members affected with chronic lymphocytic leukaemia

The excess risk of chronic lymphocytic leukaemia (CLL) in the first‐degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non‐random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B‐cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members.

Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements.

We evaluated minimal residual disease (MRD) in 23 CD5 + B-chronic lymphocytic leukemia (CLL) patients who achieved clinico-hematological remission confirmed by bone-marrow biopsy. MRD was evaluated by dual marker analysis flow-cytometry using CD5 and CD19 markers, and by the study of Ig heavy chain gene rearrangements using the fast polymerase chain reaction (PCR). According to our laboratory conditions patients were considered to be in complete phenotypic remission when total CD19+ cells were < 25% and the ratio of CD5 + CD19 + /CD19 + cells was < 25%. According to these strict criteria only 9 of the 23 patients were in complete phenotypic remission. In order to evaluate the sensitivity of the above method, PCR analysis of the configuration of the Ig heavy chain gene region was performed in 12 of these patients. Five of 7 patients in complete phenotypic remission retained a detectable monoclonal rearrangement of the Ig heavy chain gene. For the remaining 5 patients in partial phenotypic remission, only one failed to show a monoclonal band and this is probably explained by the presence of an unusual gene rearrangement. In conclusion, this study suggests that PCR is more sensitive than dual marker flow-cytometry for evaluation of residual disease and that it is indeed possible to achieve complete remission at the molecular level, in B-CLL. Nevertheless, we suggest a word of caution as this was a retrospective study, and samples were not assessed before treatment. Thus the possibility that apparent molecular remission might correspond to unusual gene rearrangements cannot be completely excluded in these cases.

Defective assembly of the B-cell receptor chains accounts for its low expression in B-chronic lymphocytic leukaemia

Summary. B‐cell chronic lymphocytic leukaemia (B‐CLL) characteristically displays low amounts of B‐cell receptor (BCR), which mainly consists of the heterodimer CD79a/CD79b bound non‐covalently with the surface immunoglobulin (SIg). This heterodimer is required for SIg expression and BCR signalling. To better define the mechanisms related to low BCR expression, we have investigated transcription, protein synthesis, assembly and transport of the BCR in B‐CLL cells. Our results demonstrated that: (1) there was no major defect in transcriptional expression of the B29 (CD79b) gene; (2) the BCR components were intracellularly detected, thus adequately synthesized, in almost all patients; (3) neither a genetic defect in the transmembrane region of SIg, which associated with CD79a/CD79b, nor a genetic abnormality in the chaperone protein calnexin that is involved in folding and assembly of the BCR were found; (4) a constant defect in the assembly of IgM and CD79b chains occurred leading to abnormal accumulation of both chains in different intracellular compartments; (5) in a majority of CLL patients all of the nascent IgM failed to be processed into mature chains and remained unsuitable for transport. These findings demonstrated that a post‐transcriptional defect located at the BCR intracellular assembly and/or trafficking levels could be involved in its low surface expression in B‐CLL.

Gene expression profiling of chronic lymphocytic leukemia can discriminate cases with stable disease and mutated Ig genes from those with progressive disease and unmutated Ig genes

Gene expression profiling of chronic lymphocytic leukemia can discriminate cases with stable disease and mutated Ig genes from those with progressive disease and unmutated Ig genes

Can isotype switch modulate antigen-binding affinity and influence clonal selection?

Four different monoclonal Ig (MIg) (IgA1κ, IgG1κ, IgG2κ and IgG4κ) displaying anti‐tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical VH and VL domains and the presence of mutations compatible with an antigen‐driven process. Surprisingly, despite complete homology in their variable domains, IgA1κ, IgG1κ, or their Fab fragments bound to a common motif recognized in β tubulin, with significant differences in affinity (IgA1κ 1.52×10–8 M, and IgG1κ 2.09×10–7 M). To substantiate these results, the VH and VL domains from IgA1κ were cloned and introduced into expression vectors containing the constant κ exon and either the μ or the γ1 constant exon, and complete recombinant IgMκ and IgG1κ were obtained. Like the IgA1κ, the IgMκ construction bound to the tubulin epitope with consistent affinity (7.7×10–9 M), whereas the IgG1κ construction displayed a significantly lower affinity (3.28×10–7 M). These results provide definitive evidence that isotype can influence binding affinity to antigen and suggest that malignant transformation occurred at the germinal center once the mutational process was achieved and the switch process was still active.

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete VH nucleotide sequence and the presence of RNA transcripts from different CH domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Novel flow‐cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal residual disease in chronic lymphocytic leukaemia

Summary. We describe a new flow‐cytometric analysis using quadruple labelling with anti‐CD19, CD20, CD5, CD79b monoclonal antibodies and sequential gating. We determined a novel criteria defined by BCD5+CD79b–/low/total BCD5+ cells ratio (BCD5+R), and compared it with the previous definition of phenotypic remission, based on CD19+CD5+ coexpression, and with complementarity‐determining region 3 polymerase chain reaction (CDR3 PCR) and clonotypic PCR (cPCR). A series of 54 peripheral blood samples from 21 chronic lymphocytic leukaemia (CLL) patients in complete haematological remission and a series of 16 from normal volunteers were analysed. In normal controls, the BCD5+R was always < 0·2. The sensitivity of the BCD5+R was 1 × 10−4vs 5 × 10−2 for CDR3 PCR and 1 × 10−5 for cPCR. Among the 54 CLL samples, 35 had a BCD5+R < 0·2 and showed polyclonal CDR3 PCR, whereas the cPCR was positive in 12 out of 20 tested. In the remaining 19 samples, BCD5+R was > 0·2, CDR3 PCR was monoclonal in 16 out of 19 and cPCR positive in 14 out 14 tested, including one out of three samples with polyclonal CDR3 amplification. Even though cPCR remains the most sensitive method to evaluate MRD, this new, sensitive and specific flow cytometric parameter, the BCD5+R, is more suitable than CDR3 PCR for routine clinical MRD assessment in CLL.

COMPLETE NUCLEOTIDE SEQUENCE OF IG V GENES IN THREE CASES OF BURKITT LYMPHOMA ASSOCIATED WITH AIDS

To investigate the role of polyclonal stimulation and antigen driven selection in the pathogenesis of acquired immunodeficiency syndrome (AIDS) related lymphomas, we studied the variable region nucleotide sequence of heavy (VH) and light (VL) chains expressed by 3 Burkitt lymphomas (BL) associated with HIV infection. Two cases expressed the VH3-30P1 gene with 88.6% and 86.7% homology when compared to their germinal counterpart, whereas the VH4-18 was rearranged in the third one (89% identity). All these genes displayed high numbers of mutations (27, 22, 28 respectively), predominating in CDR regions. The encoded light chain genes determined for cases 1 and 2 expressed the same V kappa I-018 gene. These results indicate that: 1) Although, it is difficult to address the issue of VH usage based on the limited number of cases studied, Burkitts lymphoma associated with AIDS may use a restricted repertoire of Ig genes. 2) Mutations and/or replacements predominated in CDR regions, which might suggest the occurrence of an antigen driven selection process, at least in some AIDS associated lymphomas. However, the high ratio of mutations observed in framework (FW) regions also favors the possibility that the antigen selection process is associated with polyclonal B cell stimulation.

Idiotype-pulsed dendritic cells are able to induce antitumoral cytotoxic CD8 cells in chronic lymphocytic leukaemia

Summary. Idiotypic structures of immunoglobulins from malignant B cells constitute tumour‐specific antigens, though the function of immunoglobulin‐specific CD8+ T cells in disease control and rejection remains unclear. We have studied five cases of B chronic lymphocytic leukaemia patients affected with indolent (three patients) or aggressive (two patients) disease. We showed that CD8+ T cells with major histocompatibility complex class I‐restricted cytotoxicity against autologous tumour B cells could be generated following repeated stimulations with idiotype‐pulsed dendritic cells in vitro. CD8+ T‐cell lines were able to upregulate CD69 expression and to release interferon (IFN)‐γ upon contact with the autologous B cells, though cytolytic activity was only substantiated for patients with indolent disease. The failure of cytolytic activity in patients with aggressive disease may be explained by a skewed maturation of memory CD8 cells.