Early rearrangements of genes encoding murine immunoglobulin kappa chains, unlike genes encoding heavy chains, use variable gene segments dispersed throughout the locus.

Immunoglobulin heavy-chain variable region (TH) gene segments located closest to the joining (JH) gene segments are preferentially rearranged during ontogeny, indicating that chromosomal position influences the frequency of rearrangement. In addition, certain VH gene segments are repeatedly rearranged, suggesting that the DNA sequence or structure surrounding these segments may increase the probability of rearrangement. To determine whether there is similar based rearrangement of kappa variable (V kappa) gene segments, 25 rearrangements were sequenced from murine fetal and neonatal B-cell hybridomas and from subclones of a pre-B cell line that rearranged V kappa genes during in vitro culture. Four gene segments were isolated twice and one gene segment was isolated three times, suggesting that the process that targets individual variable gene segments for repeated rearrangement operates on both the VH and V kappa loci. Based on a current map of the V kappa locus, the rearranged gene segments belong to nine families that are dispersed throughout the locus. Thus, in these cell types, V kappa rearrangements use germ-line gene segments located across the entire locus, whereas the corresponding VH rearrangements use gene segments proximal to the JH gene segments. Heterogeneity of V kappa rearrangements would add diversity to the biased pool of VH rearrangements, producing a broad repertoire of antibodies early in development.     

Immunoglobulin VH4 gene usage in B lymphoid leukaemias

Summary. V_H4 gene rearrangements occur in a similar proportion of cases of B lineage acute lymphoblastic leukaemia (ALL) and B chronic lymphocytic leukaemia (B CLL). However, there may be differences in the pattern of V_H4 gene usage between these disorders as is the case for V_H1 gene rearrangements. To examine this, we analysed the sequences of 24 PCR-amplified clonal V_H4 gene rearrangements from a series of 15 cases of ALL and nine cases of CLL. Five distinct groups of genes were rearranged, three of which (represented by V2-1, V71-2/V71-4, V4.21) have been described in rearranged form in normal B lymphoid tissues. The most frequently rearranged gene was V4.21 which is strongly associated with autoimmune reactivity. V71-2, V71-4 and V2-1 were more frequently rearranged in CLL than ALL. The remaining two groups (represented by V4.33, V4.35) have not previously been described in rearranged form. One of these, V4.35, was seen only in ALL rearrangements. Both V4.35 and a V_H1 gene, 20P3, which is also preferentially rearranged in ALL, are located at the 3'end of the V_H locus. The location of these genes suggests that their rearrangement may be developmentally regulated in ALL. The findings in this study confirm restricted repertoires of IgH gene rearrangement in ALL and CLL. Characterization of IgH repertoires provides a means of correlating these transformed B cell populations with normal B cell developmental compartments. Moreover, the distinctive repertoires in ALL and CLL may reflect important differences in the ontogenic timing and microenvironmental milieu of tumourigenesis in these disorders.     

Blastic variant of mantle cell lymphoma shows a heterogenous pattern of somatic mutations of the rearranged immunoglobulin heavy chain variable genes

Mantle cell lymphoma is a distinct clinicopathological entity associated with t(11;14) and cyclin D1 overexpression. The majority of cases show uniform morphological and phenotypic features characterized by a monotonous proliferation of small-to-medium-sized irregular B cells that express CD5 and bright surface immunoglobulin IgM and IgD. By sequence analysis of the rearranged immunoglobulin heavy chain variable genes (VH), it has been shown that these lymphoma cells carry little if no somatic mutations, as described for the fetal CD5⁺ cells or B1 cells. Besides mantle cell lymphoma with classic histological features, a morphological variant of mantle cell lymphoma with blastic features and a more aggressive clinical course has been described. To investigate whether this variant is closely related, by the cell of origin, to typical cases, we analysed the presence and the pattern of somatic mutations of the VH genes in a series of nine cases diagnosed as such. Our cases of blastic mantle cell lymphomas rearrange most frequently VH4 and VH3 family genes. In three cases there was a complete homology to published germline genes, and a near complete homology was documented in another three. In contrast, the remaining three cases showed somatic mutations in their rearranged VH genes. Mutation analysis revealed evidence for antigen selection in one of these three cases. Taken together, these data are similar to those of normal adult-type B1 cells and those described for chronic lymphocytic leukaemia (CLL) but slightly different to those reported for classic mantle cell lymphoma. It is likely that blastic mantle cell lymphoma as well as CLL originates from adult-type B1 cells. More cases will need to be studied to determine whether classic mantle cell lymphoma is different from the blastic subtype and if it arises from fetal-type B1 cells.     

Molecular analysis of VH and VL regions expressed in IgG-bearing chronic lymphocytic leukemia (CLL): further evidence that CLL is a heterogeneous group of tumors

We report the heavy (H) and light (L) chain variable (V) region sequences of cDNAs encoding the Ig receptor of two cases of CD5+ IgG- bearing CLL P87 and P103. In both CLL cases the H chain was encoded by members of the VH3 gene family. The L chain expressed by P87 belonged to the V lambda IV subgroup, whereas P103 used a member of the V kappa III subgroup. The VH3.P87 gene differed by only three nucleotides from 38P1, a VH3 gene previously cloned from a fetal liver cDNA library. Nucleotide sequence analysis demonstrated that the V kappa III.P103 gene differed by seven nucleotides from its most homologous germline counterpart, the Humkv325 gene, a highly conserved gene frequently expressed in IgM-bearing CLL. The nucleotide sequences of VH3.P103 and V lambda IV.P87 could not be reliably matched with reported germline V genes. The analysis of multiple independently obtained VH and VL cDNA clones from each tumor showed a lack of intraclonal diversification. The data show that V regions expressed in isotype-switched CD5+ CLL may be either in/near germline configuration or somatically mutated. Furthermore, these tumors, like their IgM-bearing counterparts, do not seem to undergo intraclonal diversification.     

Detection of immunoglobulin gene rearrangement in B lymphoid malignancies by polymerase chain reaction gene amplification

Immunoglobulin heavy chain gene rearrangement serves as a marker of cell lineage and clonality in B lymphoproliferative disorders. We have used polymerase chain reaction (PCR) gene amplification to detect immunoglobulin gene rearrangements involving VH251, a heavy chain variable region preferentially utilized in B lymphoproliferative disorders. Using synthetic amplimers derived from VH251 and the heavy chain joining region, under conditions of high stringency, a homogeneous VH251-specific fragment of approximately 350 bp could be amplified from leukaemic DNA. Of 53 cases of B lineage acute lymphoblastic leukaemia screened for VH251 rearrangement by PCR, 10 were positive. A background level of VH251 rearrangement could also be amplified from normal peripheral blood and bone marrow DNA, but a VH251 rearranged leukaemic clone representing 0.01% of bone marrow mononuclear cells could be readily detected. The application of PCR to detect immunoglobulin gene rearrangement involving VH251 and potentially other preferentially utilized V regions provides a sensitive method both for tracking malignant B cells and for the study of normal B cell developmental pathways through which B lineage malignancies arise.     

Developmentally restricted immunoglobulin heavy chain variable region gene expressed at high frequency in chronic lymphocytic leukemia.

During fetal development, murine and human B-lineage cells rearrange and express a highly restricted set of immunoglobulin heavy chain variable region genes (VH genes). We noted that a VH gene of the restricted human fetal repertoire, designated 51p1, potentially could encode the VH region of two human IgM rheumatoid factor proteins. These rheumatoid factors share a cross-reactive idiotype (CRI) defined by reactivity with G6, a murine monoclonal antibody that recognizes an antibody heavy chain determinant present on many human IgM autoantibodies, particularly rheumatoid factors. Recently, we found that the G6 CRI also is expressed frequently by neoplastic CD5 (Leu1) B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma. However, neoplastic CD5-negative B cells from patients with lymphomas of follicular center cell origin rarely express this CRI. Here, we report that G6-reactive leukemic cells from two unrelated CLL patients express a VH gene that shares greater than 99% homology with a rearranged VH gene previously isolated from the leukemic cell DNA of another CLL patient and that is identical to VH 51p1. Using the polymerase chain reaction, we find that this VH gene is rearranged, and presumably expressed, in the genomic DNA of all examined cases of G6-reactive CLL or small lymphocytic lymphoma. Thus these data indicate that the autoantibody-associated G6 CRI is a serologic marker for a conserved and developmentally restricted VH1 gene that is expressed at high frequency in CD5 B-cell malignancies and early B-cell ontogeny.     

Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes

The mutation status and usage of specific VH genes such as V3-21 and V1-69 are potentially independent pathogenic and prognostic factors in chronic lymphocytic leukemia (CLL). To investigate the role of antigenic stimulation, we analyzed the expression of genes involved in B-cell receptor (BCR) signaling/activation, cell cycle, and apoptosis control in CLL using these specific VH genes compared to VH mutated (VH-MUT) and VH unmutated (VH-UM) CLL not using these VH genes. V3-21 cases showed characteristic expression differences compared to VH-MUT (up: ZAP70 [or ZAP-70]; down: CCND2, P27) and VH-UM (down: PI3K, CCND2, P27, CDK4, BAX) involving several BCR-related genes. Similarly, there was a marked difference between VH unmutated cases using the V1-69 gene and VH-UM (up: FOS; down: BLNK, SYK, CDK4, TP53). Therefore, usage of specific VH genes appears to have a strong influence on the gene expression pattern pointing to antigen recognition and ongoing BCR stimulation as a pathogenic factor in these CLL subgroups.     

Adult B-cell repertoire is biased toward two heavy-chain variable-region genes that rearrange frequently in fetal pre-B cells.

Fetal pre-B cells rearrange a very restricted set of immunoglobulin variable genes for the heavy chain (VH). To determine whether the adult B-cell repertoire is similarly skewed, we first identified the genes that rearrange in pre-B cells from BALB/c mice and then determined their frequency of rearrangement in adult B cells. In fetal pre-B cell lines, two genes, VH81X from the 7183 subfamily and VHOx2 from the Q52 subfamily, comprise 75% of the rearranged alleles of an estimated 1000 genes (P less than 0.001). Sequencing analyses revealed that rearrangements involving the two genes were both productive and nonproductive. The biased rearrangement of these two VH genes persists in B-cell hybridomas from adult mice at a frequency of 22%, as determined by Southern gel analysis and RNA sequencing. The sequence of one VHOx2 rearrangement from a hybridoma shows that the rearrangement is productive, suggesting that the gene encodes an antibody that could participate in the immune response. The data indicate that the adult B-cell repertoire is not random concerning usage of individual VH genes, and it may be shaped by the unknown mechanisms that cause preferential rearrangement of certain genes early in ontogeny.     

Human heavy-chain variable region gene family nonrandomly rearranged in familial chronic lymphocytic leukemia.

We have identified a family of human immunoglobulin heavy-chain variable-region (VH) genes, one member of which is rearranged in two affected members of a family in which the father and four of five siblings developed chronic lymphocytic leukemia. Cloning and sequencing of the rearranged VH genes from leukemic lymphocytes of three affected siblings showed that two siblings had rearranged VH genes (VHTS1 and VHWS1) that were 90% homologous. The corresponding germ-line gene, VH251, was found to be part of a small (four gene) VH gene family, which we term VHV. The DNA sequence homology to VHWS1 (95%) and VHTS1 (88%) and identical restriction sites on the 5' side of VH confirm that rearrangement of VH251 followed by somatic mutation produced the identical VH gene rearrangements in the two siblings. VHTS1 is not a functional VH gene; a functional VH rearrangement was found on the other chromosome of this patient. The other two siblings had different VH gene rearrangements. All used different diversity genes. Mechanisms proposed for non-random selection of a single VH gene include developmental regulation of this VH gene rearrangement or selection of a subpopulation of B cells in which this VH has been rearranged.     

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.     

Restricted diversity of the variable region nucleotide sequences of the heavy and light chains of a human rheumatoid factor

The complete nucleotide sequences of the variable region genes of the heavy and light polypeptide chains of a human monoclonal rheumatoid factor (RF) produced from a human-mouse heterohybridoma were determined. The antibody, designated YES8c, contained V kappa III, J kappa 2, VH1, JH4, and a D gene segment of 9 amino acids. The nucleotide sequences and the deduced amino acid sequences of the light chain variable region were remarkably homologous (97-98%) to previously described RF of the Wa idiotypic family (PAY, GLO, CUR, FLO, and GAR) and to that of a V kappa III germline gene (Humkv325). The YES8c heavy chain variable region gene was most closely related to the VH1 gene of the restricted human fetal repertoire, designated 51p1, and also to 3 rearranged VH1 genes that were recently isolated from patients with chronic lymphocytic leukemia. These results suggest that variable region genes of RFs are highly conserved and that YES8c VH, as well as V kappa, may be identical to heavy and light chains expressed during early B cell development.     

Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation

To investigate whether somatic hypermutation occurs in multiple myeloma (MM) Ig VH region genes, we have cloned and sequenced the expressed VH genes from five cases of MM. The sequences were obtained after polymerase chain reaction (PCR) on total RNA isolated from the bone marrow, using 5' VH family-specific leader and 3' C gamma- or C alpha- specific primers. MM-specific CDR3 oligonucleotides were produced to isolate VH genes expressed by the malignant plasma cells. In all five cases, the productive Ig gene used the VH3 family. Extensive sequence analysis of multiple independent M13 clones showed no intraclonal variation with no evidence for ongoing somatic hypermutation in MM VH region genes. We were able to identify possible germline counterparts of the expressed VH genes in two cases. Comparison of these genes shows that the MM VH region genes have somatic mutations characteristic for an antigen-driven process. In the other three cases, no close homology could be found with published VH3 sequences. These findings implicate that, in MM, clonal proliferation takes place in a cell type that has already passed through the phase of somatic hypermutation.     

Analysis of expressed immunoglobulin heavy chain genes in familial B-CLL

In this study, we wished to determine whether familial chronic lymphocytic leukemia of B-cell phenotype (CLL) shares with sporadic B-CLL the same immunoglobulin (Ig) heavy chain variable region (VH) gene usage and occurrence of somatic mutation, to gain insight into the pathogenetic relatedness of these epidemiologically distinct forms of CLL. We therefore analyzed the expressed Ig heavy chain genes in 23 cases (11 families) of familial CLL, and compared these results with data previously reported for sporadic CLL. In addition, we assessed the relationship of the occurrence of somatic mutation to several clinical and phenotypic features. The distribution of V genes among these cases was similar to that observed in sporadic CLL: VH3 > VH1 > VH4. Thirteen of the 23 cases (57%) showed germ line VH gene sequences, whereas somatic mutations were detected in 10 cases (43%). The average mutation frequency of these latter 10 cases of was 6.7% (ranging from 1.7% to 8.8%), and evidence of antigen selection was noted in 6. Intraclonal variation, followed by clonal evolution and the appearance of a second clone over a 20-year period was observed in 1 case, suggesting that mutations can continue to accumulate after neoplastic transformation. The presence of somatic mutations correlated with age at presentation, low white blood cell (WBC) count, and low fluorescence intensity of surface CD5, and the potential significance of these relationships is discussed. Our data indicate that familial and sporadic B-CLL display a similar pattern of immunoglobulin gene usage and frequency of somatic mutation, and are consistent with a common ontogeny and immunogenetic origin for these 2 epidemiologically distinct forms of CLL. (Blood. 2000;95:1413-1419)     

Rearranged immunoglobulin heavy chain variable region (VH) pseudogene that deletes the second complementarity-determining region.

We have cloned two rearranged heavy chain variable region (VH) genes from the IgG-producing human cell line CESS. The VH gene, which is linked to the mu chain constant region (C mu) gene, has two deletions at residues 45-62 and 82A-90, the former of which corresponds closely to the second complementarity-determining region (CDR2). These results could indicate that translocation of CDR2 occurred and could give support to the argument that reassortment of the V mini-genes is involved in the generation of hypervariability during evolution. However, the rearranged pseudogene could have also arisen by fortuitous deletion. The other VH gene of CESS is an expressed form and is probably linked to the C gamma gene. The diversity region (D) segments used in these rearranged V genes are less than 38% homologous to known human germline D segments, indicating the presence of more unknown germline D segments.     

Telomere shortening in patients with plasma cell disorders

OBJECTIVES: Telomeres are essential for maintaining chromosomal integrity; their shortening is associated with chromosome instability. The aim of this work was to study telomere length (TL) on bone marrow (BM) cells from patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). METHODS: Thirty-one MM patients: 12 at diagnosis (D), 11 at relapse (R) and eight at remission (RE) and two cases with MGUS were studied. TL based on terminal restriction fragment (TRF) assay was evaluated. Cytogenetic and molecular cytogenetic analyses were performed. Telomeric associations (TAs) on BM metaphases were also studied. RESULTS: TRF analysis in total MM patients showed a mean TRF peak value (5.20 +/- 0.35 kb) shorter than those observed in controls (8.5 +/- 0.5 kb) (P < 0.001). Moreover, TRF at D and R showed a significant telomere shortening (P < 0.001), with TL restored at RE. A strong correlation with the percentage of BM plasma cell infiltration (BMPCI) (rK = -0.540; P = 0.002) was found. Patients with abnormal karyotypes (AK) had significantly shorter TRFs than that observed in MM patients with normal karyotypes (P < 0.05). TRFs in MGUS patients did not differ with respect to controls. TA analysis showed an increased percentage in MM (19.46 +/- 1.98%) with respect to MGUS (6.12 +/- 1.87%) and normal BM cells (2.00 +/- 0.93%) (P < 0.001). CONCLUSIONS: MM patients showed a significant reduction in TL (> 60% of BMPCI and AK), suggesting a probable association with clinical evolution. Moreover, our findings support the idea that telomere shortening usually leads to increased frequencies of TAs and chromosome instability.     

Molecular analysis of the leukaemic B cell in adult and childhood acute lymphoblastic leukaemia

Immunoglobulin heavy chain gene (IgH gene) rearrangements are found in the majority of cases of B-lineage acute lymphoblastic leukaemia (ALL). We have examined bone marrow samples taken at presentation or relapse from 109 patients (79 adults and 30 children) and have performed sequence analysis of the complementarity determining region 3 (CDR3) on 65 alleles from 54 patients. We aimed to define immunoglobulin heavy chain (IgH) variable segment family use and investigate biological and structural features of the B cell in adult and childhood ALL. Using the FR1 fingerprinting method, a rearranged band was identified in 70 (89%) of 79 adult ALL and in 29 (97%) of 30 childhood ALL. This study found no preferential use or selection of IgH VH genes and no statistically significant structural differences between normal and leukaemic B cells in either adult and childhood ALL. An equal proportion of amplifiable cases of adult and childhood ALL uses more than one VH family gene (24/70, 34%, and 8/29, 27.5%, respectively). There were no significant differences in the structure or size of the CDR3 region and the variable (V) or joining (J) segment use in ALL patients compared to normal B cells. We observed that the N2 region was shorter than N1 in children whereas the opposite was observed in adults (not statistically significant). The J4 segment was a more common rearrangement in children than in adults, and in both groups J4 was more frequently associated with multiple D segment VDJ rearrangements. An increase in VH6 use in leukaemic alleles compared to normal B lymphocytes (2%) was observed but it was not statistically significant in our group of patients. Amongst children and adults, in-frame CDR3 junctions occurred in 78% and 64% of rearranged alleles, respectively, compared to 75% of in-frame sequences reported by others to occur among normal B cells.     

Multiple myeloma and chronic lymphocytic leukemia: parallels and contrasts.

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) are closely related B-cell cancers. Parallel and divergent features of these diseases are reviewed. In MM, expression of multiple hemopoietic lineage-associated antigens on the malignant cells and the substantial likelihood of progression to acute myelogenous leukemia suggest transformation of a pluripotent stem cell. In CLL, transformation more likely involves a committed B-cell progenitor. Another difference is that clonal evolution with associated cytogenetic progression is common in MM but not CLL. Other data, including studies of proto-oncogenes and tumor suppressor genes, suggest that MM results both from increased proliferation and accumulation of tumor cells, whereas tumor cell accumulation is the predominant feature of CLL. These differences may be reflected in the seemingly greater role of cytokine abnormalities in MM progression. For example, osteoclast-activating properties of some cytokines account for bone involvement in MM but not in CLL. MM and CLL share common features such as stage-dependent anemia and immune deficiency. Both diseases respond to alkylating agents but vary markedly in their sensitivity to fludarabine (CLL greater than MM) and glucocorticoids (MM greater than CLL). Differences between these diseases in progression-free interval and survival may reflect different definitions of premalignant and malignant phases rather than biologic differences. Detailed comparisons between MM and CLL may provide additional insights into these and related B-cell cancers.     

Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species.

The immunoglobulin heavy-chain variable region (VH) locus in a phylogenetically primitive teleost (Elops saurus) has been characterized by a strategy that relied initially on cross-hybridization between genomic VH segments and a murine VH probe. Using a homologous (Elops) VH probe and DNA sequencing, this gene family has been shown to be complex and to contain overt pseudogenes. A homologous probe also has been used to isolate a full copy length cDNA containing constant (CH) as well as joining (JH) and VH regions. Genomic analyses using CH-, JH-, and VH-specific probes have demonstrated the presence of only a single hybridizing CH and several JH elements. JH-CH linkage is less than or equal to 3.6 kilobases (kb) and VH-CH linkage is less than or equal to 100 kb, as estimated by field-inversion gel electrophoresis. An additional VH family sharing less than 50% nucleotide identity with the prototype Elops VH sequence is described. Taken together, these results suggest that the immunoglobulin VH locus in a comparatively primitive teleost resembles the VH locus in mammals, but not that found in the more phylogenetically distant elasmobranchs. The evolutionary radiations of cartilaginous and bony fishes are associated with a dramatic change in the organization and, presumably, regulation of immunoglobulin genes. The origins of the modern VH gene locus can be traced to the primitive teleost fishes.     

Chronic lymphocytic leukemia patients with highly stable and indolent disease show distinctive phenotypic and genotypic features

Different biologic features have been associated with a more or less aggressive clinical course in chronic lymphocytic leukemia (CLL). In the present study, 20 patients with highly stable CLL observed at a single institution over a period of 10 to 23 years and who never required treatment were extensively characterized. The aim was to identify a distinct and reproducible biologic profile associated with disease stability that may be used to recognize at presentation CLL patients who are likely to have a very benign clinical course and for whom treatment is not indicated. The results obtained indicate that numerous parameters are closely associated with disease stability: a typical CLL morphology and immunophenotype, the lack of expression of the CD38 antigen, the mutated immunoglobulin (Ig) heavy (H) chain variable (V) pattern, the absence of p53 mutations, a CD4/CD8 ratio more than 1, the lack of 17p and 11q deletions and of complex karyotypic aberrations, and the occurrence of the 13q14 deletion. No case displayed the VH3-21 gene, linked in mutated CLL with a poor outcome. In addition, the VH1-69 gene associated with unmutated CLL cases was never detected. These biologic features were coupled with an indolent clinical course characterized by an unmodified clinical stage over time, and by lack of autoimmune phenomena and of major infections requiring parental antibiotics. At a time when aggressive therapeutic strategies are always more frequently used in the management of CLL, the distinctive features of patients with long-lived stable disease should be prospectively identified at presentation.