Persistent Polyclonal B-cell Lymphocytosis Research Articles

IgM interference in determination of antiphospholipid antibodies and erythrocyte sedimentation rate in persistent polyclonal B-cell lymphocytosis with binucleated lymphocytes

IgM interference in determination of antiphospholipid antibodies and erythrocyte sedimentation rate in persistent polyclonal B-cell lymphocytosis with binucleated lymphocytes

Pangenomic Study In Patients with Persistent Polyclonal Binucleated B-Cell Lymphocytosis (PPBL)

AbstractAbstract 2405 Introduction.PPBL is a rare disease characterized by a chronic, stable, persistent and polyclonal B-cell lymphocytosis, the presence of binucleated lymphocytes in the peripheral blood and a polyclonal increase in serum immunoglobulin-M. We showed that PPBL was associated with recurrent chromosomal abnormalities and a typical cytogenetic profile including additional extra isochromosome +i (3)(q10) and premature chromosome condensation (PCC). The etiology of PPBL remains unclear. The probable role of tobacco has to be elucidated. Environment factors and persistent viral infection, particularly Epstein-Barr Virus infection, could be involved. Moreover, the possibility of PPBL to evolve toward a clonal proliferation, malignant lymphoma or secondary solid cancer led us to consider PPBL as a premalignant state. Isochromosome i (3)(q10) is routinely detected by conventional cytogenetic analysis (CCA) associated with fluorescent in situ hybridization (FISH) in 71% of the cases. In the peripheral blood, the polyclonal expansion of B-cells is variable, usually low with a mean of 49% of the lymphocytes (7–83%). Material and methods.In order to study more extensively cytogenetic abnormalities, we used Affymetrix™ Cytogenetics Whole-Genome 2.7M Arrays® in 7 typical PPBL patients. The DNA was extracted from peripheral CD19+ B-cells and CD19− cells purified using Miltenyi™ technology (AutoMACS Pro Separator®). The patients had a median age at 49 years (40–59), with a sex ratio at 2:7, smoker in 7/7. Using CCA/FISH, we detected +i (3)(q10) and PCC in 5/7 and 2/7 patients respectively. CD19+ B-cells were separated by immunomagnetic cell sorting with a purity of 95% (88% to 98%). We performed DNA arrays on CD19+ and CD19− cells in 4 patients, CD19+ cells in 2 patients and CD19− cells in 1 patient. Results.(1) Recurrent gene copy number (GCN) gains were detected on the long arm of chromosome 3 (3q) and identified only in CD19+ cells. Recurrent GCN gains were observed in 552/595 (92.8%) genes of 3q. GCN gains concerned either the totality of 3q or only telomeric part of 3q (3q13.3-3qter) or non consecutive genes on 3q. Moreover, we observed that GCN gains did not involve all the CD19+ B-cells (mosaicism phenomenon). (2) We observed in CD19+ B-cells other recurrent GCN aberrations, including 5 gains on Xp (3 patients) and 16 losses on 6q (2 patients). Non recurrent GCN aberrations involving the whole genome, except chromosomes 5 and 22, confirmed the presence of genetic instability in all patients. (3) Out of the genes amplified on 3q, a few genes have been reported to be implicated in hematological diseases (MLF1/LPP/ATR/BCL6). Interestingly, we identified with a high frequency partial or complete amplification of MDS1 and EVI1 complex locus (MECOM)(3q26.2). Conclusion.(1) +i (3)(q10) identified by CCA is not a true complete duplication of 3q and can be associated with other recurrent GCN aberrations on chromosomes 6q and Xp. (2) Whereas MDS1 rearrangement (inversion or translocation) has been reported in cases of malignant myeloid disease (MDS, AML) as a poor prognosis factor, the role of MDS1 in lymphoid malignancies has to be elucidated. We are getting MDS1 quantitative PCR and direct sequencing. (3) We confirmed genetic instability in PPBL motivating a long term follow-up of PPBL patients. Disclosures:No relevant conflicts of interest to declare.

Histopathological and molecular features of persistent polyclonal B‐cell lymphocytosis (PPBL) with progressive splenomegaly

Five cases of persistent polyclonal B-cell lymphocytosis (PPBL) with progressive splenomegaly are reported; three were splenectomized. BCL2/IGH rearrangements were found in three cases; HLA-DRB1*07 in all. Bone marrow (BM) trephines showed a moderate lymphoid infiltrate with intrasinusoidal distribution resembling a splenic marginal-zone lymphoma. Splenic white pulp revealed an enlargement of the marginal-zone area; red pulp was infiltrated by the same lymphocytes engulfing the sinuses. Splenic and BM B-lymphocytes were CD79a(+)/CD20(+)/IgM(+)/IgD(+)/bcl-2(+)/CD27(+)/DBA.44(-)/CD31(-) and polyclonal by immunophenotype/polymerase chain reaction. PPBL features an expansion of splenic marginal-zone B-lymphocytes, which infiltrate BM sinusoids and circulate in the blood with no evidence of clonality, even in cases with progressive splenomegaly.

Selective defect of anti-pneumococcal IgG in a patient with persistent polyclonal B cell lymphocytosis

Background Persistent polyclonal B cell lymphocytosis (PPBL) is a rare condition characterized by increased IgM and large excess of B cells with an IgD + CD27 + phenotype. In normal individuals, these cells play a central role in the defense against pneumococcal infection. So far, few studies have characterized humoral immune responses in PPBL patients. We therefore measured IgG directed against S. pneumoniae antigens in a 51 yr-old woman with PPBL before and after vaccination with a pneumococcal 23-valent polysaccharide vaccine. Methods Antibodies against pneumococcal antigens were measured first with an overall immunoassay using microplates coated with the 23-valent pneumococcal vaccine. A serotype-specific test was also performed according to the WHO consensus protocol. Results Despite a large number of IgD + CD27 + cells, our patient had low baseline titers of IgG directed against pneumococcal antigens and did not significantly respond to a 23-valent polysaccharide vaccine against S. pneumoniae. On the contrary, she had good titers of IgG directed against tetanus toxoid. Conclusion IgM + IgD + CD27 + cells which accumulate in this patient with typical PPBL patient failed to perform IgG isotype switch after a polysaccharide vaccine. The potential mechanisms and relationships with the main features of PPBL are discussed. Further studies on a larger number of similar patients are needed.

Polyclonal B-cell lymphocytosis with binucleated lymphocytes (PPBL).

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare and recently described entity. The review of the literature show PPBL is diagnosed predominantly but not exclusively in women, usually smokers. PPBL is recognized by a moderate, chronic and absolute lymphocytosis (>4 × 10(9)/l) in the peripheral blood. In 10% of cases without lymphocytosis, the PPBL diagnosis has to be suggested by peripheral blood examination showing in all cases atypical binucleated lymphocytes. A polyclonal serum IgM is also associated and HLA-DR7 expression is present in most cases. Contrary to B-cell chronic lymphoproliferative disorders (B-CLPD), peripheral B cells are polyclonal with kappa and lambda light-chain expression and no clonal rearrangement of immunoglobulin heavy chain genes is usually demonstrated. The detection of an extra isochromosome for the long arm of chromosome 3 +i(3)(q10) has to be considered as a specific marker of PPBL. We performed conventional cytogenetic analysis (CCA) in 111 patients with typical PPBL we followed-up more than 4 years. +i(3q) was detected in 34% (33/98), PCC in 8% (8/98) and both abnormalities in 31% (30/98). CCA showed neither +i(3q) nor PCC in 28% (27/98). Fluorescence in situ hybridization (FISH) was also performed in 84 cases and +i(3q) was detected in 71% (60/84). When combining both procedures in 84 patients, +i(3q) was detected in 17 patients with negative CCA and was confirmed in 43 patients with positive CCA. CCA and FISH were both negative in 24 cases. Whether patients with PPBL are at increased risk of hematological malignancy remains unclear. After a median follow-up of 4.4 years, most PPBL patients presented a stable clinical and biological course. Six patients died from pulmonary cancer, myocardial infarction, cerebral aneurysm rupture or diffuse large B-cell lymphoma. Two patients had IgM monoclonal gammopathy of undetermined significance (MGUS) at the time of PPBL diagnosis and two other patients developed IgM MGUS respectively 12 and 22 years after PPBL diagnosis. A malignant non Hodgkin's lymphoma (NHL) appeared in 3 additional patients: two patients presented diffuse large B cell lymphoma and 1 patient a splenic marginal zone lymphoma. In conclusion, the possibility of PPBL to evolve toward a clonal proliferation, malignant lymphoma or secondary solid cancer lead us to consider PPBL not as a benign pathology. We recommend a careful and continued clinical and biological long-term follow-up in all PPBL patients.

Polyclonal B-cell lymphocytosis with hairy cell appearance: hairy B-cell lymphoproliferative disorder

A 72-year-old woman was referred to our hospital for evaluation of leukocytosis revealed by a medical examination. Her physical examination demonstrated no splenomegaly and no palpable lymph nodes. Her white cell count was 10,900/microl with atypical lymphocytosis (84.5%). Her hemoglobin concentration was 10.4 g/dl, and platelet count 151,000/microl. On peripheral blood smears, the atypical lymphocytes had a hairy cell-like appearance, and phase-contrast microscopic and transmission electron microscopic findings revealed the lymphocytes had many long surface microvilli. Flowcytometric analysis of peripheral blood lymphocytes identified expanded B-lymphocytes as having the IgG+, CD5- CD10- CD11c+ CD19+ CD20+ CD23- CD25- and CD103- cell surface phenotype. Serum electrophoresis disclosed polyclonal elevation of IgG and IgM (2620 mg/dl and 840 mg/dl, respectively). No light-chain restriction and a polyclonal VH gene rearrangement pattern indicated the polyclonal proliferation of B cells. The patient was a nonsmoker and had HLA-DR4, as in previous reports which have suggested an association between hairy B-cell lymphoproliferative disorder (HBLD) and HLA-DR4. No chromosome 3 abnormality was observed. These findings were consistent with the characteristics of HBLD, but differed in some respects from those of persistent polyclonal B-cell lymphocytosis (PPBL). Therefore, we diagnosed this patient as having HBLD.

Expression profiling of persistent polyclonal B-cell lymphocytosis suggests constitutive expression of the AP-1 transcription complex and downregulation of Fas-apoptotic and TGFβ signalling pathways

Expression profiling of persistent polyclonal B-cell lymphocytosis suggests constitutive expression of the AP-1 transcription complex and downregulation of Fas-apoptotic and TGFβ signalling pathways

Long-term follow-up of 111 patients with persistent polyclonal B-cell lymphocytosis with binucleated lymphocytes

Long-term follow-up of 111 patients with persistent polyclonal B-cell lymphocytosis with binucleated lymphocytes

Combined automated cell and flow cytometric analysis enables recognition of persistent polyclonal B-cell lymphocytosis (PPBL), a study of 25 patients

Persistent polyclonal B-cell lymphocytosis (PPBL) is an extremely rare disorder, which occurs almost exclusively in smoking women and is characterized by a lymphocytosis with circulating binucleated lymphocytes. We analyzed 25 PPBL patients with respect to immunophenotype and by adaptive cluster analysis system (ACAS). Furthermore, HLA type, presence of Epstein-Barr virus (EBV) DNA in B cells, and clinical data were evaluated. Overall, the median percentages of B cells in PPBL patients with expression of CD5(dim), CD23(dim), CD25, CD27, and FMC7 were 21%, 38%, 16%, 74%, and 93%. Compared to normal controls, ACAS revealed a subset of nucleic-acid-rich lymphocytes located between the regular lymphocyte and regular monocyte region. Sixteen (64%) of 25 patients carried a HLA DR7 phenotype. Quantitative real-time polymerase chain reaction analysis did not detect relevant amounts of EBV DNA in circulating B cells of any patient. During a median follow-up of 5 years, a single patient developed lymphoplasmacytic lymphoma. The abnormal morphology and frequent, albeit dim, expression of CD5 and CD23 in PPBL may result in erratic diagnostic assignment of this benign disorder. However, incorporation of immunophenotyping and ACAS into the diagnostic algorithm allows recognition of PPBL in routine analysis and its differentiation from malignant B cell lymphoproliferative diseases. We found that an infection of a significant percentage of PPBL cells by EBV is unlikely. The observation of malignant lymphoma in a single patient implicates that evolution into a clonal malignant transformation may occasionally occur in PPBL.

High incidence of ancestral HLA haplotype 8.1 and monoclonal incomplete DH–JH immunoglobulin heavy chain gene rearrangement in persistent polyclonal B-cell lymphocytosis

High incidence of ancestral HLA haplotype 8.1 and monoclonal incomplete DH–JH immunoglobulin heavy chain gene rearrangement in persistent polyclonal B-cell lymphocytosis

Asymptomatische 32-jährige Raucherin mit persistierender Lymphozytose

A 32 year old female smoker (20 pack years) presented with an asymptomatic lymphocytosis of 13,000/nl and splenomegaly. The patient's blood smear showed an absolute lymphocytosis with 65% atypical lymphocytes. A total of 1% of the lymphocytes were bilobulated. Bone marrow histology and immunphenotyping of blood and bone marrow excluded leukemia and non-Hodgkin's lymphoma. IgH-CDR-3 PCR analysis revealed a polyclonal pattern. In summary, a persistent polyclonal B-cell-lymphocytosis (PPBL) was diagnosed. The exact etiology of PPBL is still unclear, however, it is associated with a polyclonal raise in the lymphocyte count of CD27+IgD+-memory-B-lymphocytes due to a defect in apoptosis signaling and leukocyte homing to secondary lymphoid tissues. An association with cigarette smoking is obvious since all patients are smokers. From all published cases, only two developed a malignancy with an uncertain association with PPBL. We have been monitoring our patient for 6.5 years without any evidence of the development of a lymphoma.

Persistent polyclonal B-cell lymphocytosis: a case report

Persistent polyclonal B-cell lymphocytosis: a case report

Gene Expression Profiling of Persistent Polyclonal B-Cell Lymphocytosis Using Whole Genome Microarrays.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare B-cell disorder characterised by elevated serum levels of polyclonal IgM and persistent lymphocytosis of often bi-nucleated B-cells. The molecular basis of this disorder however remains poorly understood. We therefore used whole genome expression microarrays (Affymetrix U133 plus2.0) to compare the gene expression profile of 14 cases of PPBL with normal lymphocyte populations (6 B cell (CD19+) and 3 T cell (CD3+) samples). PPBL patients (10 female, 4 male) had a median age of 43 (range 28–63) and all were smokers with an average lymphocyte count of 5.34 x 107/ml (range 3.7–9) at time of presentation. Eight of these cases had +i(3q) and eight cases exhibited PCC, four cases displayed both chromosomal abnormalities. Normal lymphocyte populations were obtained from two pools of 12 individuals and individual donors.Microarray data were first RMA pre-processed and then expression levels of patient samples' genes compared with levels of both normal B cell and T cell samples. ANOVA (P<0.01) was used to identify differentially expressed genes. Thirty-six genes were found to be up-regulated and 157 genes were down-regulated in all patient samples by at least 2-fold.Components of the AP-1 transcription complex (FOS, FOSB and JUN) were found to be highly up-regulated in all PPBL samples with an average of 7.2, 7.9 and 31.6 fold expression respectively. The cell cycle regulatory molecule CDC27 was also found to be up-regulated in all patient samples (8.8-fold). Down-regulated genes of interest included the B-cell specific transcription factor BACH2 (3-fold), tumor-suppressor SMAD4 (3.6-fold) and apoptotic genes DRAK1 (3-fold) and CAS (5-fold). These data are currently being validated by quantitative RT-PCR.We propose that over-expression of AP-1 may have significance in PPBL as increased expression of components of AP-1 have previously been linked with lymphoproliferative disorders including Hodgkin's lymphoma and splenic marginal zone lymphoma. The underlying reason for over-expression of AP-1 in PPBL remains unknown but two characteristic features of this disease could provide clues; firstly AP-1 up-regulation has been demonstrated to be tobacco-induced in animal models and secondly the LMP1 protein of EBV can result in constitutive expression of the AP-1 complex.

Histopathologic and Molecular Features of Persistent Polyclonal B-Cell Lymphocytosis Challenging the Benign Nature of the Disorder.

Persistent polyclonal B-cell lymphocytosis (PPBL) is an unusual expansion of B lymphocytes, whose immunophenotype resembles that of memory B cells. The physiopathology of PPBL is unknown and whether this syndrome represents a pre-malignant or benign disease is unclear. Aiming at further defining the histological features of BM involvement and at challenging the polyclonal nature of PPBL, we reviewed 6 cases of PPBL who underwent BM biopsy. There were 3 males and 3 females, with a median age at diagnosis of 35 years (range 17–46). The median lymphocyte count was 5.3 × 109/L (5.1–8.3). One patient had been splenectomized 10 years earlier and 2 were splenomegalic. During the follow-up (median 20 months, range 5–138), 1 showed progressive spleen enlargement and 2 additional patients developed splenomegaly. Screening for hepatitis B, C and HIV was negative. PB morphology showed 2 to 32% of cleaved lymphocytes. Immunophenotype documented 21 to 70% B lymphocytes in both PB and BM aspirate, all positive for CD19, CD22, CD79b, FMC7, CD18 and CD11a, negative for CD5, CD23, CD10, with no Ig light chain restriction. CD25 was expressed in 4 cases. PCR analysis of the IgH gene configuration of PB and BM cells showed a polyclonal picture in all cases. On Giemsa-stained sections of BM trephines, BM was normocellular in 5 cases and slightly hypocellular in 1. A mild lymphoid infiltrate was visible, mainly interstitial with occasional small lymphoid aggregates. An intrasinusoidal pattern was seen in 4/6 cases. Immunohistochemistry confirmed the interstitial infiltration by B cells (CD79a+, CD20+, IgM+, IgD+, DBA44−/+, CD5−, AnnexinD1−) in all cases and highlighted the intrasinusoidal pattern in 4 in which a splenic marginal zone lymphoma (MZL)-like morphology was described. By IgH gene PCR on BM tissue, a clonal rearrangement was detected in 2/4 cases with intrasinusoidal infiltration; in 2 cases DNA quality was poor. The 4 cases showed splenomegaly at diagnosis or during the follow-up. Splenectomy was performed in 1 patient due to progressive enlargement of the spleen over 9 years. White pulp was normal and red pulp was expanded by an infiltration of small lymphocytes with an intrasinusoidal distribution. Splenic B lymphocytes were CD79a+, CD20+, IgM+, IgD+, DBA44−, as in the BM biopsy, with low proliferation activity and proven clonality by IgH-PCR. In the remaining 2 cases, in which no intrasinusoidal lymphoid pattern was evident, clonal IgH rearrangement was absent and no splenomegaly has developed. We confirm the interstitial lymphoid infiltration of BM trephine in all cases of PPBL, with an evident intrasinusoidal component in some. The detection of IgH rearrangements on lymphocytes infiltrating the BM from bioptic samples and the absence of clonality in PB lymphocytes and in the BM aspirate, possibly due to the intrasinusoidal distribution, have not been described. These findings suggest that at least some of the cases described as PPBL are pre-malignant disorders close to splenic MZL, justifying the occurrence of progressive splenomegaly over time. Prolonged follow-up of patients with PPBL and an accurate biologic work-up of the BM, are therefore warranted.

HLA-DRB1 Analysis in Hairy B-Cell Lymphoproliferative Disorder (HBLD): A Japanese Type of Benign Persistent Polyclonal B-Cell Lymphocytosis.

Two types of benign persistent polyclonal B-cell Lymphocytosis have been reported: persistent polyclonal B-cell lymphocytosis (PPBL) in Western countries and hairy B-cell lymphoproliferative disorder (HBLD) in Japan. PPBL is characterized by B lymphocytosis with binucleated lymphocytes, polyclonal IgM, isolated splenomegaly, female sex, cigarette smoking and the presence of HLA-DR7 allele. Most patients with HBLD have atypical lymphocytes with microvilli and prominent membranous ruffles, serum polyclonal IgG, and splenomegaly. Only 9 patients with HBLD have been reported to date, and all of these patients are Japanese. Here, we report three new cases of HBLD. We performed HLA-DRB1 subtyping since the association between HBLD and HLA-DR4 has been suggested: 4 of 5 HBLD patients examined demonstrated the HLA allele. We also analyzed HLA-DRB1 alleles in a patient with hairy cell leukemia (HCL) and 3 with HCL Japanese variant (HCL-JV) to examine the possibility that polyclonal cell expansion in HBLD may be followed by transformation to these lymphoproliferative disorders. If the transformation were to occur, the patients should share the same HLA-DR allele.Expanded B-lymphocytes in all HBLD cases displayed CD5− CD10− CD11c+ CD19+ CD20+ CD23− CD25− and CD103− cell surface phenotype and all patients exhibited a polyclonal increase in serum IgG. Phase-contrast microscopic and scanning electron microscopic examinations demonstrated lymphoid cells with many long surface microvilli. Analysis of immunoglobulin VH gene rearrangements showed a polyclonal pattern. Therefore, we diagnosed these patients as HBLD. HLA-DRB1 subtype analysis strengthened the close relation between HBLD and HLA-DR4: 2 patients having HLA-DRB1*0405 and one HLA-DRB1*0407. In the meanwhile one each patient with HCL and HCL-JV had HLA-DRB1*0405 allele.Although the exact mechanism underlying the association between HBLD and HLAB1*04 needs to be elucidated, the presence of this HLA might be useful to establish correct diagnosis of the disease, and thus avoid patient exposure to unnecessary treatment. One each patient with HCL and HCL-JV has HLA-DRB1*0405, but a definitive conclusion cannot yet be drawn due to high HLA-DRB1*04 frequency in Japanese population (25.4%).

Persistent polyclonal B-cell lymphocytosis: a case report

Persistent polyclonal B-cell lymphocytosis: a case report

High Incidence of Ancestral HLA Haplotype 8.1 and Incomplete DH-JH Immunoglobulin Rearrangement in Persistent Polyclonal B-Cell Lymphocytosis.

Four women aged from 48 to 51 years with persistent polyclonal B-cell lymphocytosis (PPBCL) were studied for the clonality of B-cell population and HLA haplotype. All patients were smokers, one presented an immune deficiency syndrome due to decreased IgG and IgA levels, the others were asymptomatic. All patients presented serum increase of polyclonal IgM (5.55 – 8.12 g/L), absolute peripheral lymphocytosis ranging from 6100 to 10500/μL with elevated proportion of B cells, the percentage of CD5/CD19 lymphocytes < 5%, and binucleated/bilobulated lymphocytes accounting for 5–9% of nucleated cells on the blood smear. In one patient, cytogenetic study revealed trisomy 3 in 4 out of 40 metaphases, +18 in 2 cells, and i(18q), +15, t(13,14) in single cells. In the remaining patients no clonal chromosomal abnormalities were found. All patients were HLA typed by PCR based sequence specific primer amplification. HLA allele and haplotype incidence in patients' group were compared to healthy population of 286 ethnically matched (Caucasian, Polish) controls. DRB1*07 allele was found in 2/4 patients, seems than to be elevated than in general Polish population but a small number of patients does not allow to perform relevant statistical tests. Moreover we found significantly more frequent B*08-DRB1*03 fragment of ancestral HLA 8.1 haplotype (8.1 AH) in PPBCL patients (3/4) than in control group (47/286) (OR=15.3; 95% CI 1.55–150; p=0.02). Peripheral blood mononuclear cells from all PPBCL patients were also studied for monoclonality in immunoglobulin (IGH VH-JH, incomplete DH-JH, IGK, IGL) and crosslineage TCR (TCRB, TCRG, TCRD) genes rearrangements according to BIOMED-2 protocol. The multiplex PCR reactions were followed with heteroduplex analysis of PCR products. We found polyclonality of lymphocytes population in all tests except incomplete DH7-JH IGH rearrangements in 2 patients. The described gene rearrangement was not related to other upstream DH or downstream JH gene segments. The HLA incidence we found is distinct from previously described elevated incidence HLA DRB1*07 allele and may suggest another genetic background of PPBCL pathogenesis. Moreover, our finding of high prevalence of 8.1 AH, known to be more frequent in autoimmune diseases, and to worsen the prognosis in some infectious diseases and non-Hodgkin lymphomas, as well as of the incomplete DJ monoclonality in 2/4 patients, warrant further studies aiming to determine the relationship between PPBCL, immune disorders and true lymphoproliferative diseases.

Functional T-cell anergy in a case of persistent polyclonal B-cell lymphocytosis

The T-cell population of a patient with persistent polyclonal B-cell lymphocytosis (PPBL) presenting with an intermittent Epstein-Barr virus (EBV)-associated disease was studied. Unstimulated T-cells did not express CD40 ligand (CD40L), whereas activation with IL-2 led to expression of this costimulatory molecule. CD40L expression was inhibited upon incubation with the supernatant of an EBV-positive B-cell line (SM) which had been grown spontaneously from the patient's peripheral blood cells. The supernatant of SM cells effectively inhibited cytotoxic T-cells. Elevated levels of IL-10, TNF-alpha and soluble CD40 were found in the supernatant of SM cells. Additionally, enhanced levels of LMP-1 protein were detected.

Intravascular bone marrow accumulation in persistent polyclonal lymphocytosis: a misleading feature for B-cell neoplasm

Persistent polyclonal B-cell lymphocytosis is usually reported in young smoking women. Whether this syndrome represents a premalignant or benign disease remains unclear. Indeed, because of the association of Bcl-2/IgH rearrangement and cytogenetic abnormalities, such cases may be misdiagnosed as the leukemic phase of a non-Hodgkin's lymphoma. We report eight new cases of persistent polyclonal B-cell lymphocytosis, which displayed a misleading bone marrow histological pattern, that is, intravascular B-cell infiltrate, constantly associated with Bcl-2 immunohistostaining, as seen in some lymphoma. We also show the absence or low expression of adhesion molecules on persistent polyclonal B-cell lymphocytes, suggesting that migration abnormalities might lead to bone marrow and peripheral blood accumulation. Although most cases presented multiple Bcl-2/IgH gene rearrangements and appeared to be polyclonal, oligoclonal expansion was identified in one out of eight patients, yet was not associated with clinical aggressiveness. The occasional reports of oligoclonal IgH and Bcl-2/IgH rearrangements in this disorder suggest that polyclonal expansion may be followed by the emergence of a predominant clone. However, the benign clinical course and lack of biological evolution in most cases imply that it is mandatory to distinguish this disorder from other malignant lymphoproliferations, so that unnecessary aggressive therapy can be avoided.

Analysis of expressed V(H) genes in persistent polyclonal B cell lymphocytosis reveals absence of selection in CD27+IgM+IgD+ memory B cells.

Persistent polyclonal B cell lymphocytosis (PPBL) is a hematological disorder diagnosed predominantly in women, characterized by a polyclonal increase in the number of peripheral blood B lymphocytes. Abnormality of the B cell population was evidenced by the finding of multiple bcl-2/Ig gene rearrangements and an additional long-arm chromosome within a significant proportion of B cells. To gain further insight about the developmental status of B lymphocytes in PPBL, analysis of cell surface Ig receptors was undertaken. An important expansion of the CD27+IgM+IgD+ B cell population was noted in PPBL patients (n=4). When investigated by PCR, pattern of heavy chain variable region (VH) genes usage in patients (n=6) was shown tobe similar to that observed in healthy individuals (n=3). In-depth investigation was then conducted through cloning and sequencing of individual VH genes in three of those patients. They were mostly found to be mutated (21/29), correlating with the observed increase in CD27 expression, a marker of memory B cells. Altogether, these data clearly point out to the exact nature of the expanding B cell subset in patients. Finally, analysis of the repartition of recombinant versus silent mutations in framework regions (FR) of Ig genes showed no evidence of positive antigenic selection following somatic hypermutation. Thus, we suggest that a lack of response to physiological signals responsible for the elimination of low affinity memory IgM+IgD+ B cells in germinal centers could play an important role in the development of PPBL.