Epstein-Barr virus (EBV) gene expression in lymphoid B cells during acute infectious mononucleosis (IM) and clonality of the directly growing cell lines

We examined the patterns of viral gene expression in acute infectious mononucleosis (IM) patients and the clonality of the directly growing EBV-carrying cell lines. Both low- and high-density EBV-carrying B cells obtained from the patients' tonsils expressed EBNA1, EBNA2 and LMP1. Like LCLs and immunoblastic B-cell lymphomas, the in vivo EBV-carrying low-density cells used only the latency III program for viral gene expression. The in vivo EBV-carrying high-density B cells used both the latency I program, as indicated by the QUK-, and the latency III program, as indicated by the YUK-EBNA1. This suggests that the lymphoid tissues contained not only proliferating immunoblasts but also cells programmed for latent viral persistence in vivo. EBV-carrying cells that grew directly into permanent cell lines in the presence of virus-neutralizing antibody and a late viral inhibitor were polyclonal, as indicated by JH rearrangement. Two of the high-density-derived lines had identical JH and TR patterns, indicating a common parental origin. Our investigation indicates that EBV-carrying cells divide and survive in a fully competent immune system during the outbreak of acute IM. Int. J. Cancer 71:345-349, 1997. © 1997 Wiley-Liss Inc.     

The leukemic B-cell population of patients with monoclonal lymphocytosis of undetermined significance (MLUS) are functionally distinct from the chronic lymphocytic leukemia (CLL) derived cell population.

Some patients with CLL survive for decades with a stable lymphocytosis without other signs of progression of the disease. This condition has been termed monoclonal lymphocytosis of undetermined significance (MLUS). The aim of the present study was to search for functional differences between the monoclonal B-cell population of CLL (n = 3) and MLUS (n = 5) patients. MLUS derived B-cell populations were susceptible to Epstein-Barr virus (EBV) infection measured as the production of EB nuclear antigen (EBNA) whereas CLL derived cells were resistant. In 4 out of 5 MLUS patients, lymphoblastoid cell line (LCL) like cell-clumps were formed, but not in CLL. The clonal B-cell population from 2 of 5 MLUS patients was immortalized by EBV (LCL restricted to the expression of one Ig light chain) while no cell line emerged from the CLL patients. Phorbol esters induced cell to cell adhesion of MLUS and normal B cells but not of CLL derived cells. This study further enlarges previous observations and strengthens the assumption that MLUS clonal B cells are functionally close to normal B cells while CLL B cells display various functional abnormalities.     

Epstein barr virus (EBV)-carrying cells of a chronic lymphocytic leukemia (CLL) subpopulation express EBNA1 and LMPS but not EBNA2 in vivo

We have previously described an exceptional CLL patient, P.G., whose leukemic cell population contained a small fraction of Epstein-Barr virus (EBV)-carrying cells. These cells grow directly into permanent cell lines in vitro. Using RT-PCR analysis, we now show that the in vivo EBV-carrying CLL cells expressed EBNAI, LMPI, LMP2a and 2b, but not EBNA2, in 4 of 4 blood samples obtained during the last 3 years of the patient's life. Our data also show that the CLL cells used a promoter in the F/Q, but not the W or C, region. This is consistent with the fact that CLL cells resemble resting lymphocytes rather than immunoblasts. Expression of LMPI and LMP2b differs from the exclusive EBNAI and LMP2a expression of normal resting B cells, however, and corresponds to the state defined as latency II. This form of latency was until now detected only in EBV-carrying non-B cells in vivo. Our data show that a B-cell subtype can also show this expression pattern in vivo.     

Clonality and methylation status of the Epstein-Barr virus (EBV) genomes in in vivo-infected EBV-carrying chronic lymphocytic leukemia (CLL) cell lines.

Directly growing Epstein-Barr virus (EBV)-carrying cell lines were established from a chronic lymphocytic leukemia (CLL) patient (PG) on repeated occasions. The lines carried the same ring chromosome 15 as the leukemia cells in vivo and were similarly trisomic for chromosome 12. They all showed the same JH rearrangement, indicating that they had arisen from the same B-cell progenitor. They also had the same single EBV-terminal repeat (TR), indicating that they had been generated by a single EBV infection event. It may be surmised that a single CLL cell had been infected by EBV in vivo and established itself subsequently as a subclone within the CLL population. This subpopulation persists in vivo but does not appear to expand with time. After explantation, it transforms into lymphoblastoid cells and proliferates selectively as immortalized lines. The leukemia-representative CLL lines were phenotypically indistinguishable from the B95-8 virus-transformed normal diploid cells of the patient, established in parallel by in vitro infection. They grew as typical LCL clusters and expressed the same B-cell activation markers. The methylation status of EBV-DNA was different in the CLL lines and the B95-8-virus-transformed LCLs. When Hpall- and Mspl- digested DNA was probed with BamHI C, E, H and W fragments, the CLL lines showed a mixture of methylated and unmethylated restriction fragments as in certain EBV-carrying Burkitt lymphoma (BL) lines. In contrast, the EBV-DNA of B95-8 virus-transformed normal diploid cells was completely unmethylated, as in other LCLs.     

The B cell antigen receptor in atypical chronic lymphocytic leukemia with t(14;19)(q32;q13) demonstrates remarkable stereotypy

The t(14;19)(q32;q13) is a recurrent chromosomal translocation reported in a variety of B‐cell leukemias and lymphomas, including chronic lymphocytic leukemia (CLL). CLL cases associated with t(14;19) often have atypical morphologic and immunophenotypic features and unmutated immunoglobulin heavy chain (IGH) variable region (V) genes, associated with an aggressive clinical course. We analyzed IGHV somatic mutation status and gene use in 11 patients with t(14;19)‐positive CLL. All cases were unmutated, and the IGHV genes in 10 cases showed minimal deviation from germline sequences. In 7 of 11 patients, we found homologous heavy chain rearrangements using IGHV4‐39; light chain analysis revealed identical IGKV1‐39 use. Corresponding V‐(D)‐J sequences demonstrated remarkable stereotypy of the immunoglobulin heavy and kappa light chain complementarity determining region 3 (H/K CDR3) genes. These findings raise the possibility that specific antigen drive is involved in the clonal development and/or selection of t(14;19)(q32;q13)‐positive CLL cells. Our findings support the hypothesis that stimulatory signals through specific antigen receptors may promote the expansion of either CLL precursor cells or CLL clones that harbor distinct chromosomal abnormalities.     

B-CLL cells with unusual properties

In studies concerning the interaction of B-CLL cells and Epstein-Barr virus (EBV), we encountered one patient whose cells had several unusual properties. In addition to the B-cell markers, the CLL cells expressed the exclusive T-cell markers CD3 and CD8 and carried a translocation t(18,22)(q21;q11), involving the bcl-2 and Igλ loci. The patient represents the 4th reported CLL case with this translocation. The CLL cells could be infected and immortalized by the indigenous and by the prototype B958 virus in vitro. The T-cell markers were not detectable on the established lines. In all experiments the immortalized lines originated from the CLL cells. Their preferential emergence over virus-infected normal B cells may be coupled to the high expression of the bcl-2 gene due to the translocation. In spite of the sensitivity of CLL cells to EBV infection in vitro, no EBNA-positive cells were detected in the ex vivo population. In vitro, we could generate cytotoxic function in T-lymphocyte cultures which acted on autologous EBV-infected CLL cells. Therefore we assume that if such cells emerged in vivo they were eliminated by the T-cell response. © 1997 Wiley-Liss, Inc.     

bcl-2 rearrangement detected by pulsed-field gel electrophoresis (PFGE) in B-chronic lymphocytic leukemia (CLL) cells

B-chronic lymphocytic leukemia (CLL) is characterized by an accumulation of long-lived, resting B cells expressing the Bcl-2 protein. However, less than 10% of the CLL patients shows bcl-2 gene rearrangement in blood cells, using traditional Southern blotting analysis. In the present study, rearrangement of thebcl-2 gene in CLL cells was studied by pulsed-field gel electrophoresis (PFGE). With this method, large DNA fragments (>50–10,000 kb) could be analyzed. Blood CLL cells from 9 of 9 patients and 2 of 2 CLL cell lines showed rearranged bcl-2 gene. In comparison, healthy blood B cells and lymphoblastoid cell lines (LCLs) established from normal peripheral blood lymphocytes of the patients showed only germ line configuration. Thus, the possibility of restriction fragment length polymorphisms (RFLPs) in this gene could be excluded. The primary cell involved in CLL might be a progenitor B cell that has accidentally rearranged the bcl-2gene. As a consequence, such cells express stable amount of Bcl-2 protein and do not enter apoptosis. During prolonged survival, such cells may acquire secondary changes including chromosomal translocations and mutations. Int. J. Cancer 76:909–912, 1998.© 1998 Wiley-Liss, Inc.     

In-vitro infection of chronic lymphocytic leukemia cells by Epstein-Barr virus (EBV)

We sought to determine the potential of infecting lymphoid cells from patients with chronic leukemia (CLL) with Epstein-Barr virus (EBV) by testing for EBV receptors (EBVR) by flow cytometry, assessing for infectability of these cells by culturing with B95-8-derived virus, and staining for EB nuclear-associated antigens (EBNA) at various times post-infection. EBVR were present on 54-91% of lymphoid cells in seven cases of CLL and on 46% of prolymphocytic leukemia cells. Dynamic changes regarding EBNA positivity, morphology, and viability occurred post-infection with the virus. On day 2 only a few EBNA-positive lymphoblasts were observed. On days 11-21 positivity increased from 2 to 34% of cells. Simultaneously, the viable cell number declined to approximately 1/10th of original number. A significant proportion of the EBNA-positive cells corresponded to the original CLL cells. In 3 of 7 cases of CLL a Pan T-cell phenotype was demonstrated by Leu-1 monoclonal antibody testing. The infected cells did not react with two monoclonal antibodies, EBV-CS 1 and 4, which react with B-cell lymphoblastoid cell lines (B-LCL). Moreover, the B-LCL derived at 1-2 months post-infection of CLL cells did not express the Leu-1 antigen, but expressed EBV-CS 1 or 4 defined antigens. In the prolymphocytic leukemia, 64% of the cells showed EBNA positivity on day 7 and giant cells with huge round or multiple nuclei appeared which were EBNA-positive. CLL and prolymphocytic leukemia cells can be infected as demonstrated by EBNA-positivity. This infection does not lead to immediate transformation, but evokes lymphoblast and multinucleated giant cell production prior to the death of cells.     

Concomitant or Sequential Chronic Lymphocytic Leukemia (CLL) and Chronic Myeloid Leukemia (CML): A Molecular and Clinical Survey

A 75-year-old man presented with B-CLL followed by a CML. We retrospectively analyzed the frozen BM and PB lymphocytes that evidenced 2 distinct clonal proliferations: one is a B CD5⁺/CD19⁺ lymphocyte population, without BCR-ABL fusion; the other is the CD19⁺ mononuclear cell population expressing traces of BCR-ABL, 3 years before the occurrence of CML. We suggest that (1) the emergence of the Ph⁺ clone after the treatment of CLL is probably related to RFC-induced immunosuppression, and (2) the long DFS of CLL might be due to the current TK inhibitor treatmentFull AbstractIntroductionThe coexistence of B-cell chronic lymphocytic leukemia (B-CLL) and chronic myeloid leukemia (CML) is a rare event. This coexistence has led to speculation of the clonal origin of these neoplasms. In some cases, 2 independent clones were demonstrated.Patients and MethodsA 75-year-old man presented with splenomegaly and enlarged lymph nodes. Immunophenotype (kappa, CD19⁺, CD5⁺, CD23⁺) was compatible with B-CLL. Bone marrow examination showed infiltration by small lymphocytes next to a myeloid and megakaryocytic hyperplasia. Cytogenetic analysis of the marrow showed 46XY, del(16q). Philadelphia chromosome (Ph) was negative. The patient was diagnosed with B-CLL and treated by 6 RFC (rituximab/fludarabine/cyclophosphamide). He reached a molecular remission (MCR), but 3 years after the treatment for CLL, he developed hyperleukocytosis (26000/μL), mainly myeloid cells. Bone marrow smear demonstrated a myeloid hyperplasia with predominance of immature progenitors. The Ph was clearly evidenced, and CML was confirmed. The patient thus received thus imatinib 400 mg/day orally with hematologic but only partial molecular remission at 18 months. A Y25.3H mutation was detected, and the patient was switched to dasatinib with a successful evolution. The CLL remained in MCR during this 5-year period.ResultsTo know whether CML is a secondary event induced by RFC treatment or the emergence of a latent CML clone, we analyzed retrospectively the frozen BM and PBL lymphocytes. The results were consistent with the view that there are 2 distinct clonal proliferations: one is a clonal B CD5⁺/CD19⁺ lymphocyte population, without BCR-ABL fusion; the other is the CD19⁺ mononuclear cell population expressing traces of BCR-ABL, 3 years before the occurrence of CML.ConclusionOur data provide evidence for a separate clonal origin for each disorder, raising the hypothesis that (1) the emergence of the Ph⁺ clone after the treatment of CLL is probably related to RFC-induced immunosuppression, and (2) the long disease-free survival of CLL might be due to the current tyrosine kinase inhibitor treatment.     

Consistency of chromosomal aberrations in chronic B-lymphocytic leukemia. A longitudinal cytogenetic study of 41 patients

Serial chromosome analyses with a mean of 3.7 samplings during a mean interval of 4.2 years (range, 1.5 to 8.6 years) were performed on B-cell mitogen-activated chronic B-lymphocytic leukemia (CLL) cells from 41 patients. Twenty-four of these patients (59%) had progressive disease. Clonal chromosomal aberrations were found in 28 patients; 12 had an extra chromosome 12. Thirty patients (73%), 17 with and 13 without clonal aberrations, retained their karyotype throughout the study, although six lost minor subclones. In five patients (12%), a clonal aberration was found only once. Six patients (15%) showed changes of the karyotype. One treated patient with multiple aberrations acquired another monosomy. Another patient with multiple aberrations and prolymphocytic transformation gained a marker chromosome. One treated patient with an initially normal karyotype acquired two independent clonal aberrations. Three patients lost one subclone but retained another clone that increased in frequency. In two cases, clonal changes were associated with clinical changes. The chromosomal aberrations are mostly established already at diagnosis and mark the disease of the CLL patient. Cytogenetic analysis at any time is representative and useful in the prognosis prediction.     

Atypical lymphocytes in B-cell chronic lymphocytic leukemia and trisomy 12 studied by conventional staining combined with fluorescence in situ hybridization

Trisomy 12 is one of the most frequent chromosomal abnormalities in B-cell chronic lymphocytic leukemia (CLL), and is predominantly found in CLL with atypical morphology (aCLL). It has been suggested that the atypical morphology might be a feature of the abnormal trisomy 12 clone, but so far it has been difficult to allocate chromosomal aberrations to individual leukemic cells identified by cytomorphology. We therefore wanted to use our MGG/FISH method, which combines fluorescence in situ hybridization (FISH) and standard cytomorphology, to study if the trisomy 12 clone in CLL was restricted to lymphocytes with atypical morphology. Peripheral blood specimens of four patients with aCLL were studied using a DNA probe against the pericentromeric region of chromosome 12. Trisomy 12 was found in 10-34 % of the lymphocytes. In three patients, the proportion of atypical and typical lymphocytes with trisomy 12 was quite comparable, and so was the percentage of atypical cells with lymphoplasmacytoid morphology and those with cleaved nucleus showing trisomy 12. Only one patient differed, since we found an overrepresentation of trisomy 12 among the atypical lymphocytes. However, this could be fully explained by the diluting effect of contaminating T-cells after chemotherapy. The results of the present study show that despite the strong association of trisomy 12 and atypical morphology in CLL, this chromosomal abnormality is not confined to lymphocytes with atypical morphology, but is also found in typical CLL cells. This supports that both cell types have the same clonal origin and that different cell morphology cannot be explained alone by the acquisition of an additional chromosome 12.     

The establishment of Epstein-Barr virus nuclear antigen-positive (SP-50B) and Epstein-Barr virus nuclear antigen-negative (SP-53) cell lines with t(11;14)(q13;q32) chromosome abnormality from an intermediate lymphocytic lymphoma

Two lymphoma cell lines, SP-50B and SP-53, were established from peripheral blood of a 58-year-old woman with leukemic conversion of intermediate lymphocytic lymphoma. These cell lines grew in suspension with or without forming clumps of cells. SP-50B was morphologically similar to the common Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines and was positive for EB virus nuclear antigen (EBNA), whereas SP-53 closely resembled the patient's lymphoma cells and was negative for EBNA. Both cell lines expressed the same phenotypic markers as original lymphoma cells (CpIg+, SmIg+, OKIa1+, Leu12+) and possessed t(11;14)(q13;q32) chromosome translocation. These results indicate that although morphologically different, SP-50B and SP-53 were both derived from patient's lymphoma cells. The long-term cultivation of EBNA-positive and EBNA-negative B-cell lymphoma lines from a single donor has not been previously reported. These cell lines would provide useful tools for studying the oncogenic role of EB virus and bcl-1 oncogene that is located on chromosome 11q13.     

Characterization of chronic lymphocytic leukemia (CLL) cells with low density of surface membrane bound immunoglobulins (smIg)

The study consists of 6 CLL patients with leukemic blood lymphocytes lacking T-cell characteristics and with smIg on only a very small fraction of the cells detected by microscopy or FACS analysis after direct immunofluorescence (IFL) staining. Using B-cell specific monoclonal antibodies all leukemias were found to be of the B-cell type. SmIg, mainly of IgD-class and the monotypic light chain, was detected on a large number of cells in all cases when using direct IFL. No lymphocyte clone was judged to be of the pre-B-cell type or represented fully differentiated terminal B-cells. CLL cells from the individual cases probably represent intermediate B-cell maturation steps. The results lend no support to the suggestion that this subtype of CLL necessarily belongs to an early stage of differentiation just beyond the pre-B-cell level.