Search

Introduction: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice., Methods: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls., Results: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good., Conclusions: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies., (© 2020 International Clinical Cytometry Society.)

Epstein-Barr virus (EBV) preferentially infects epithelial cells and B lymphocytes and sometimes T and NK lymphocytes. Persistence of EBV-infected cells results in severe lymphoproliferative disorders (LPDs). Diagnosis of EBV-driven T or NK cell LPD and chronic active EBV diseases (CAEBV) is difficult, often requiring biopsies. Herein, we report a flow-FISH cytometry assay that detects cells expressing EBV-encoded small RNAs (EBERs), allowing rapid identification of EBV-infected cells among PBMCs. EBV-infected B, T, and/or NK cells were detectable in various LPD conditions. Diagnosis of CAEBV in 22 patients of Caucasian and African origins was established. All exhibited circulating EBV-infected T and/or NK cells, highlighting that CAEBV is not restricted to native American and Asian populations. Proportions of EBV-infected cells correlated with blood EBV loads. We showed that EBV-infected T cells had an effector memory activated phenotype, whereas EBV-infected B cells expressed plasma cell differentiation markers. Thus, this method achieves accurate and unambiguous diagnoses of different forms of EBV-driven LPD and represents a powerful tool to study their pathophysiological mechanisms., Competing Interests: Disclosures: B. Terrier reported personal fees from AstraZeneca, GlaxoSmithKline, Roche/Chugai, and Vifor Pharma outside the submitted work. L. Galicier reported personal fees from Lilly and VIREOTEAM and nonfinancial support from EUSA Pharma, Overcome, and Janssen-Cilag outside the submitted work. No other disclosures were reported., (© 2020 Fournier et al.)

Objectives: Lymphocytosis may represent either a lymphoproliferative disorder (LPD) or a reactive process. The absolute lymphocyte count (ALC) threshold for further evaluation of lymphocytosis is not well established., Methods: We prospectively performed flow cytometry on blood samples from patients 50 years or older with ALCs of 4.0 × 109 cells/L or greater without a history of an LPD., Results: Monoclonal B-cell populations were found in 34 (19.1%) of 178 cases, with incidence increasing with age. In patients younger than 75 years, no monoclonal B-cell population was identified in patients with ALCs less than 4.4 × 109 cells/L, while such clones were found below and above this threshold in patients 75 years and older., Conclusions: These findings support a threshold for smear review and flow cytometry no lower than 4.4 × 109 cells/L in patients younger than 75 years and a threshold as low as 4.0 × 109 cells/L in patients 75 years and older., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We describe the case of a 23-month-old male infant with Epstein-Barr virus (EBV)-associated lymphoproliferative disorder, which mimicked the recurrence of EBV-associated hemophagocytic lymphohistiocytosis. Chemotherapy with dexamethasone, etoposide, and cyclosporine resolved fever, hepatosplenomegaly, and pancytopenia. However, on day 81 of illness, the patient developed similar symptoms. Plasma EBV-DNA levels markedly increased again, but no T-cell clonality was observed. B cells were identified to be infected with EBV. He was successfully treated with rituximab, dexamethasone and etoposide. When recurrence of EBV-associated hemophagocytic lymphohistiocytosis is suspected, performing tests to identify the infected cells will enable accurate understanding of the clinical condition, resulting in proper treatments.

B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.

Observation of binucleated lymphocytes on a blood film is a cornerstone event to assess persistent polyclonal B-cell lymphocytosis diagnosis (PPBL). But seldom case reports mentioned their finding in other contexts, mainly B-cell lymphoproliferative neoplasms such chronic lymphocytic leukemia or in reactive circumstances as multiple sclerosis undergoing natalizumab treatment. We aimed to check if some particular morphologic features were more specificaly linked to PPBL, reviewing blood films of series of patients affected by PPBL or harbouring binucleated lymphocytes whatever the diagnosis was. We found that binucleated cells in PPBL were heterogeneous but mostly midde-sized cells, asymetricaly bilobed with nucleoli, and with a moderately basophilic cytoplasm. Though mainly observed in PPBL patients, all that criteria remained unspecific. Conversely, the morphologic features of the other lymphoid cells were of good clinical value to affirm PPBL, either monocytoid or hyperbasophilic cells being systematically found. We think that a substantial percentage of binucleated lymphocytes, harbouring morphologic features such as described, and in a context of monocytoid and/or hyperbasophilic lymphocytes is predictive of PPBL diagnosis.

Objective: Primary SS is characterized by an increased risk of lymphoma in patients with prelymphomatous manifestations (i.e. myoepithelial sialadenitis or mixed cryoglobulinaemia). Serum B-lymphocyte stimulator (s-BLyS) levels in SS-related B-cell lymphoproliferative disorders were studied by integrating the results with the disease activity score and with molecular analyses of B-cell expansion in the salivary glands., Methods: Seventy-six primary SS patients (with or without lymphoma or prelymphomatous manifestations), 56 HCV-related cryoglobulinaemic vasculitis patients and 55 controls were studied. s-BLyS and molecular analyses of B-cell expansion in the salivary gland tissues were performed. Patients with SS and persistent parotid swelling underwent parotid biopsy., Results: s-BLyS differed between SS subgroups, higher levels being documented in patients with lymphoma or prelymphomatous manifestations vs SS without [1.85 (0.45-4.12) ng/ml vs 1.12 (0.56-1.98) ng/ml; P < 0.0001]. s-BLyS levels significantly correlated with the European League Against Rheumatism (EULAR) SS disease activity index (r = 0.62, P < 0.0001, Spearman's test). Clonal B-cell expansion in the salivary glands, but not polyclonal B-cell expansion, was associated with higher s-BLyS levels [1.98 (0.45-4.12) ng/ml vs 1.15 (0.56-3.25) ng/ml; P = 0.013)]., Conclusion: Higher s-BLyS levels and tissue clonal B-cell expansion characterize SS with B-cell lymphoproliferative disorders, even at prelymphomatous stages. This subgroup of SS patients showed the highest EULAR SS disease activity index scores. This represents a biologic rationale for targeting both clonal B-cell expansion and s-BLyS overproduction in SS.

Background: Detection of absolute and relative lymphocytosis in otherwise asymptomatic elderly patients is very common in the primary care setting and frequently results in referral for screening of lymphoproliferative disorders. Since many B-cell chronic lymphoproliferative disorders (B-CLPD) are indeed asymptomatic at diagnosis in most patients with lymphocytosis, no sign of such a disorder is usually detected. Currently, specific guidelines for screening of patients with lymphocytosis are lacking. We investigated the practicability and clinical value of a single colour CD19 count performed by a primary care laboratory in order to improve the diagnostic follow-up of patients with lymphocytosis in a primary care laboratory., Methods: The capability of detecting monoclonal B-cell lymphocytosis and B-CLPD by CD19, was first confirmed in patient samples with known B-CLPD. Next, in a previously defined geographic area, a CD19 count was performed on all samples for patients aged ≥40 years with relative or absolute lymphocytosis but without neutropenia. Clinical follow-up, with a median of 4 years, was performed using both a survey among the requesting general practitioners and by analysis of the records of the referral hospitals within the borders of the defined area., Results: A total of 520 cases with asymptomatic lymphocytosis were identified. In all cases, the CD19 count was performed; 207 (40%) showed increased values and 313 (60%) showed normal values. An increase in CD19 proved highly sensitive for detection of B-CLPD (98%, 95% CI; 94%-100%) with a high positive predictive value (57%, 95% CI; 50%-63%). The area under curve, the receiver-operating characteristic curve of the CD19 count (0.93, 95% CI; 0.91-0.96), was significantly higher compared to the absolute lymphocyte count (0.86, 95% CI; 0.83-0.89), especially in patients with moderate lymphocytosis., Conclusions: This study indicates that the CD19 count, performed by a primary care laboratory, is feasible and a promising tool for initial screening of lymphocytosis to discriminate B-CLPD from benign causes of lymphocytosis.

Background: The success of treatment using monoclonal antibodies in oncology is influenced by, among other factors, the level of target antigen expression on tumor cells. The authors analyzed the intensity of the CD52 antigen expression in patients with chronic lymphoproliferative diseases and compared them with B-lymphocytes of a healthy population and CD34(+) cells in peripheral blood stem cells (PBSC) grafts., Methods: Recently diagnosed and previously untreated patients with B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), or small lymphocytic lymphoma (SLL) were evaluated and compared with control group and CD34(+) cells. The intensity of CD52 was expressed in molecules of equivalent soluble fluorochrome units (MESF) and antibody-binding capacity (ABC)., Results: In the group of patients with B-CLL, the CD52 level on tumor cells (245 x 10(3) MESF; 107 x 10(3) ABC) was significantly lower than on B-lymphocytes of the control group (446 x 10(3) MESF; 194 x 10(3) ABC; P < 0.001) and SLL tumor cells (526 x 10(3) MESF; 229 x 10(3) ABC; P < 0.001). The CD52 antigen was expressed on a majority of CD34(+) cells, but its expression intensity was low (101 x 10(3) MESF; 44 x 10(3) ABC)., Conclusions: Our data demonstrate differences in the intensity of the CD52 antigen expression between B-lymphocytes and tumor lymphocytes of B-CLL patients, and between B-CLL and SLL tumor cells. CD52 antigen is expressed at low level on CD34(+) cells., (Copyright 2007 Clinical Cytometry Society.)

B-cell posttransplantation lymphoproliferative disorder (B-PTLD) is a rare but severe complication of transplantation, with no consensus on best treatment practice. This prospective trial, the first to test a treatment for PTLD, was designed to evaluate the efficacy and safety of rituximab in patients with B-PTLD after solid organ transplantation (SOT). Forty-six patients were included and 43 patients were analyzed. Patients were eligible if they had untreated B-PTLD that was not responding to tapering of immunosuppression. Treatment consisted of 4 weekly injections of rituximab at 375 mg/m2. At day (d) 80, 37 (86%) patients were alive, and the response rate was 44.2%, including 12 complete response/unconfirmed complete response (CR/CRu). The only factor predictive of a response at d80 was a normal lactate dehydrogenase level (P = .007, odds ratio [OR] = 6.9). At d360, responses were maintained in 68% of patients, and 56% of patients were alive. The overall survival rate at 1 year was 67%. We conclude that rituximab is effective and safe in PTLD, with stable responses at 1 year. The response rate and overall survival might be improved by combining rituximab with other treatments.

The correlation dimension (D2) is a mathematical tool that quantifies dimensional properties of fractals. We set out to determine whether immunoglobulin light chain restriction (LCR) in B-cell lymphoproliferative disorders (BCLPDs) could be identified using the D2. The D2 was calculated from flow cytometry data files (FCS 2.0) from a training set of peripheral blood specimens from 22 patients with and 23 patients without BCLPD. A cutoff value derived from the training set was applied to a validation set of 69 patients with and 64 patients without BCLPD. In the training set, all BCLPDs had a D2 of less than 0.72 (CD19-gated kappa-lambda data). When this cutoff was applied to the validation set, the D2 had sensitivity and specificity values of 88% and 98%, respectively. The D2 can identify LCR in peripheral blood flow cytometry data. It offers operator-independent data analysis and may be useful in objectively quantifying differences in data distribution.

Viral infection is an important cause of morbidity and mortality in the post-allograft period. Recently, a new therapeutic approach was developed in post-transplant lymphoproliferative disorder (PTLD) induced by Epstein-Barr virus (EBV): the anti-CD20 monoclonal antibody or rituximab. We performed a single-center study on the treatment effectiveness of rituximab in three EBV-induced PTLD and evaluated biologic data, such as T and B lymphocytes count, during PTLD development and treatment. Before PTLD treatment, blood cell profile showed a severe T lymphopenia with a progressive increase of CD8+ cells and B lymphopenia. Secondly, during treatment, there appeared a T response, as in primary EBV, and a regressive B lymphopenia.

Bone marrow (BM) is accepted as the tissue of choice for the detection of monoclonal populations in leukemias and lymphomas; however, obtaining BM can be painful and traumatic for the patients. Although it is possible to detect clonality in peripheral blood (PB) samples, there are no reports comparing the results observed from BM with those from PB. Lymphoblastic leukemias and lymphomas are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementarity Determining Regions (CDR). Of these, CDR3 is unique for each lymphocyte and therefore it can be used as a tumour-specific marker in these malignant disorders. Among the 104 patients from whom we obtained pre-treatment paired samples of PB and BM, 94 (90.4%) showed concordant results. Similarly, at the end of treatment, 40 of 44 patients (90.9%) showed this concordance. During treatment only 24 patients were monitored and monoclones disappeared in 12 patients; in the other half, they persisted either partial or totally. We demonstrate that the detection and monitoring of monoclonal populations in the PB, in comparison with BM, was achieved with a statistical sensitivity of 90% and specificity of 92%., (Copyright 2003 John Wiley & Sons, Ltd.)

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

Mechanisms involving the in vitro effect of rituximab in cells from 55 patients with B-cell lymphoproliferative disorders were investigated. No cytotoxic effect was observed when cells were incubated with rituximab alone, but in the presence of human AB serum rituximab induced complement-dependent cell death (R-CDC). A cytotoxic effect was observed in cells from 9 of 33 patients with B-cell chronic lymphocytic leukemia, 16 of 16 patients with mantle-cell lymphoma, 4 of 4 patients with follicular lymphoma, and 2 of 2 patients with hairy-cell leukemia. R-CDC was observed in cells from patients expressing more than 50 x 10(3) CD20 molecules per cell, and directly correlated with the number of CD20 molecules per cell. Preincubation with anti-CD59 increased the cytotoxic effect of rituximab and sensitized cells from nonsensitive cases. Neither cleavage of poly-ADP ribose polymerase (PARP) nor activation of caspase-3 was observed in R-CDC. In addition, no cells with a hypodiploid DNA content were detected and R-CDC was not prevented by a broad-spectrum caspase inhibitor, suggesting a caspase-independent mechanism. Incubation with rituximab in the presence of AB serum induced a rapid and intense production of reactive oxygen species (ROS). R-CDC was blocked by the incubation of cells with N-acetyl-L-cysteine (NAC) or Tiron, 2 ROS scavengers, indicating that the cytotoxic effect was due to the generation of superoxide (O) radicals. In conclusion, the results of the present study suggest that CD20, CD59, and complement have a role in the in vitro cytotoxic effect of rituximab, which is mediated by a caspase-independent process that involves ROS generation.

Distinguishing leukemic phases of B-cell disorders in peripheral blood smears is well recognized to be difficult in some cases since it depends on subtle and subjective criteria. In order to quantify cytological features and to assess objective descriptions, a morphometric analysis was performed on 83 peripheral blood smears of B-cells disorders (n = 77) and healthy donors (n = 6). Using standardized May-Grunwald Giemsa staining, standardized image acquisition system and well defined microscopic fields, we have analyzed lymphoid cells, measuring morphometric and color parameters. By combining seven relevant morphometric criteria (the nuclear shape, the cellular shape and area, the nucleo-cytoplasmic ratio, the nuclear red/blue ratio, the cytoplasmic green/blue ratio and the proportion of cells with nucleolus), we have established a score that could range from a minimum of -3 (large B-CLL type) to a maximum of +8 (large MCL type): negative scores corresponds to different types of B-CLL (n = 30), including "atypical B-CLL" (n = 6), the score zero correspond to healthy donors (n = 6) used as baseline, the positive score values correspond to +1 for Follicular lymphoma (n = 2), +3 for Splenic Lymphoma with Villous Lymphocytes (n = 12), +4 for Hairy Cell Leukemia (n = 7), for Hairy Cell Leukemia-variant (n = 2), +6 for B-prolymphocytic leukemia (n = 6) and +7 and +8 for most Mantle Cell Lymphoma (n = 18). Testing T-cell disorders samples (n = 10) using the same protocol, the profile is different and cannot be confused with B-cell diseases. Our scoring system indicates that measurement of some common morphologic features in standardized conditions provides objective criteria to characterize those diseases and might be helpful for diagnosis.

Severe T-cell immunodeficiency after solid organ or bone marrow transplantation may result in the uncontrolled outgrowth of latently Epstein-Barr virus-infected B cells, leading to B-lymphoproliferative disorder (BLPD). Given the potentially important pathogenic role of IL-6 in BLPD, it was tested whether the in vivo neutralization of IL-6 by a monoclonal anti-IL-6 antibody could contribute to the control of BLPD. Safety and efficacy were assessed in 12 recipients of transplanted organs who had BLPD refractory to the reduction of immunosuppression over 8 days. Five patients received 0.4 mg/kg per day. The next 7 patients received 0.8 mg/kg per day. Treatment was scheduled to last 15 days. It was completed in 10 patients, and in the other 2 patients was discontinued early (days 10 and 13, respectively) because of disease progression. Treatment tolerance was good, and no major side effects were observed. High C-reactive protein levels were found in 9 patients before treatment but were normalized under treatment in all patients, demonstrating efficient IL-6 neutralization. Complete remission (CR) was observed in 5 patients and partial remission (PR) in 3 patients. Relapse was observed in 1 of these 8 patients in whom remission was observed. This relapse was unresponsive to treatment. Disease was stable in 1 patient, but it progressed in 3 patients. Seven patients are alive and well. Two patients died because of disease progression, and 3 patients died while in CR (chronic rejection in 2 patients and BLPD sequelae in 1 patient). These data suggest that the anti-IL-6 antibody is safe and should be further explored in the treatment of BLPD.

Thirty three B-chronic lymphoproliferative disorder (B-CLD) patients [22 with B-chronic lymphocytic leukemia (B-CLL), 5 with small lymphocytic lymphoma (SLL) and 6 with lymphoplasmacytic lymphoma (LPL)] with anaemia (Ht <32%) of no other cause but their disease, received recombinant human erythropoietin (r-HuEPO). The treatment protocol provided r-HuEPO in a dose of 150 U/kg s.c. thrice weekly for 3 mo. After 1.5 mo of r-HuEPO administration, if response was not satisfactory, r-HuEPO dose escalation was utilised by giving incremental doses of 50 U/kg more than the previous dose up to a maximum dose of 300 U/kg tiw. After maximal response, half of the responding patients discontinued therapy, while the other half received maintenance therapy at a dose of 150 U/kg s.c./w. Oral iron was given throughout the study. Pretreatment EPO levels were determined in all patients. A complete response (CR) was defined when Ht was >38% and a partial response (PR) when there was an increase of the Ht >6% from the initial value was achieved. Sixteen of the 22 B-CLL patients had Rai stage III disease and 6 stage IV, with a median duration of anaemia 27 months (6-38); twelve of them were receiving chlorambucil while the rest were on no treatment. Of the SLL and LPL group, 4 patients had Ann Arbor stage III disease and 7 stage IV with a median duration of anaemia 24 months (5-36); 8 patients were on chlorambucil. Complete response was achieved in 50% of the B-CLL group and 54% of the SLL and LPL group, with an overall response rate of 77% and 81% respectively. All patients on maintenance therapy had a continuous response, while all patients, in whom rHuEPO was discontinued, relapsed. No correlation was found between patients: with low or high pretreatment serum EPO levels; those receiving concomitant therapy or not; those with B-symptoms or not; those with a non-diffuse or diffuse bone marrow infiltration pattern; and with splenomegaly or not. Life quality was significantly improved and no major side effects were encountered. We conclude from our study that r-HuEPO is very effective in correcting disease-related anaemia in B-CLD, resulting in down-staging of Rai stage III patients and that maintenance therapy is necessary. Whether the correction of anaemia improves patients' overall survival, still remains to be seen.

We studied 40 patients with CD5- B-cell lymphoproliferative disorders (B-LPDs) presenting in blood or bone marrow and 28 control patients with CD5+ B-cell chronic lymphocytic leukemia (CLL). Fifteen study patients had morphologic features typical of CLL. The 15 patients with CD5- CLL were older and had lower absolute lymphocyte counts and more advanced-stage disease at diagnosis than controls. Ten study patients had morphologic features suggesting mantle cell lymphoma (MCL); 3 were later given a diagnosis of MCL based on lymph node biopsy results. The 10 patients with CD5- MCL were older and at a more advanced stage than CLL control patients. The remaining 15 study patients were given the following diagnoses: circulating non-Hodgkin lymphoma, 5; splenic lymphoma with villous lymphocytes, 5; lymphoplasmacytoid lymphoma, 3; and CLL/pro-lymphocytic leukemia, 2. For the patients with CD5- B-LPDs with morphologic features and manifestations resembling CLL, we prefer the term CD5- CLL variant because of clinical and immunophenotypic differences. Patients with CD5- B-LPDs with atypical nuclear morphologic features may represent the leukemic phase of MCL. Since CD23 is expressed in most patients with CD5- B-LPD, its use in subclassifying these disorders seems limited.

Two receptors for tumor necrosis factor (TNF) with different molecular weight (75-Kd and 55-Kd) and binding affinity have been recently discovered. To investigate the distribution and the functional role of these receptors on leukemic B cells from hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia (B-CLL) patients, we evaluated: (1) the cytofluorimetric pattern of uncultured and cultured leukemic B cells incubated with utr-1 and htr-9 monoclonal antibodies (MoAbs), which specifically recognize the 75-Kd and 55-Kd TNF receptors (TNFR), respectively; (2) the effect of TNF-alpha and TNF-beta on leukemic B cells in an in vitro proliferation assay; (3) the role of anti-TNFR MoAbs on TNF-alpha and TNF-beta-driven B-cell growth; and (4) the proliferative effect of utr-1 and htr-9 MoAbs on in vitro cultured leukemic cells. Our study shows that the high affinity (75-Kd) but not the low affinity (55-Kd) TNFR molecules are expressed on freshly isolated leukemic B cells recovered from HCL and B-CLL patients. The expression of these receptors was neither upregulated nor downregulated by different stimuli, including TNF-alpha, TNF-beta, B-cell growth factor, and interleukin-2. TNF-alpha efficiently triggers the proliferation of HC and, to a lesser extent, the growth of B-CLL cells. TNF-beta was also able to transduce the proliferative signal in HCL, but not in B-CLL patients. TNF-alpha- and TNF-beta-driven B-cell proliferation was inhibited by the preincubation of leukemic B cells with utr-1 but not htr-9 MoAb. Moreover, anti-75-Kd, but not anti-55-Kd TNFR MoAb, was able to trigger the proliferation of leukemic B cells, and in particular of HC. These results show that leukemic B cells from patients with HCL and B-CLL are equipped with a fully functional high affinity TNFR.

A further case of persistent polyclonal B-cell lymphocytosis is reported. This recently identified distinct clinicopathologic entity is frequently associated with the presence of the HLA-DR7 antigen. It follows a benign course and has so far been reported only in women smokers. The disorder is characterized by mild chronic peripheral lymphocytosis, the presence of characteristic binucleate lymphocytes on peripheral blood smears, and a polyclonal increase in serum IgM. In some cases, lymphadenopathies and/or splenomegaly are observed. Surface marker studies of peripheral lymphocytes demonstrate the polyclonal B-cell nature of this entity.

A hybridoma-derived monoclonal antibody, produced by immunization with the Burkitt's tumor-derived B-lymphoblastoid cell line, B35M, was previously shown to detect a 68,000 dalton surface membrane protein, BL2, on the surface of peripheral blood B cells, which is absent from thymocytes, T cells, and granulocytes. In this study, we investigated the expression and distribution of BL2 on benign and malignant human lymphoid cells. Indirect immunofluorescent assay with this monoclonal antibody demonstrated that BL2 is expressed by cells within the fetal liver and by a variable proportion of lymph node, tonsil, and spleen B cells, but not by T cells. The neoplastic cells isolated from 18 T-cell malignancies were BL2- . BL2 was was heterogeneously expressed by a variable proportion of the malignant cells in 29/32 cases of B-chronic lymphocytic leukemia and 33/38 cases of B-cell lymphomas, but appeared to be lost in the terminal stages of B-cell differentiation, as myeloma plasma cells were BL2- . BL2 expression was not limited to B cells of a particular surface immunoglobulin isotype. Immunofluorescent staining for BL2 in cryostat tissue sections demonstrated that the majority, but not all, germinal center and interfollicular Ia+ (non-T) cells are BL2+. These findings suggest that BL2 is a B-cell lineage-specific differentiation marker that may be useful in the study of B-cell ontogeny and in defining subgroups of the B-cell malignancies.

Using a sensitive flow cytometric method ("kappa-lambda analysis"), we have found monoclonal B lymphocytes in the blood of 71 of 91 patients with non-Hodgkin's lymphoma. The presence of the B lymphocytes was independent of the histologic subclassification of the patient's disease. When we performed simultaneous analysis of the surface light-chain type in tumor tissue obtained by biopsy, the apparent light-chain type of the blood monoclonal cells corresponded with that of the tumor in 21 of 23 patients (P = 0.03). There was no correlation of the presence of these cells in the blood with morphologic evidence of bone-marrow involvement by lymphoma, but there was a strong correlation with clinical staging. Studies performed during prolonged clinical remission showed that whereas 16 of 25 patients with nodular non-Hodgkin's lymphoma had persistence of monoclonal lymphocytes, none of the 14 patients with diffuse histiocytic lymphoma in remission had these findings (P less than 0.005). Our analysis for B-cell clonal excess demonstrates the persistence of circulating monoclonal lymphocytes during complete remission in patients with forms of lymphoma that have a high probability of relapse, but we did not find these cells in patients in remission from categories of lymphoma in which prolonged remission is associated with cure. It is possible that the circulating monoclonal lymphocytes in patients with lymphoma are malignant cells, and their disappearance or persistence after remission may have prognostic importance.