hTERT expression and prognosis in B-chronic lymphocytic leukemia.

BACKGROUND: In B-chronic lymphocytic leukemia (B-CLL), there is a need for molecular markers to predict the evolution of this heterogeneous disease in individual patients. The level of expression of the human telomerase reverse transcriptase (hTERT) gene has been associated with disease aggressiveness in human cancers. The purpose of the present study was to examine the prognostic significance of hTERT expression in B-CLL. PATIENTS AND METHODS: We used real-time reverse transcription-PCR to quantitate the amount of hTERT transcripts in mononuclear blood cells from 90 B-CLL patients. In addition, samples were analyzed for somatic mutations in the immunoglobulin V (IgV) genes. RESULTS: The expression of hTERT gene was detected in 59% of patients. The level of expression increased with advancing B-CLL stage (P=0.0064). Patients expressing hTERT showed significantly shorter survival than hTERT-negative patients (P=0.000034), irrespective of the disease stage. On average, the level hTERT mRNA expression was seven-fold higher in the poor-prognosis B-CLL group with unmutated IgV than in the Ig-mutated group (P<10(-7)). The level of hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL in 89% of cases. CONCLUSION: Our data indicate that hTERT expression in B-CLL may serve as a molecular prognostic marker.

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

In B-CLL IgV(H) genes mutational status is a major prognostic factor. Since sequencing of IgV(H) 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.

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 V(H) nucleotide sequence and the presence of RNA transcripts from different C(H) 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.

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

Four different monoclonal Ig (MIg) (IgA1kappa, IgG1kappa, IgG2kappa and IgG4kappa) displaying anti-tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical V(H) and V(L) domains and the presence of mutations compatible with an antigen-driven process. Surprisingly, despite complete homology in their variable domains, IgA1kappa, IgG1kappa, or their Fab fragments bound to a common motif recognized in beta tubulin, with significant differences in affinity (IgA1kappa 1.52x10(-8) M, and IgG1kappa 2.09x10(-7) M). To substantiate these results, the V(H) and V(L) domains from IgA1kappa were cloned and introduced into expression vectors containing the constant kappa exon and either the mu or the gamma1 constant exon, and complete recombinant IgMkappa and IgG1kappa were obtained. Like the IgA1kappa, the IgMkappa construction bound to the tubulin epitope with consistent affinity (7.7x10(-9) M), whereas the IgG1kappa construction displayed a significantly lower affinity (3.28x10(-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.

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. Among familial cases, VH gene segment utilization proved non-random and diverged from the frequencies previously reported among unrelated patients with CLL. Specifically, although the 4-34 and 5-51 gene segments were found repeatedly, the 1-69 and 4-39 gene segments were used sparingly and the 3-23 gene segment presented with increased frequency. Following the pattern detected in studies of unrelated patients, the single 1-69 expressing CLL contained an unmutated H chain sequence and included a long HCDR3 interval. In contrast, 3-23 containing H chains all used JH4, retained at most 93% homology with germline sequence, and included only short HCDR3 intervals. The vast majority of the CLL variable domains contained a high degree of somatic mutation and exhibited an excess of replacement mutations in the CDR intervals. These findings suggest that familial CLL cases may preferentially derive from B-cell progenitors that have responded to antigen.

Analysis of the B-cell receptor B29 (CD79b) gene in familial chronic lymphocytic leukemia.

The B-cell antigen receptor (BCR) comprises membrane Igs (mIgs) and a heterodimer of Igalpha (CD79a) and Igbeta (CD79b) transmembrane proteins, encoded by the mb-1 and B29 genes, respectively. These accessory proteins are required for surface expression of mIg and BCR signaling. B cells from chronic lymphocytic leukemia (B-CLL) frequently express low to undetectable surface Ig, as well as CD79b protein. Recent work described genetic aberrations affecting B29 expression and/or function in B-CLL. Because the prevalence of CLL is increased among first degree relatives, we analyzed the B29 gene in 10 families including 2 affected members each. A few silent or replacement mutations were observed at the genomic level, which never lead to truncated CD79b protein. Both members of the same family did not harbor the same mutations. However, a single silent base change in the B29 extracellular domain, corresponding to a polymorphism, was detected on 1 allele of most patients. These results indicate that the few mutations observed in the B29 gene in these patients do not induce structural abnormalities of the CD79b protein and thus do not account for its low surface expression in B-CLL. Furthermore, genetic factors were not implicated, because identical mutations were not observed among 2 members of the same family.

Detection of minimal residual disease in B chronic lymphocytic leukemia (CLL).

UNLABELLED: In the absence of specific chromosomal translocations the best method for detecting minimal residual disease (MRD) in B cell malignancies is based on the uniqueness of immunoglobulin (Ig) genes rearrangement. We here report a very sensitive method for assessing MRD in complete hematological remission (CHR) chronic lymphocytic leukemia (CLL) patients as defined by the international workshop on CLL (IWCLL). PATIENTS: Twelve CLL patients in CHR and complete phenotypic remission (CPR) were included in the study. Eight of them received Fludarabine (FDR), one was treated by Chop regimen, and the remaining 3 were rescued by polychemotherapy followed by autologous bone marrow transplantation (ABMT). METHODS: DNA extracted from peripheral blood lymphocytes (PBL) of each patient was amplified with VH family specific and framework 3 primers in 5' and a consensus JH primer in 3', before treatment and sequentially after the CPR completion. When no clonal rearrangement could be detected by this assay, the CDR3 sequence specific probe of the clone was used as the 3' primer, associated to the VH family specific primer in 5'. PCR products were analyzed by classical procedures in agarose and/or acrylamide gels. RESULTS: Mixtures of leukemic cells and normal PBL showed detection of a single leukemic cell among more than 10(5) normal cells. Four out of the 12 patients achieved molecular remission (MR) when employing CDR3 amplification. All 3 autografted patients were in MR, whereas only one out of the 9 patients treated by chemotherapy alone achieved MR. When using a clone specific probe, a clonal signal was observed in all cases but one (ABMT). Results presented here confirm that MR may be achieved in a few cases of B-CLL. Further studies are needed to determine the exact relationship between MRD and clinical outcome.

VH gene expression in hairy cell leukaemia.

Hairy cells are characterized by their typical morphology and expression of specific surface antigens. Although their B-cell origin is now confirmed, their exact position in B-cell development remains unclear. To better define the origin of hairy cells, we analysed the immunophenotype and the Ig VH nucleotide sequence of seven cases of hairy cell leukaemia (HCL). Six of them were typical HCL and the remaining case corresponded to a variant HCL. Analysis of sequenced VH genes revealed that the VH1 family was used in one case, VH2 in one, VH3 in two, VH4 in two and VH5 in one. No preferential usage of VH genes was observed in this small series. In five cases high rates of somatic mutations were observed, with a predominance of mutations and replacements in CDR regions for three. indicating that these cells originate from cells that have been exposed to the hypermutation mechanism. The distribution of mutations in our small series provides some evidence of a selective mutational process.

VH gene expression in CD5 positive and CD5 negative B cell chronic lymphoid malignancies.

In this review, we report analyses of VH genes in mature B cell malignancies generally or occasionally bearing CD5 antigen such as B CLL, MCL, SLVL and PLL. In the majority of cases, B CLL and MCL use VH genes in germline configuration. However in some cases a higher rate of random mutations is observed. These differences are not related to CD5 expression but are accounted by Ig phenotype, since less mutations are observed in CLL cases expressing membrane mu delta, when compared to forms exclusively expressing membrane mu. PLL and SLVL cases display mutated V genes independently of CD5 expression. Although there is some evidence that CD5+ B cells constitute a separate lineage, the possibility that CD5 constitutes an activation marker cannot be ruled out. Indeed, CD5- B cells can be induced to differentiate into CD5+ B cells and VH gene analyses showed no significative differences between CD5+ and CD5- B cell lymphoproliferative disorders. In this review we have tried to examine B cell chronic malignancies on the basis of phenotype and VH gene usage. Thus we propose a tentative classification where these disorders are allocated according to these characteristics.

Structural and affinity studies of IgM polyreactive natural autoantibodies.

Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.

High frequency of somatic mutations in the VH genes expressed in prolymphocytic leukemia.

Prolymphocytic leukemia (PLL) is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed lg heavy chain variable region (VH) genes in eleven cases of de novo PLL Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%), with preferential use of the V3-23 gene (50% of the VH3 genes). All sequences from expressed VH genes diverged from their putative germline counterpart, and in eight cases the divergence was greater than 5%. In seven cases, which expressed the V3-23 gene and VH4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B-PLL cells display a skewed repertoire of lg VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.

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, Burkitt's 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.

Analysis of VH gene expression in CD5+ and CD5- B-cell chronic lymphocytic leukemia.

In contrast to highly mutated follicular lymphomas and multiple myelomas, chronic lymphocytic leukemias (CLLs) frequently express VH genes in germline configuration. It is currently unclear whether this difference is related to the expression of CD5 or to the differentiation stage of the B cell when malignant transformation occurs. We have studied the VH sequence of 11 cases of CD5- B-CLL to address the question whether CD5- B-CLL are derived from naive pregerminal B cells (low mutation pattern) or from germinal center-derived memory B cells (high mutation pattern). Among the 12 detected rearrangements (2 distinct rearrangements in 1 case) VH1 family was found in 2, VH2 in 2, VH3 in 4, and VH4 in 4. Nine different VH genes were detected among the 12 rearrangements, including 2 cases expressing V1-69 (51p1) and 1 case expressing V4-39 (VH4.18), previously reported to be overexpressed in CD5+ B-CLL. A higher mutation pattern, following a random distribution, was observed when compared with classical CD5+ B-CLL. However, as reported in normal B cells, these results appeared to be related to membrane Ig phenotype (less mutations in membrane mu delta-expressing forms in leukemias expressing exclusively membrane mu). Overall, the differences found when comparing the mutational profile with classical CD5+ B-CLL were not clearcut and might be explained more by the membrane isotype (mu v mu delta) than by CD5 expression.

Skewed rearrangement of the VH4-21 gene during pre-B acute lymphoblastic leukemia.

Thirty-six pre-B acute lymphoblastic leukemias (ALL) were studied for VH family expression. Among the 35 detected rearrangements, VH1 family genes were expressed in 7, VH2 in 1, VH3 in 18, VH4 in 6 and VH6 in 3. This expression is close to that expected according to the complexity of the system. The complete sequence of the 6 VH4 genes was examined in order to determine whether there is a skewed rearrangement of individual genes in this family. Our results indicate rearrangement of VH4-21 in 3 cases, 71-4 in one, 58P2 in one case and probably of a new germinal VH4 gene for the sixth case. All the genes were displaying an almost complete homology with their germinal VH counterparts. The 6 sequenced genes associated with 6 different D gene segments displaying a close homology with their germinal counterpart. JH4 segment was expressed in 3 cases and JH6 in the remaining 3. These results associated with previous results obtained by others indicate that there is skewed rearrangement of the VH4-21 gene in pre-B ALL. It is presently unknown whether this phenomenon is the consequence of a selective process or whether it reflects what normally occurs in the normal human functional repertoire, which could be more limited than the germline repertoire.

V gene usage by seven hybrids derived from CD5+ B-cell chronic lymphocytic leukemia and displaying autoantibody activity.

We report here the complete heavy and light chain variable region sequences of seven heterohybridomas derived from CD5+ chronic lymphocytic leukemia (CLL) B lymphocytes and displaying natural autoantibody activity. The three hybrids displaying a polyreactive pattern of binding used VH4 family members, ie, the VH4-18 gene in germinal configuration in two cases and a VH4 gene with 90% homology with VH4-21 for the third one. A hybrid expressing anti-Sm activity used a VH3 family member with 95.26% homology with the 30P1 gene. The three hybrids exclusively displaying rheumatoid factor activity expressed VH1 family genes: 51P1 gene for two (in germinal configuration in one, and with 93.2% homology in the other), whereas the third one used the V1-3b gene (98.8% homology). Definitive homology with known germline D segments was found for four of the seven hybrids (DN2 in 3 and DLR4 in 1) and JH use appeared to be random. The three hybrids displaying polyreactive activity expressed V kappa I, V lambda III, and V lambda II genes, all in germinal configuration. Among the three hybrids with rheumatoid factor activity, two used the same V kappa II gene with, respectively, 98% and 96% homology with a gene previously described; the third used a V lambda I gene in germinal configuration. Finally, the clone with anti-Sm activity used a V lambda III gene having 97% homology with a germinal gene. Overall, these results attempt to establish the relationship between frequent self-reactivity observed in CD5+ B-CLL and V gene usage. For VH genes, they confirm overexpression of the 51P1 gene in B-CLL and suggest nonstochastic use of two VH4 genes (4-21 and 4-18). For VL genes, available information is too scarce to lead to firm conclusions.