Isolation of a normal B cell subset with a Burkitt-like phenotype and transformation in vitro with Epstein-Barr virus

Epstein-Barr virus (EBV) is causally linked with endemic Burkitt's lymphoma (BL), a tumor whose homogeneous cell surface phenotype suggests derivation from a particular subset of activated germinal centre B cells in vivo. Endemic BL also shows an unusual form of EBV infection with down-regulation of certain of the virus latent proteins which are constitutively expressed when EBV infects and transforms normal resting B cells in vitro. Here we question whether this virus:cell interaction is unique to malignant BL cells or whether it might be reproduced by in vitro infection of those particular germinal centre cells displaying the BL-like phenotype. Firstly, we show by biochemical means that a subset of normal tonsillar B cells does indeed express the globotriaosylceramide glycolipid BLA and the common acute lymphoblastic leukaemia antigen CALLA, 2 important markers of the BL phenotype. Secondly, using 2-colour immunofluorescence labelling with anti-BLA and anti-CALLA monoclonal antibodies (MAbs), 4 subsets of low buoyant density tonsillar B cells (BLA+ CALLA+, BLA+ CALLA-, BLA- CALLA+, BLA- CALLA-) have been separated by means of a FACS and tested for their susceptibility to EBV-induced growth transformation in a limiting dilution assay. The BLA+ CALLA+ (i.e., BL-like) subset contained the highest proportion of cells already actively in cycle in vivo and gave the lowest yield of transformants, perhaps reflecting the greater efficiency with which EBV transforms resting target cells. Of the cell lines established from the BLA+ CALLA+ population, a significant number retained BLA expression but CALLA was always lost. In 2 further respects, these lines resembled conventional in vitro transformants rather than lines of BL type; thus the cells expressed cellular "activation" antigens (CD23, CD39, CD30, Ki-24) characteristic of the lymphoblastoid phenotype and contained the full spectrum of EBV latent proteins.     

Studies of immunologic markers in malignant lymphomas: diagnostic and prognostic relevance

Immunological markers important for the diagnosis of the following subtypes of Non-Hodgkin lymphomas (NHL) are discussed: lymphocytic lymphomas (i.e.: B-CLL, immunocytic NHL, prolymphocytic- and hairy cell leukemia), NHL derived from follicular center cells (centrocytic, centroblastic/centrocytic and centroblastic NHL), lymphomas with blastoid appearance (various forms of lymphoblastic and "large cell" NHL) and disorders of T lymphocytes (NHL) of peripheral T cells and T gamma lymphocytosis with neutropenia). In these disorders, the patterns of leukocyte surface antigens like T1 (CD5), Tac (CD25), CALLA (CD10), FMC7, Tü1 and Tü33 as well as staining for the terminal transferase (TdT) and of dendritic follicular cells have been examined by investigating specimens of a large group of patients. Apart from the diagnostical value, these evaluations have contributed to a better understanding of normal B cell differentiation whereas the role of marker studies as independent prognostic factors requires additional prognostic studies. Finally, the value of immunoperoxidase or alkaline phosphatase staining of cryostat sections of bone marrow biopsies is emphasized, which allowed the diagnosis and classification of 74% of 92 NHL studied by this technique.     

Immunologic markers in non-Hodgkin's lymphoma

The majority of non-Hodgkin's lymphomas (NHLs) are of B-cell lineage, with less than 20% of cases being of T-cell lineage. The B-cell NHLs phenotypically correspond to normal cells in the mid stages of normal differentiation. More specifically, by their expression of B-cell activation antigens, these tumors are the neoplastic counterparts of normal activated B cells. The follicular lymphomas--including the small cleaved, mixed small and large cell, and large cell types, as well as the small noncleaved cell (Burkitt's) lymphomas--represent malignant expansions of normal germinal center B cells by their expression of pan-B cell antigens, B-cell activation antigens, and CD10 (CALLA). The diffuse lymphomas also correspond to normal activated B cells. The small lymphocytic lymphomas express the low-affinity IL-2 receptor and CD5, both of which are induced on normal B cells following mitogen stimulation. The other diffuse B-cell NHLs similarly express activation antigens and resemble "transformed" B cells. The T-cell NHLs generally correspond to normal activated CD4+ T cells. These tumors--which include most peripheral T-cell lymphomas, cutaneous T-cell lymphomas, and HTLV-I-associated adult T-cell leukemias/lymphomas--express antigens induced on activated T cells, including IL-2 and transferrin receptors (CD25 and CD71, respectively), as well as HLA-DR. The lymphoblastic lymphomas, which are generally of T-cell lineage, phenotypically correspond to stages of intrathymic differentiation, often by their coexpression of CD4 and CD8, as well as expression of CD1. It remains controversial whether the immunophenotype of lymphoblastic lymphoma differs significantly from T-cell acute lymphoblastic leukemia. Since immunologic heterogeneity of NHL was first observed, attempts have been made to employ the data as a prognostic variable. Early studies suggested that lineage derivation or expression of markers of proliferating cells affected outcome in NHL. However, these reports were often retrospective, included various histologies, and did not treat patients uniformly. More recent prospective studies with relatively uniformly treated patients, predominantly involving DLCL, suggest that certain immunologically defined subgroups may have significantly different clinical outcomes. However, additional clinical studies will be necessary before treatment options are based upon immunologic markers.     

Diagnostic specificity of the monoclonal anti-CALLA antibody VIL-A1 in leukemia and malignant lymphoma

VIL-A1 is an anti-CALLA antibody which binds efficiently and exclusively to CALLA positive cells. When the cell type specificity of VIL-A1 is studied in acute leukemias and lymphomas, results show that in those leukemias which could be characterized by cytochemical and morphological methods, VIL-A1 reactivity was specific for cells of lymphoid origin. It can therefore be assumed that VIL-A1 positive AUL cells (in this case 4 out of 9 patients) are also lymphoid in origin. In no case were AML blasts found to be positive with this antibody. Seventy-four per cent of the 88 ALL patients were positive (L1 + L2) whereas none in the L3 subgroup were positive, and 48% of CML patients in blastic crisis were positive. Of the low grade non-Hodgkin malignancies, only CB/CC was positive, distinguishing it from the CC type which was negative. Of the high grade lymphomas IB was found to be negative, while the others showed a heterogeneous picture which was not related to other immunological parameters.     

Serial transplantation and characterization of a canine diffuse large cell lymphoma grafted in nude mice.

A continuous canine lymphoma cell line (DL. 24N) was established by serial transplantation from a diffuse large cell lymphoma into athymic nude mice, yielding subcutaneous tumours at the injection site associated with lymph node metastasis. The transplanted tumour cells, of canine origin as assessed by the karyotype, appear comparable to the large non-cleaved and immunoblastic human cells, as did the initial tumour cells. Immunological studies show reactivity with antibodies to dog immunoglobulins, DT-2 and Thy-1, to MHC-class II antigens, and to both CD10 (CALLA) and CD21 (CR2) human antigens. Such a cell line, showing similarities with some of the human diffuse large cell lymphomas, will thus provide a new tool for further comparative studies of malignant lymphomas.     

Cell origin of 18 patients with non-T, non-B acute lymphoblastic leukemia

Cell surface markers of 18 patients with non-T, non-B acute lymphoblastic leukemia (ALL) were analysed with a panel of monoclonal antibodies (McAbs) by indirect immunofluorescence. The results showed that the cells of 13/18 patients originated from immature B cell or B progenitors. The cells of 12/18 patients expressed B cell antigens (CD 19+ or CDw 40+). It was suggested that antibodies directed to these two surface markers could help each other in the diagnosis of ALL of B cell origin. Diagnosis might be further improved if anti-CD19 and CDw 40 McAb be used at the same time. CD 9 antigen was expressed on immature B cell surface. 13/14 patients with B-CLL did not react with anti-CD 9, but 14/18 with non-T, non-B ALL were positive. Cells of 7/18 patients expressed CALLA (CD 10), HLA-DR and CD 19/CDw 40, but did not express T cell associated antigens (CD1, CD2, CD3, CD7), so CALLA(+)-ALL also belongs to B cell lineage.     

Characterization of thymocytes expressing the common acute lymphoblastic leukemia antigen

We studied the relationship between CALLA + thymocytes and two known markers of T-lymphocyte differentiation, Tdt and the sheep erythrocyte receptor. Thymocytes were studied using double fluorochrome analysis (with monoclonal anti-CALLA antibody and anti-Tdt) before and after E rosette separation. We found that approx. 4% of unseparated thymocytes were CALLA + and that most CALLA + cells were also Tdt+. After E rosettes separation CALLA + Tdt + cells were found mostly in the ER- fraction (20% of ER- cells) while only 1.0% of ER + cells were CALLA +. The expression of CALLA on ER? Tdt + thymocytes suggests that CALLA may defined cells early in T-cell differentiation.     

T cell receptor gene rearrangements in B-precursor acute lymphoblastic leukemia correlate with age and the stage of B cell differentiation

Children with ALL diagnosed at less than 2 years of age have a poor prognosis when compared with older children. In an effort to identify biologic features of ALL in children less than 2 that might explain this difference, we performed extensive immunophenotypic and molecular genetic analyses on a series of patients. For comparison purposes patients were divided into four groups: CALLA- (CD10-) infants less than 2 years of age at diagnosis (n = 10), CALLA- children greater than 2 years of age at diagnosis (n = 10), CALLA+ infants (less than 2 years, n = 21) and CALLA+ children (older than 2 years, n = 21). No immunophenotyping differences in CALLA- or CALLA+ subgroups were identified when cases less than 2 were compared with cases greater than 2 years of age at diagnosis. The most interesting results were in the CALLA- group where 94% of the samples expressed the B cell antigen CD19 but 27% co-expressed CD7. Double labeling experiments confirmed leukemic blast cells co-expressed CD19 and CD7. The double-labeled cells represent either leukemic conversion of a precursor cell which has not yet committed to B or T cell lineage or aberrant expression of these antigens. Molecular genetic studies demonstrated that all samples, regardless of the patients' age or immunophenotype, had rearrangement of the Ig heavy chain gene. The most striking molecular results were in CALLA- patients; in patients less than 2 at diagnosis neither the beta- nor the gamma-chain gene of the T cell receptor (TCR) was rearranged, whereas DNA from 5 of 10 patients over the age of 2 demonstrated beta- or gamma-chain TCR gene rearrangements. The percentage of CALLA+ cases under the age of 2 years with rearrangements in TCR genes is less than that found in CALLA+ cases over the age of 2 years. The finding of no TCR rearrangements in CALLA- ALL and a decreased number of gamma-TCR rearrangements in CALLA+ cases under the age of 2 suggest that age may affect TCR gene rearrangements in lymphoblasts. The molecular differences in TCR gene rearrangements do not appear to correlate with the response to therapy.     

Non-Hodgkin's lymphoma phenotyping: problems in the use of heterologous and monoclonal antibodies

Cell suspensions or frozen sections of lymph node biopsies from 32 patients with non-Hodgkin's lymphoma (NHL) were studied for sheep erythrocyte (E)-binding under three conditions (Estandard, EAET, Egravity), Fc and C receptors, immunoglobulin (Ig) heavy and light chain class and reactivity with heterologous antisera to T cells (T-LCL), HLA-D (Ia-like) and common acute lymphocytic leukemia (c-ALL) antigens. Selected B and T cell lymphomas were also tested for reactivity with the monoclonal antibodies OKT 3, OKT 4, OKT 6, OKT 8, OKT 11A, Leu-1, Leu-2a, Leu-3a, Leu-4 and Leu-7. There were 26 B and 6 T lymphomas. Most B lymphomas were mu+ (81%), kappa+ (77%) and 31% were mu+ delta+. One of the T lymphomas arose in a patient with antecedent follicular small-cleaved (B) cell lymphoma. The most accurate marker for characterizing the immunologic phenotype in NHL was the clonal excess of kappa+ or lambda+ cells. Neither Estandard, EAET, Egravity or T-LCL were consistently reliable as sole reagents in identifying T-cell lymphomas, their individual scores often being lower than those of monoclonal pan-T cell reagents. HLA-D (Ia-like) antigen was noted in 89% of B and 50% of T lymphomas. The corresponding values for c-ALL antigen were 12 and 33%, respectively. The comparative scores in T-lymphomas between OKT 4 and Leu-3a for "helper-inducer" (HE) cells and OKT 8 and Leu-2a for "suppressor-cytotoxic" (SU) cells were not uniformly consistent. Four T lymphomas had a mixed HE/SU cell phenotype, one was HE, and another SU. Anti-T reactivity was detected in the neoplastic follicles of six of seven follicular lymphomas. The percentage of anti-T reactive cells within positive neoplastic follicles was usually small (5-15%) and of the same order as that noted within reactive lymphoid follicles (5-30%). High numbers (50-100%) of cells from five small lymphocytic B, three diffuse small cleaved cell B and six T cell lymphomas were also positive with one or more anti-T reagents, suggesting the presence of cross-reactive antigens that make phenotyping of lymphomas with monoclonal antibodies problematic. Reactivity with the monoclonal antibody Leu-7 (HNK-1), a putative NK-specific reagent, was seen in one of five B and three of five T lymphomas.     

Anatomical distribution of CALL antigen expressing cells in normal lymphatic tissue and in lymphomas

Anatomical distribution of common acute lymphoblastic leukemia antigen (CALLA) was studied in lymphomas as well as in normal lymphatic organs using the monoclonal antibody VIL-A1. Twelve lymphomas were labelled by VIL-A1. Three of the 12 tumours also had T-cell marker, six lymphomas also showed immunoglobulin staining and only three tumours were pure CALLA lymphomas. Tonsils showed a distinct CALLA labelling of many germinal centre cells and of singular cells in interfollicular T-cell regions. Children's thymuses showed rare distinctly labelled cells in the cortex and medulla and slightly more cortical cells stained faintly by VIL-A1. Foetal thymuses of about the twelfth week of gestation contained many heavily labelled cells. The findings are discussed as evidence for the presence of CALLA on immature B as well as T lymphocytes. They favour the idea of CALLA as a common lymphocyte differentiation antigen although other possibilities of interpretation are also discussed.     

Immunophenotypic similarities of mediastinal clear-cell lymphoma and sinusoidal (monocytoid) B cells

Using 14 well-defined (clustered) monoclonal antibodies (MAbs) against B-cell restricted/associated differentiation and activation (CD) antigens and 12 mediastinal clear-cell lymphomas (MCCL), 46 follicular-center-cell lymphomas (FCCL), and 20 non-neoplastic lymph nodes--including toxoplasmic and HIV-associated lymphadenitis--were immunohistochemically examined to determine the histogenesis of MCCL. Antigenically, MCCL was characterized as CD5-, CD10-, CD19+, CD20+, CD21-, CD22+, CD30-, CD37+, CDw40+, and by a frequent expression of CD11c and CD23, while other antigens were inconsistently expressed. The antigenic profiles of MCCL and FCCL showed statistically significant differences in 4/14 distinct antigens. When the neoplastic cells of both tumor groups were compared with morphologically defined normal B-cell types, the overall resemblance of their immunophenotypes was even closer between MCCL and sinusoidal (monocytoid) B cells than between FCCL and follicular-center B cells. We conclude that MCCL is a lymphoma type distinct from FCCL, most probably representing a highly malignant neoplasm corresponding to sinusoidal B-cell reaction.     

CD5 expression in a lymphoma of the mucosa-associated lymphoid tissue (MALT)-type as a marker for early dissemination and aggressive clinical behaviour.

Mucosa associated lymphoid tissue (MALT) developing in response to chronic infection or autoimmune stimuli has been recognized as a possible site of origin for a distinct type of B-cell lymphoma. While preferentially occurring in the stomach, MALT-type lymphomas can be found in virtually all organs. MALT-type lymphomas normally follow an indolent course, with a tendency to remain localized at their site of origin for a prolonged period of time. Histologically, MALT-type lymphomas are heterogeneous covering a cytological spectrum ranging from centrocyte-like cells to smaller lymphoid cells or monocytoid B-cells. Usually a small number of transformed blasts are also present. Immunohistochemically, the malignant cells express markers of B-cell lineage, but are distinct from follicular lymphomas (which express CD10), mantle cell lymphomas (expressing cyclin D1 and CD5) and small lymphocytic lymphoma, which express CD5 and CD23. In contrast to the usual phenotype CD20+CD10-CD5-Cyclin D1-, scattered reports in the literature have documented expression of CD5 in marginal zone B-cell lymphomas of MALT-type. However, these cases are rare, and aberrant CD5-expression has been thought to be a marker for early dissemination and aggressive behavior in some patients, while other reports have also found CD5 expression in localized indolent MALT-type lymphomas. We report a patient with a CD5+ MALT-type lymphoma following an aggressive clinical course without histological progression who relapsed only 18 months after local radiotherapy at the initial localizations (conjunctiva of the right upper eye lid and hypopharynx), and showed a rapid generalization to the contralateral conjunctiva, mediastinal lymph nodes and the esophagogastric junction. Our case lends further support to the notion that CD5+ MALT-lymphomas arising in the head-and-neck area and/or the ocular adnexa might be characterised by an aggressive clinical course.     

Expression of B-cell-specific markers in different Burkitt lymphoma subgroups

Forty-three Burkitt lymphoma (BL) lines were examined for the expression of 5 monoclonal antibody (MAb)-identified B-cell-specific markers and immunoglobulin production. All (13) EBV-negative BL lines were CALLA+ LB-1-, whereas 30 EBV-carrying lines showed a more heterogeneous pattern. In the EBV-negative lines, the follicle mantle zone markers BA-1 and 35.1C5 were expressed concordantly, at a different level in each line. This coordination was disrupted in EBV-carrying lines. In the EBV-negative lines, there was also an inverted correlation between the expression of 35.1C5 and the germinal center marker BLA, suggesting that some etiologically important event, perhaps the translocation, had fixed the cells at different stages of their transition from one zone to the other. This inverted relationship was also disrupted in the EBV-carrying lines, suggesting that EBV can interfere with the maturation program of the BL cell. This conclusion was also supported by a comparison between 5 EBV-negative BL lines and their EBV-converted sublines. All converted lines have undergone marker changes, but the degree and nature of these changes was different for each EBV-BL line. Both the coordinated expression of BA-1 and 35.1C5 and the inverted relationship between CALLA and LB-1 were disrupted in several other convertants. We have reexamined our previous finding (Ehlin-Henriksson and Klein, 1984) that the majority of the variant translocation-carrying BL lines were CALLA- LB-1+, in contrast to the majority of the typical translocation carriers that were mostly CALLA+ LB-1-. All II EBV-negative lines were CALLA+ LB-1-, irrespective of the type of translocation. Among the EBV-carrying lines, 4 of 17 typical (8;14) translocation carriers were CALLA- LB-1+, whereas 7 of the 12 variant translocation-carrying lines were CALLA- LB-1+. The remaining two expressed both antigens to some extent. The difference is statistically significant at the 0.03 level.     

Distinctive Pattern of Expression of Activation and Resting B Cell Antigens on Normal and Neoplastic Human B Cells: Immunophenotypic Heterogeneity in Some Lymphomas

The pattern of expression of four B cell antigen systems on mature human B cells and B cell lymphomas were studied. The L30 antigen was detected on small resting B cells, while the B cell activation antigens, CD10, CD25 and L29, were expressed differentially on activated B cells. The multiparameter-flowcytometric analysis of these four antigens revealed that mature B cells changed their pattern of expression in an activation-stage specific manner. Thus, the presence of L30, CD10, CD25 and L29 on mature human B cells correlated with distinct B cell populations at a particular stage of activation. Histo-pathologically well defined B cell lymphomas were also studied for the expression of these four antigens. Burkitt's lymphoma and diffuse small cleaved lymphoma were found to have an heterogeneous expression of these antigens, suggesting that certain types of non-Hodgkin's lymphoma (NHL) are immunophenotypically heterogeneous, and that this heterogeneity may reflect a different biology and behavior in vivo.     

Karyotypic patterns in acute mixed lineage leukemia

We performed cytogenetic and immunologic studies of blast cells from 13 children with acute mixed lineage leukemia (AMLL) to discern patterns of chromosome alteration and antigen expression that would assist in classification of this disease entity. Six patients with 11q23 translocations--including four with the t(11;19), one with the t(9;11), and one with the t(1;11)--were characterized by a young age and hyperleukocytosis. A B cell-associated antigen (CD19) and HLA-DR antigens were expressed by blast cells from all patients; only one case was positive for the common acute lymphocytic leukemia antigen (CALLA, CD10). A myeloid-associated antigen (CD13) was expressed by blast cells from one patient at diagnosis and from another at relapse; it was also expressed by cells from the remaining four patients after brief in vitro culture without addition of differentiating agents. Four patients with t(9;22)(q34;q11) were characterized by an older age and hyperleukocytosis. Each of these cases was positive for CD13, CD19, and HLA-DR, and three were positive for CALLA. The 11q23 translocation was associated with CALLA- ALL marked by a myeloid phenotype, whereas the t(9;22) occurred in cases of acute myeloid leukemia with a CALLA+ lymphoid phenotype. One case had a 7q35-q36 translocation, which involves the region of the T cell receptor beta-chain gene. Our results suggest that karyotypic alterations can be used to refine the classification of AMLL.     

In situ follicular lymphoma: pathologic characteristics and diagnostic features

The diagnosis of in situ follicular lymphoma (FL) is feasible when immunohistochemical characterization is carried out and genetic abnormalities are assessed. We usually use a selected diagnostic panel of antibodies (CD10, CD20, CD23, BCL2, BCL6, and Ki67) in lymph nodes with follicular hyperplasia only when we analyze an unexplained lymphadenopathy. Molecular studies, for example, fluorescence in situ hybridization analysis for t(14;18), are restricted to doubtful cases in which immunohistochemistry data are ambiguous. Immunohistochemically, the involved follicles show strongly positive staining for BCL2 and CD10. The BCL2+ cells are confined only to germinal centers and are not seen in the interfollicular region or elsewhere in the lymph node. The BCL2 staining in the abnormal follicles is notable for its high-level and uniform intensity. In situ FL may be associated with overt FL or with lymphomas other than FL or with other malignancies. The crucial point relies on distinguishing in situ FL arising in asymptomatic patients from cases with presence of lymphoma at the same or other sites. Other open questions remain on the frequency with which in situ FLs occur and the frequency of concomitant systemic disease.     

The expression of the peripheral cannabinoid receptor on cells of the immune system and non-Hodgkin's lymphomas

The peripheral cannabinoid receptor CB2 is expressed highly on normal human B-lymphocytes. C-terminal specific anti-CB2 antibody recognises a non-phosphorylated inactive receptor on na?ve and resting B-lymphocytes. Another, N-terminal specific CB2 antibody, primarily recognises B-cells present in the germinal centres of secondary follicles in lymph nodes. We hypothesise that N-terminal specific CB2 antibody recognises activated CB2 receptors. In this study, we showed using these antibodies, that expression of CB2 is generally absent on T-lymphocytes in reactive, non-malignant human lymphoid tissues. Applying single and dual immunohistochemistry, CD23(+) follicular dendritic cells and a small but significant subpopulation of CD68(+) macrophages showed positive staining with the N-terminal specific CB2 antibody but not with the C-terminal specific CB2 antibody. This may indicate the presence of an active CB2 receptor on these cells with possible involvement in immunomodulation. In contrast to the low expression on normal T-cells, abundant levels of CB2 protein were present on T-non-Hodgkin's lymphomas (NHL). Moreover, in many B-NHL, high CB2 protein expression was found as well. In contrast to the distinct expression patterns in normal immune tissues using the two different CB2 antibodies, NHL specimens in general stained positively with both. We conclude that CB2 receptor expression pattern may be abnormal in NHL.     

BLA.36: a glycoprotein specifically expressed on the surface of Hodgkin's and B cells.

Anti-BLA.36 is an antibody that recognizes a glycoprotein with an apparent molecular weight of 36 kilodaltons, termed B lymphocyte antigen (BLA.36). By using an immunochemical staining technique, BLA.36 was found to be specifically expressed on Hodgkin's and human B cell lines including early B progenitor cells. Other cell lines representing T cell lymphomas, non-B large cell lymphomas, melanomas and carcinomas were consistently negative. BLA.36 is distinct from the previously identified antigens of hematopoietic cell lineage. The specificity of expression of BLA.36 in tissue sections mirrored that of cell lines. In normal tissues, BLA.36 was detectable predominantly on cells in the germinal center and mantle zone of reactive follicles in lymph nodes and spleens. In hematopoietic malignancy, the antigen was expressed on the surface of Reed-Sternberg cells, mononuclear Hodgkin's cells and also on malignant cells of B cell lineage. BLA.36 was also observed on lymphoid cells of 10 to 24 week fetal liver: a double-antibody-staining method revealed that these BLA.36-positive cells also contained immunoglobulin mu heavy chain consistent with identification as early B cells. Under these conditions, T lymphocytes, histiocytes, granulocytes, macrophages, stromal cells in lymphoid tissue, and both normal and neoplastic epithelial cells were consistently negative for the expression of the antigen, with the single exception of a variable proportion of Kupffer cells in normal liver. The antibody has already established its usefulness for the identification of Reed-Sternberg and Hodgkin's cells, and also normal and malignant B lymphocytes in frozen as well as formalin-fixed tissue sections. Furthermore, binding of F(ab)2 fragments of anti-BLA.36 to antigen-positive cell lines specifically inhibited the proliferation of cells. Such an effect was eliminated by the removal of the antibody from the culture-medium, suggesting a possible growth-related function of the antigen in Hodgkin's and B cells.     

Aberrant antigen expression detected by multiparameter three color flow cytometry in intermediate and high grade B-cell lymphomas.

The aberrant expression of antigens (Ag) in lymphoproliferative disorders may cause a diagnostic problem when single parameter immunohistochemical assays are performed on frozen or paraffin sections because coexpression by relevant cells is not determined. This aberrant expression also raises the question as to whether mixed lineage (biphenotypic) lymphoid proliferations exist. Marrow (6) and extramedullary (20) tissues from 26 patients with diffuse, intermediate and high grade, B-cell lymphomas (IWF E=1, F=1, G=19, H=1 and J=4) were analyzed with 19 markers using 3-color flow cytometry. The percentages (%) of patients with double Ag coexpression in at least 20% of the CD19+ or CD20+ lymphoma cells were: stem cell (SC) Ag: CD10 = 58 and CD34 = 15; T-cell Ag: CD2 = 38, CD5 = 19 and CD7 = 19; myeloid (My) Ag: CD13 = 19 and CD33 = 8. The corresponding % with unusual triple Ag coexpression in at least 10% of the CD19+ B-cells were SC+T+ Ag: CD10CD2 = 50, CD10CD5 = 27, CD10CD7 = 38, CD34CD2 = 31, CD34CD5 = 19 and CD34CD7 = 27; T+T+ Ag: CD2CD5 = 35, CD2CD7 = 42 and CD5CD7 = 31; T+My+ Ag: CD2CD13 = 35 and CD2CD33 = 12; and My+My+ Ag: CD13CD33 = 12. Ten of 12 lymphomas tested showed clonal immunoglobulin (Ig) heavy chain gene rearrangements in the absence of clonal T-cell receptor (TCR) gene rearrangements. None (0%) of the My Ag positive cases showed immunoreactivity for myeloperoxidase. We conclude that the anomalous T and My Ag expression seen in the above B-cell lymphomas is not indicative of mixed lineage proliferation but represents the aberrant expression of these antigens by the malignant cells.     

Expression of CD29 on lymphoma cells and/or CD36 on microvascular endothels correlates with high serum LDH level in diffuse large B-cell lymphomas (DLBCLs) and is frequent in de novo CD5-positive DLBCLs

Our previous study demonstrated that CD29 and CD36 mRNAs were overexpressed in de novo CD5-positive diffuse large B-cell lymphomas (CD5+ DLBCLs) compared with CD5-negative (CD5-) DLBCLs. CD29 was expressed on lymphoma cells, and CD36 on microvascular endothels. In the present study, we analyzed the clinicopathological significance of CD29 and microvascular CD36 (mvCD36) expression in many cases of DLBCLs. CD29 and CD36 expression was examined by immunohistochemistry on frozen sections of 159 specimens, consisting of 120 cases of CD5-DLBCL, 14 cases of de novo CD5+ DLBCL, 11 cases of follicular lymphoma, 8 cases of mantle cell lymphoma, and 6 cases of reactive lymphoid hyperplasia. Three cases of CD5- intravascular large B-cell lymphomas (IVLs) with hepatic intrasinusoidal infiltration were also examined. CD29+ and/or mvCD36+ DLBCL cases had significantly higher serum LDH levels compared with CD29- mvCD36- DLBCL cases. The number of CD29+ and mvCD36+ cases, and the number of CD29+ or mvCD36+ cases, were significantly higher in de novo CD5+ DLBCLs than in CD5- DLBCLs. The frequencies of CD29 and mvCD36 expression were low in follicular lymphomas, and mvCD36 was negative in reactive lymphoid hyperplasias. Liver biopsies of three IVL cases showed that CD29 was positive on lymphoma cells, and, that CD36 was expressed on hepatic sinusoids. The present study supports the notion that CD29 and/or mvCD36 expression manifests the aggressive feature of a subset of DLBCLs because of its correlation with high serum LDH levels. Furthermore, CD29 and mvCD36 expression should be evaluated in relation to the intravascular growth pattern of lymphoma cells.