Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors.

To better understand the stage(s) of differentiation reached by B-type chronic lymphocytic leukemia (B-CLL) cells and to gain insight into the potential role of antigenic stimulation in the development and diversification of these cells, we analyzed the rearranged VH genes expressed by 83 B-CLL cells (64 IgM+ and 19 non-IgM+). Our results confirm and extend the observations of a bias in the use of certain VH, D, and JH genes among B-CLL cells. In addition, they indicate that the VH genes of approximately 50% of the IgM+ B-CLL cells and approximately 75% of the non-IgM+ B-CLL cells can exhibit somatic mutations. The presence of mutation varies according to the VH family expressed by the B-CLL cell (VH3 expressers displaying more mutation than VH1 and VH4 expressers). In addition, the extent of mutation can be sizeable with approximately 32% of the IgM+ cases and approximately 68% of the non-IgM+ cases differing by > 5% from the most similar germline gene. Approximately 20% of the mutated VH genes display replacement mutations in a pattern consistent with antigen selection. However, CDR3 characteristics (D and JH gene use and association and HCDR3 length, composition, and charge) suggest that selection for distinct B cell receptors (BCR) occurs in many more B-CLL cells. Based on these data, we suggest three prototypic BCR, representing the VH genes most frequently encountered in our study. These data suggest that many B-CLL cells have been previously stimulated, placing them in the "experienced" or "memory" CD5(+) B cell subset.

Examples of in vivo isotype class switching in IgM+ chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5+ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur. To test the hypothesis that in vivo isotype class switching occurs in IgM+ B-type CLL cells, we analyzed the PBMC of 19 CLL patients for the presence of transcripts encoding the rearranged CLL V(H)DJ(H) associated with either gamma or alpha H chains. The molecular data indicate that approximately 50% of B-CLL patients have amplifications of IgM+ B cells that undergo an isotype class switch. Switching to IgA appears to occur more often than to IgG; also, switching can involve different IgG subclasses in individual patients. In many instances, these CLL-related gamma and alpha transcripts are much more plentiful than those of normal B cells that produce the same isotype. These switched transcripts do not reveal evidence for the accumulation of significant numbers of new V(H) gene mutations. The cellular data indicate that B cells with lesser amounts of surface membrane IgD and higher IgM/IgD ratios are more likely to undergo this switching process. Furthermore, B cells expressing IgG and IgA of the same idiotype or V(H) family and the same CDR3 length as those of the CLL IgM+ clone can be identified in the blood of patients studied using multiparameter immunofluorescence analyses. Collectively, these data suggest that not all members of a B-CLL clone are frozen at the surface membrane Ig-expressing stage of B cell maturation, and that some members can switch to the production of non-IgM isotypes. The occurrence of switching without the accumulation of V gene mutations indicates that the processes of differentiation and diversification are not linked.

Cytokine gene expression in neoplastic B cells from human mantle cell, follicular, and marginal zone lymphomas and in their postulated normal counterparts.

Cytokines may promote tumor growth by paracrine and/or autocrine pathways. Little information is available because malignant cells differ from their normal counterparts for the cytokine repertoire they express. Here we have investigated by reverse transcription-PCR the expression of 22 cytokine genes in neoplastic B lymphocytes from six patients with mantle cell lymphoma, 10 with follicular lymphoma, and 5 with marginal zone lymphoma and in their normal counterparts, i.e., naive, germinal center, and memory B cells, purified from tonsils. The overall profiles of cytokine gene expression in neoplastic B cells and in the corresponding normal B-cell subsets were similar, but some "holes" in the repertoire of malignant versus normal B lymphocytes were detected. Different "hole" combinations were identified consistently in mantle cell lymphoma, follicular lymphoma, and marginal zone lymphoma, thus representing molecular fingerprints of each individual lymphoma entity.

Immunoglobulin V region gene use and structure suggest antigen selection in AIDS-related primary effusion lymphomas.

Primary effusion lymphoma (PEL) is a lymphoproliferation of B cells infected by Kaposi's sarcoma-associated herpes-virus/human herpesvirus-8 and reflecting a late stage of B cell differentiation close to plasma cell. Apart from viral infection, the pathogenesis of PEL is currently unclear. The aim of the present study was to investigate the role of antigen stimulation and selection in the evolution of PEL. In order to assess the specific variable heavy (VH) and light (VL) genes used by PEL and to define the heavy and light chain isotypes expressed by these lymphomas, immunoglobulin (Ig) genes from seven AIDS-related PEL were sequenced (three cell lines and four primary samples). Most of the samples (five out of seven) used lambda light chain genes; the majority of these (n = 4) belonged to the V lambda 3 family. Two cases expressed mu chains, whereas gamma chains were found in two cases. In all cases, significant deviations from the presumed germline counterpart were found in both the expressed VH and VL genes. Statistical evidence for antigen selection was evident in four out of seven samples studied. Evidence for selection was more frequent in the light chain genes than in the heavy chain genes. Collectively, these data indicate that PEL originate from mature, antigen-experienced B cells and bear implications for the pathogenesis and histogenesis of this lymphoma.

On-line urea monitoring: a further step towards adequate dialysis prescription and delivery.

The aim of this study is to present a clinical experience carried out with a new device designed to measure on-line Urea Nitrogen concentration in the effluent dialysate. The Biostat 1000 Urea Monitor (Baxter Healthcare, Dirfield, Ill, USA) was utilized in the present study. The monitor is based on the principle that multiple urea measurements in the dialysate effluent from the dialyzer, permit to built a double exponential regression leading to the urea kinetic parameters of the dialysis session. Data obtained with the Urea Monitor were, in the present study, compared with those obtained by direct measurements carried out in blood and dialysate and by the collection of the whole amount of spent dialysate. The monitor provided an accurate value of predialysis BUN without any blood drawing. Urea kinetics were established from multiple dialysate measurements and no blood drawing was necessary. The double pool kinetics were taken into account and Kt/V, PCR and SRI% obtained were comparable to those obtained from direct measurement. Since a projected value of Kt/V can be obtained, the monitor could represent a potential source of information to detect possible filter and machine dysfunction, as well as high rate of recirculation.

N-(4-hydroxyphenyl)retinamide promotes apoptosis of resting and proliferating B-cell chronic lymphocytic leukemia cells and potentiates fludarabine and ABT-737 cytotoxicity.

The in vitro effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide) on primary B-cell chronic lymphocytic leukemia (CLL) cells from previously untreated CLL patients were investigated. 4HPR promoted the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation and was accompanied by drop of Mcl-1 protein expression. The latter was not attributable to transcriptional downregulation but to protein degradation mediated by jun N-terminal kinase activation, and likely by NF-kB downregulation and Noxa upregulation. CLL cells stimulated in vitro with CD40L did not increase 4HPR chemoresistance if activation was accompanied by proliferation. Intra-patient analysis confirmed that the proliferating pool of CLL cells was more sensitive to the cytotoxic action of 4HPR than the activated but resting CLL subpopulation. The different 4HPR susceptibility of the two subpopulations was associated with higher Noxa expression in proliferating CLLs. Combination experiments revealed that 4HPR strongly potentiated ABT-737 cytotoxicity, especially in proliferating CLL cells that displayed amplified chemoresistance to ABT-737 alone. Synergic cytotoxicity was also demonstrated in combination with fludarabine, in both resting and stimulated CLL samples. This study entitles 4HPR to be assayed as a chemotherapeutic adjuvant for the treatment of CLL.

Analysis of the hepatitis B virus genome and immune response in HBsAg, anti-HBs positive chronic hepatitis.

Although the development of antibodies against the hepatitis B virus surface antigen generally leads to the clearance of the infecting virus, anti-HBs reactivity has been reported in patients with chronic hepatitis. In the present study we analyzed the viral genome and the antibody specificity in a series of serum samples collected from a patient who seroconverted to anti-HBs during interferon therapy without clearing HBsAg. The appearance of an anti-HBs response was accompanied by the emergence of a pre-S1 defective viral genome. However, the wild-type adw2 molecular species remained largely dominant during follow up. The patient's antibody response to the surface viral antigens was directed towards the heterologous y subdeterminant and the pre-S1 fragment deleted in the variant hepatitis B virus. These results suggest that the selection of the escape viral mutant does not play a major role in viral persistence.

Functional and molecular characterization of tumour-infiltrating lymphocytes and clones thereof from a major-histocompatibility-complex-negative human tumour: neuroblastoma.

Neuroblastoma (NB) is a major-histocompatibility-complex(MHC)-negative neuroectodermal tumour that is often infiltrated with lymphocytes. A detailed characterization of NB-associated tumour-infiltrating lymphocytes (TIL) has never been carried out. Here we have investigated the immunophenotype and the cytotoxic activities of TIL from nine and seven NB patients respectively. Furthermore, the T cell receptor (TcR) variability and the patterns of cytokine gene expression of fresh versus recombinant (r) interleukin (IL)-2-cultured TIL were studied in four NB cases. The results obtained showed the following: (1) freshly isolated TIL were comprised of a mixture of CD4+ and CD8+ T cells partially expressing HLA-DR and/or CD25. The CD4/CD8 ratio ranged from 0.5 to 5 in the different cases. Upon culture of TIL with rIL-2, an increased proportion of CD56+ and CD8+ lymphocytes was consistently observed; (2) IL-2-expanded TIL lysed natural-killer(NK)sensitive and lymphokine-activated-killer(LAK)-sensitive target cell lines; (3) reverse-transcriptase/polymerase-chain-reaction (RT-PCR) experiments showed that most TcR V beta genes were expressed both in fresh and in cultured TIL, suggesting that such cell populations were polyclonal; (4) interferon gamma, IL-4, IL-5, tumour necrosis factor (TNF) alpha, IL-8, IL-10 mRNA and, to a lesser extent, IL-2 mRNA were expressed by cultured TIL, as assessed by RT-PCR; the corresponding tumour samples consistently contained TNF alpha, IL-8 and IL-10 mRNA, whereas IL-2 and IFN gamma mRNA were faintly expressed in some NB tumours and IL-4 and IL-5 mRNA were never detected. A total of 90 clones were subsequently raised from IL-2-expanded TIL from six NB patients; 87/90 clones were of T cell lineage with a CD4+ or CD8+ immunophenotype, whereas the 3 remaining clones were of NK cell origin. Upon triggering of the CD3-TcR complex, 64% CD4+ and 77% CD8+ T cell clones killed the murine P815 mastocytoma cell line. Virtually no T cell clone lysed a LAK-sensitive NB cell line whereas 15% CD4+ and 17% CD8+ clones mediated NK-like activity against the K562 cell line. Finally, the patterns of cytokine production by CD4+ clones were roughly consistent with those of a T helper (TH) 1 profile and similar to those observed in CD8+ clones.

Toxoplasma gondii-specific CD4+ T cell clones from healthy, latently infected humans display a Th0 profile of cytokine secretion.

Human Toxoplasma gondii (Tg)-specific T cell clones were raised by infecting peripheral blood mononuclear cells (MNC) from two healthy, latently infected individuals with Tg trophozoites. All of the clones had a CD4+ immunophenotype and produced simultaneously interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 upon mitogen or antigen stimulation. Tg-specific T cell clones were classified as T helper of type 0 (Th0) since most of them released roughly comparable amounts of IFN-gamma and IL-4. In some clones, a trend to an increased production of IFN-gamma following antigen-specific as compared to non-specific stimulation was observed. The Th0 phenotype was also expressed by T cell clones that had been raised from bulk cultures performed in the presence of IL-4 or IFN-gamma. All of the Tg-specific T cell clones were cytolytic in a non-specific assay which involves the triggering of the CD3-T cell receptor (TcR) complex. Some clones specifically lysed an autologous lymphoblastoid cell line (LCL) that had been infected with Tg trophozoites. Finally, most of the Tg-specific T cell clones produced IL-10, irrespective of whether they had been raised from bulk cultures incubated in the presence or absence of IL-4 or IFN-gamma. Taken together, these findings suggest that Tg-specific Th0 helper cell clones from healthy, latently infected individuals, beside activating toxoplasmacidal mechanisms through IFN-gamma release, might limit the magnitude of the immune response of the parasite by killing Tg-infected antigen-presenting cells and by releasing IL-10.

Synovial fluid T cell clones from oligoarticular juvenile arthritis patients display a prevalent Th1/Th0-type pattern of cytokine secretion irrespective of immunophenotype.

The aim of the present study was to investigate the patterns of cytokine production by T cell clones raised from in vivo activated synovial fluid (SF) mononuclear cells (MNC) of five patients with oligoarticular juvenile arthritis (JA). Freshly isolated SF T cells were cultured in vitro with low dose recombinant IL-2 and subsequently cloned by limiting dilution. Sixty-four clones were obtained from the five patients studied. Fifty-nine clones were TCR alpha/beta+, either CD4+ (n = 43) or CD8+ (n = 15). The remaining five clones were TCR gamma/delta+, CD4-, CD8-. Clone immunophenotypes differed in the individual patients. Forty-four T cell clones were stimulated with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and supernatants tested for the presence of IL-2, IL-4, IL-5 and interferon-gamma (IFN-gamma) by ELISA or bioassays. Cytokine mRNA accumulation was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). Most of 44 clones tested released large amounts of IFN-gamma irrespective of the immunophenotype. Of these, 27 were classified as Th1-type and 17 as Th0-type based upon the IFN-gamma/IL-4 ratio in culture supernatants. Finally, when 10 representative T cell clones were tested for pro- and anti-inflammatory cytokines, gene expression by RT-PCR, all of them were found to express the granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha), IL-10 and transforming growth factor-beta 1 (TGF-beta1) genes, and half of them IL-6 and IL-8 mRNA. In conclusion, T cell clones, that represent the progeny of in vivo activated SF T cells from oligoarticular JA patients, display heterogeneous immunophenotypes, but all share the ability to produce large amounts of IFN-gamma, with a predominant Th1/Th0 pattern. The expression of pro- and anti-inflammatory cytokine genes in these clones suggests that in vivo activated SF T cells modulate joint inflammation in a complex fashion.

A novel human homologue of the SH3BGR gene encodes a small protein similar to Glutaredoxin 1 of Escherichia coli.

Glutaredoxins (GRXs) are ubiquitous GSH-dependent oxidoreductases, which catalyze the reduction of protein-glutathionyl-mixed disulfides and are considered to play an important role in the enzymatic regulation of redox-sensitive proteins. In this paper, we describe the identification and characterization of a new human homologue of the SH3BGR gene, named SH3BGRL3 (SH3 domain binding glutamic acid-rich protein like 3). SH3BGRL3 is widely expressed and codes for a highly conserved small protein, which shows a significant similarity to Glutaredoxin 1 (GRX1) of Escherichia coli and is predicted to belong to the Thioredoxin Superfamily. However, the SH3BGRL3 protein lacks both the conserved cysteine residues, which characterize the enzymatic active site of GRX. This structural feature raises the possibility that SH3BGRL3 could function as an endogenous modulator of GRX biological activity. EGFP-SH3BGRL3 fusion protein expressed in COS-7 cells localizes both to the nucleus and to the cytoplasm. The SH3BGRL3 gene was mapped to chromosome 1p34.3-35.

Recombinant tumor necrosis factor enhances the locomotion of memory and naive B lymphocytes from human tonsils through the selective engagement of the type II receptor.

Recent studies performed in mice knocked out for the tumor necrosis factor (TNF ), the lymphotoxin-alpha, or the type I TNF receptor (R), genes have shown that these animals display gross defects in germinal center (GC) formation, suggesting that members of the TNF and TNFR superfamilies are involved in the control of B-cell migration. Based on these premises, we have here investigated the effects of human recombinant (r) TNF on the polarization and locomotion of tonsillar B cells. rTNF increased the spontaneous polarization and locomotion of unfractionated tonsillar B lymphocytes in a dose-dependent manner by inducing a true chemotactic response. Memory (IgD-, CD38(-)) and naive (IgD+, CD38(-)), but not GC (IgD-, CD38(+)) B cells purified from total tonsillar B lymphocytes, showed a significantly higher locomotion in the presence than in the absence of rTNF. Accordingly, type I and II TNF receptors (TNFRs) were detected by flow cytometry on the surface of memory and naive, but not GC, B lymphocytes. Blocking experiments with monoclonal antibodies to type I or II TNFR showed that rTNF enhanced the spontaneous chemotaxis of memory and naive B cells through the selective engagement of type II TNFR. Finally, the TNF gene was found to be expressed in memory, naive and GC B lymphocytes; the cytokine was released in culture supernatants from the three B-cell subsets after stimulation. These data may support the hypothesis that human TNF is involved in the paracrine and perhaps autocrine control of B-cell migration in secondary lymphoid tissues.

Cell activation via CD44 occurs in advanced stages of squamous cell carcinogenesis.

Squamous cell carcinoma (SCC) derives from dysplastic or metaplastic stratified epithelia. The process of squamous cell carcinogenesis has been investigated for the potential role of the adhesion molecule CD44, whose standard form (CD44s) and isoforms generated by alternative splicing of variant exons are known to display altered expression during tumorigenesis in other systems. We have utilized an in vitro correlate of squamous cell carcinogenesis, in which progression stages from normal squamous epithelium to dysplastic lesions and to SCC are represented by primary cultures of normal keratinocytes, by human papilloma virus-immortalized keratinocytes (UP) and by HPVimmortalized/v-Ha-ras transfected tumorigenic keratinocytes (UPR). We investigated expression of CD44 and of variant isoforms, from mRNA to intracellular and surface protein levels, and found no relationship between expression of CD44 and stages of squamous cell carcinogenesis. However, when the function of CD44 was analyzed as Ca(2+) mobilization ability upon monoclonal antibody binding and crosslinking, signal transduction via CD44 was found only for the neoplastic stage (UPR cells). Ca(2+) mobilization was completely independent of density of surface CD44. We have performed similar analyses in an in vitro model of SCC in which four squamous tumor cell lines and UPR cells were sorted according to increasing resistance to external cytotoxic stimuli, i.e. starving conditions, treatment with the retinoid N-(4-hydroxyphenyl)retinamide and cytolytic activity of effector lymphokine-activated killer cells. No relationship between expression of CD44 and level of cell resistance against external cell death-inducing stimuli was found, while CD44-mediated Ca(2+) mobilization ability was restricted to the highly resistant tumor cell lines. Our results indicate that the role(s) of CD44 in squamous cell proliferative disorders can be evinced from the functional features of the molecule, rather than from its phenotypic repertoire.

The patterns of IL2, IFN-gamma, IL4 and IL5 gene expression in Hodgkin's disease and reactive lymph nodes are similar.

BACKGROUND AND OBJECTIVE: The lymph nodes involved in classic Hodgkin's disease (HD), i.e. mixed cellularity (MC) and nodular sclerosis (NS) subtypes, usually contain few (1-2%) Reed-Sternberg (RS) cells scattered in a background of lymphocytes, eosinophils, plasma cells and neutrophils. CD4+ T-lymphocytes are increased in number, express activation markers and cluster around RS cells. The presence of eosinophilia in most HD patients and the presence of hyper-IgE in a subset of them may suggest that activated lymph node T cells release large amounts of IL5 and IL4, respectively. METHODS: The expression of four T-cell-associated cytokine genes, i.e. interleukin (IL)2, IL4, IL5 and interferon (IFN)-gamma, in frozen sections of 14 HD (7 MC, 7 NS) and 10 reactive lymph nodes was investigated by qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). T-cell clones were also raised from purified CD4+ lymphocytes of one HD lymph node and one reactive lymph node and tested for IL2, IL4, IL5 and IFN-gamma secretion in culture supernatants by immunoassays. RESULTS: The transcripts of all the cytokine genes were detected in every lymph node irrespective of the HD or reactive nature. HD or reactive lymph node-derived CD4+ T-cell clones released the four cytokines according to a predominant T-helper (Th)0-type pattern. In more than half of the lymph nodes of either HD or reactive nature, there was a predominance of IL4 over IFN-gamma mRNA production (Th2-type pattern). In the remaining HD or reactive lymphadenopathies, either a balanced IL4/IFN-gamma mRNA ratio (Th0-type pattern) or a predominance of IFN-gamma over IL4 mRNA expression (Th1-type pattern) was observed. INTERPRETATION AND CONCLUSIONS: The overall pattern of cytokine gene expression in classic HD is similar to that detected in reactive lymph nodes. Further studies are needed to determine whether differences in the absolute concentrations of cytokines released in HD versus reactive lymph nodes and the long-standing course of HD versus the self-limiting nature of reactive adenopathies may explain certain peculiar features of HD, such as eosinophilia, for example.

Cytokine gene expression and T-cell proliferative responses in lymph node mononuclear cells from children with early stage human immunodeficiency virus infection.

BACKGROUND AND OBJECTIVES: The immunologic events taking place in secondary lymphoid tissue from children with early stage human immunodeficiency virus (HIV) infection are poorly understood. The aim of this study was to investigate cytokine gene expression and proliferative responses in lymph node (LN) biopsies from five children with early stage HIV infection, in the context of LN morphology and viral load. DESIGN AND METHODS: The design of the study was approved by the local Ethical Committee. Cytokine gene expression was studied in LN biopsies and in paired peripheral blood (PB) samples from HIV-infected children by reverse transcriptase-polymerase chain reaction. T-cell proliferation was assessed by 3H-thymidine incorporation. Viral burden in germinal centers was assessed by video densitometric analysis following immunohistochemical staining for HIV p24. RESULTS: Interleukin (IL)-2, IL-4 and IL-5 mRNA were not detected in any LN or PB sample from HIV-infected children. Interferon (IFN)-gamma mRNA was found only in CD8+ cells. IL-12 p35, IL-10, transforming growth factor-(TGF)-beta1, regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and IL-16 transcripts were detected in all samples. Proliferation of LN and PB mononuclear cells to polyclonal mitogens and soluble (recall and HIV-related) antigens was impaired as compared with the responses in a group of age-matched healthy controls. INTERPRETATION AND CONCLUSIONS: Changes in cytokine gene expression and T-cell proliferative responses are already detectable in lymph nodes from HIV-infected children at an early stage of disease.

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions.

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.

Cellular immunophenotypic studies were performed on a cohort of randomly selected IgM(+) B-chronic lymphocytic leukemia (B-CLL) cases for which Ig V(H) and V(L) gene sequences were available. The cases were categorized based on V gene mutation status and CD38 expression and analyzed for treatment history and survival. The B-CLL cases could be divided into 2 groups. Those patients with unmutated V genes displayed higher percentages of CD38(+) B-CLL cells (>/=30%) than those with mutated V genes that had lower percentages of CD38(+) cells (<30%). Patients in both the unmutated and the >/=30% CD38(+) groups responded poorly to continuous multiregimen chemotherapy (including fludarabine) and had shorter survival. In contrast, the mutated and the <30% CD38(+) groups required minimal or no chemotherapy and had prolonged survival. These observations were true also for those patients who stratified to the Rai intermediate risk category. In the mutated and the <30% CD38(+) groups, males and females were virtually equally distributed, whereas in the unmutated and the >/=30% CD38(+) groups, a marked male predominance was found. Thus, Ig V gene mutation status and the percentages of CD38(+) B-CLL cells appear to be accurate predictors of clinical outcome in B-CLL patients. These parameters, especially CD38 expression that can be analyzed conveniently in most clinical laboratories, should be valuable adjuncts to the present staging systems for predicting the clinical course in individual B-CLL cases. Future evaluations of new therapeutic strategies and drugs should take into account the different natural histories of patients categorized in these manners.