Localization of immunological complexes fixing beta1C (C3) in germinal centers of lymph nodes.

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the beta(1C) component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit beta(1C), IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, beta(1C), and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for beta(1C), IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the beta(1C) component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.

Use of a novel colony assay to evaluate the cytotoxicity of an immunotoxin containing pokeweed antiviral protein against blast progenitor cells freshly obtained from patients with common B-lineage acute lymphoblastic leukemia.

We report a novel colony assay for B-lineage progenitor cells in acute lymphoblastic leukemia (ALL). The primary plating efficiency of blast progenitors freshly obtained from common B-lineage ALL patients varied between 0.09 and 2.63%. Morphological, cytochemical, and immunological analyses of cells from day 7 colonies provided the evidence that they are B-lineage lymphoblasts. Immunological marker analyses of cultured blasts using BA-2 (anti-CD9), BA-3 (anti-CD10), BA-1 (anti-CD24), and B43 mAb have allowed us to define two distinct immunological groups. The first group had BA-2+, BA-3+, BA-1+, B43+ marker profiles, consistent with the phenotype of uncultured bone marrow blasts. The second group differed in that the cells in the blast colonies were BA-3 (anti-CD10)-negative, although many of the cells in the bulk population were BA-3+ before culture. Blasts from both groups were able to proliferate and form secondary colonies when recultured. A pan-B immunotoxin was synthesized by linking B43, a human B cell-specific mAb, to pokeweed antiviral protein (PAP). This study showed that B43-PAP can effectively eradicate leukemic progenitor cells freshly obtained from patients with common B-lineage ALL. B43-PAP eliminated greater than 99.96% of blast progenitors under conditions in which only minimal inhibition of normal bone marrow progenitor cells (CFU-GM, CFU-E, CFU-MK, CFU-GEMM) was observed. Our results establish that the surface determinant recognized by B43 is expressed on B-lineage progenitor cells in ALL, and that these cells are sensitive to PAP at the ribosomal level. To our knowledge, B43-PAP is the first IT to prove effective against common B-lineage ALL cells.

B and T lymphocytes in primary immunodeficiency disease in man.

B- and T-cell populations in 32 patients with different forms of primary immunodeficiency disease were studied. The B-cells in peripheral blood were investigated with respect to surface immunoglobulins by means of immunofluorescence. The T-cell function was studied utilizing quantitation of proliferative response to phytochemagglutinin (PHA)(1) and delayed allergy to various antigens. In 10 patients lymph node lymphocytes were also evaluated 11 male children with infantile x-linked agammaglobulinemia were divided into two subgroups. One did not show immunoglobulin spots on peripheral blood lymphocytes at all, the other contained a very low percentage of IgM- and occasionally IgA bearing lymphocytes. Eight patients with common variable immunodeficiency had moderately decreased percentages of peripheral blood and lymph node lymphocytes with surface immunoglobulins, but these patients lacked immunoglobulin secreting cells. Four cases of isolated IgA deficiency had normal or high percentages, and two cases of ataxia-telangiectasia had high percentages of lymphocytes with IgA in so called receptor distribution in both peripheral blood and lymph nodes. In three patients with infantile combined immunodeficiency that had been corrected by marrow transplantation, the percentages of Ig-bearing lymphocytes increased to normal or high levels together with establishment of functional T-cell population and ultimate secretion of serum immunoglobulins. One case of Di George syndrome reconstituted by fetal thymus transplant showed gradual decrease of B lymphocytes in circulation parallel to restoration of T-cell population.

B lymphocytes in untreated patients with malignant lymphoma and Hodgkin's disease.

B and T lymphocytes in 37 untreated patients with malignant lymphoma and Hodgkin's disease were studied. B cells in the peripheral blood were investigated with respect to surface immunoglobulins and in a few patients with respect to intracytoplasmic immunoglobulins by means of immunofluorescence. T cell function was studied by direct phytohemagglutinin (PHA) microtest (from the same sample of whole blood), mixed lymphocyte culture (MLC), and by delayed hypersensitivity to various antigens. In the 13 patients with Hodgkin's disease the histologic subtype was nodular sclerosis in nine, lymphocyte predominant in two, mixed cellularity in two. Only one of these patients had disseminated disease (stage IV); he showed impaired cellular immunity, a very low percentage of B cells and low levels of serum immunoglobulins. Of the remaining patients with Hodgkin's disease, with one exception, normal percentages but rather low absolute numbers of B lymphocytes per mm(3) of blood were found. One patient with a low percent and low absolute number of B lymphocytes showed very high serum IgG. Of 24 patients with non-Hodgkin's malignant lymphoma, seven (29%) showed monoclonal B cell proliferation in the peripheral blood (five mukappa, two gammakappa). By morphologic criteria, 14 patients had involvement of bone marrow, five of these had involvement of peripheral blood. Four of the latter five patients showed marked increases in percentages and absolute numbers of B lymphocytes in the peripheral blood reflecting the monoclonal proliferation. In three additional patients monoclonal proliferation of lymphocytes was found by immunofluorescence although the blood smears appeared morphologically normal. Serum immunoglobulin abnormalities without monoclonal B cell proliferation in the peripheral blood were observed in six patients.

Oligoclonal T cell receptor gene rearrangements in blood lymphocytes of patients with acute Epstein-Barr virus-induced infectious mononucleosis.

Gene rearrangement studies were performed on blood lymphocytes from eight patients with acute Epstein-Barr virus-induced infectious mononucleosis. The diagnosis in each case was based on characteristic clinical, hematologic, and serologic findings. The blood lymphocytes in each patient consisted predominantly of CD8+ T cells. EBV DNA was detected in seven patients by Southern blot analysis (EBV Bam HI W probe, Bam HI). A germline configuration was found for the immunoglobulin heavy and light chain genes (JH probe, Bam HI and Eco RI; C kappa probe, Bam HI; and C lambda probe, Eco RI). T cell receptor gene rearrangements were detected with J gamma and J beta 1 + 2 probes. Using a J gamma probe with two different restriction enzymes (Bgl II and Eco RI), the blood from each patient showed several bands corresponding to the polyclonal pattern previously described in the blood of normal individuals. Using J beta 1 + 2 probes with two different restriction enzymes (Bgl II and Bam HI), each case showed from 3 to about 12 extragermline bands of varying intensity and in different locations from case to case. In addition, each case showed relative deletion of the J beta 1 germline band. This oligoclonal pattern of T cell receptor gene rearrangements has not been previously reported in benign or malignant T cell populations.

Heterogeneity of cultured leukemic lymphoid progenitor cells from B cell precursor acute lymphoblastic leukemia (ALL) patients.

Colony assays were performed for 50 patients with B cell precursor acute lymphoblastic leukemia (ALL). Blast colony formation was observed for 33 patients, and the plating efficiency (PE) showed a marked interpatient variation, which indicates a pronounced biological heterogeneity at the level of leukemic progenitor cells. Notably, the mean PE of leukemic B cell precursors from patients with a pseudodiploid or near-diploid karyotype with structural chromosomal abnormalities (SCA) was significantly higher than the mean PE of normal diploid or hyperdiploid cases. All patients who had SCA involving 7p13, 11q23-24, or 12p11-13, and patients with a Philadelphia chromosome had high PE values. The S phase percentage, expression of CD19 antigen, and relapse status were also correlated with PE. Significantly, colony blasts had slightly different surface marker profiles in each case and were common ALL antigen negative in 33% of cases, which indicates the existence of a marked immunological heterogeneity at the level of leukemic progenitor cells.

Monoclonal antibody MCS-2 as the marker of phorbol diester-induced myeloid differentiation in acute undifferentiated leukemia.

Leukemic cells from 32 cases of acute leukemia were cultured in vitro with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to study their differentiative potential. Three cases of acute undifferentiated leukemia (AUL) were studied intensively. We found that culturing of leukemic cells with TPA can induce changes in cell surface antigens. In particular, MCS-2, a "pan" granulocyte/monocyte marker, was inducible in vitro in AUL and in acute myelogenous leukemia, while it was not inducible in acute lymphoblastic leukemia. BA-2 (recognizing the Mr 24,000 protein) and TA-1 (recognizing the Mr 170,000 and Mr 95,000 proteins) were also inducible in cases of AUL, acute myelocytic leukemia, and acute monoblastic leukemia, although these antigens are not limited only to leukemias of the myelomonocytic lineage. Our studies also indicate that undifferentiated cells could be induced to nonspecific esterase and sometimes to chloroacetate esterase reactivity while losing terminal deoxynucleotidyl transferase. Morphological studies in these cases revealed cytological maturation following TPA treatment. In most cases, these changes were also partially inducible by culturing cells in medium alone or with the addition of dimethyl sulfoxide but not to the extent that was demonstrated by TPA. Our studies showed that MCS-2 is a very good, specific marker of acute nonlymphocytic leukemia. A potential use for TPA to aid in the subclassification of patients with AUL is also suggested.

Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients.

The lymphoproliferative processes that developed in five renal transplant recipients were studied in an attempt to characterize and classify them morphologically. Nine surgical specimens, hematological material on all patients, and autopsy specimens from three patients were available. Studies performed included: conventional histopathology; evaluation of cell markers (immunoglobulins and sheep erythrocyte, complement, and Fc receptors) and cytoplasmic immunoglobulins (peroxidase-antiperoxidase technique); ultrastructural examination; and karyotype analysis. The lymphoid lesions in our patients shared marked cytological polymorphism (small and large cells, of both follicular center and "medullary" type) and polyclonal B-cell features, which indicated a common reactive nonneoplastic origin. However, other features, such as morphological atypia of the immunoblasts, extensive necrosis, chromosomal aberrations, and an incipient monoclonal component suggested the development of lymphoma in some of these lesions. In contradistinction, the abundance of typical immunoblasts was a feature that seemed to correlate with the clinical activity of the disease rather than with the biological malignancy. The multiplicity of B-cell types and the presence of a follicular center cell component with diffuse distribution, as well as the extensive necrosis in the malignant forms, seem to differentiate morphologically the lymphoproliferative processes arising in transplant recipients from both the hyperplasias and the lymphomas developing in immunologically normal hosts. For the former, we propose the terms of "polymorphic diffuse B-cell hyperplasias" and "polymorphic B-cell lymphomas."

Correlations between immunological phenotype and karyotype in malignant lymphoma.

We have correlated immunological characteristics and karyotypic abnormalities from lymphomas in 118 patients. T-lymphomas differed significantly from B- and non-B-, non-T-lymphomas in having more normal metaphases, trisomy 19, and breaks at 1q21, 2q21, 3q27, 4q21, and 17q21 (P less than or equal to 0.03). Non-T-lymphomas had breaks in 18q in one-half the cases, but only one of 11 T-lymphomas had such breaks (P = 0.02). Among B-lymphomas, specific chromosome abnormalities were associated with the type of immunoglobulin heavy but not light chain expressed. A break at 14q22 or q24 was associated with surface delta mu-immunoglobulin (P = 0.02); trisomy 22 or a break in 22q and a break at 2q32 was associated with surface gamma-immunoglobulin (P less than 0.001); and trisomy 12 and a break at 2p13 was associated with cytoplasmic gamma-immunoglobulin (P less than 0.01). Among B-lymphomas, several cytogenetic abnormalities were associated (P less than or equal to 0.02) with expression of CD24 or CD9 surface antigens. Lack of CD24 was associated with breaks in 2p25, 5q, and 6q21; CD9 was associated with a break at 6q15. Associations with a specific immunological phenotype were not identified for cytogenetic abnormalities involving a band to which genes encoding immunoglobulin or the T-cell receptor have been localized. Breaks were common at 14q32, the genomic site of the immunoglobulin heavy chain loci, in B-, non-B-, non-T-, and T-lymphomas. In T-lymphomas this may be because this is the site of the AKT1 oncogene. Breaks were uncommonly found at the light chain loci or the genomic sites encoding the T-cell receptor. However, the recurring breakpoints associated with T-lymphomas were commonly found on chromosomes to which genes coding for various T-cell antigens have recently been provisionally assigned.

Nonrandom chromosome abnormalities in lymphoma.

G-banded chromosomes were studied from involved lymph nodes or other tumor masses in 94 patients with malignant lymphoma. Clonal chromosome abnormalities were identified in 91 patients including all 81 B-lymphomas but only 6 of 9 T-lymphomas. Many recurring chromosome abnormalities were found. Most common numerical alterations involved gains of chromosomes 12 (19% of patients), 18 (13%), 7 (12%), and 21 (10%). Structural abnormalities, which were more frequent than numerical alterations, most commonly involved chromosome regions 14q (71% of patients), 18q (36%), 6q (31%), 1p (24%), and 8q (19%). Seven recurring translocations were identified, and all except one involved 14q32. The most frequent were t(14;18)(q32;q21) in 22 patients, t(8;14)(q24;q32) in 9 patients, and t(1;14)(q42;q32) in 3 patients. Deletions most frequently involved the long arm of chromosome 6 at band q21 (11 patients) or q23 (7 patients). The common recurring chromosome abnormalities were correlated with histology (International Working Formulation for Clinical Usage) and immunological phenotype. Four abnormalities were significantly associated with specific histologies. Eighteen (82%) patients with t(14;18)(q32;q21) were follicular. Similarly, 82% of patients with del(6)(q21) were large cell lymphomas. Lymphomas with trisomy 7 were either diffuse large cell or follicular, while patients with t(8;14)(q24;q32) were either diffuse large cell or small noncleaved cell. A significant association with immunological phenotype was seen for t(14;18) only. All patients were either B- or complement lymphomas, and the heavy chain(s) was more commonly gamma and less frequently delta mu than among the total B-lymphoma population. We conclude that essentially all lymphomas have cytogenetic abnormalities; further study is required to determine their significance. Particularly, it will be of interest to see if oncogenes are found in the regions of these chromosome abnormalities.

Use of monoclonal antibodies, morphology, and cytochemistry to probe the cellular heterogeneity of acute leukemia and lymphoma.

A combined immunological, morphological, and cytochemical approach to the study of malignant cells in patients with acute leukemia and lymphoma is presented. Newly produced monoclonal antibodies that bind to antigens of human mononuclear cells (TA-1), or B-lymphocytes (BA-1) were used to study malignant cells from patients with acute lymphoblastic leukemia (ALL). acute myelocytic leukemia, acute myelomonocytic leukemia, and chronic lymphocytic leukemia. Results in lymphoid leukemia-lymphoma patients were compared with other immunological markers and indicate that the major groups of ALL and childhood non-Hodgkin's lymphoma are T-ALL, pre-T-ALL, pre-B-ALL, B-ALL, and non-T, non-B-ALL. In addition, each major group had multiple phenotypes when analyzed with seven immunological markers including the erythrocyte rosette receptor, surface immunoglobulin, cytoplasmic immunoglobulin M, the early lymphocyte-acute lymphoblastic leukemia antigen, monoclonal antibody TA-1, monoclonal antibody BA-1, and a monoclonal antibody against HLA-DR. While immunological heterogeneity was demonstrable within each group, distinct biological behavior was observed, with T-ALL and B-ALL generally presenting as "lymphomas" and the others presenting as "leukemias." Morphological analysis using the French-American-British classification provided independent information in the definition of groups with differing clinical behavior. Cytochemical analyses demonstrated focal paranuclear staining of leukemia cells with acid phosphatase in 73% of T-ALLs and 6% of non-T, non-B-ALLs.

Late-developing Philadelphia chromosomes in a case of T-cell acute lymphoblastic leukemia.

A child with T-cell acute lymphoblastic leukemia (ALL) is presented who at relapse acquired two Philadelphia chromosomes (Ph). Molecular studies at relapse revealed a rearrangement of the major breakpoint cluster region (M-bcr) on chromosome 22. No rearrangements of the immunoglobulin heavy chain or T-cell beta receptor gene loci were demonstrated. This case supports the hypothesis that leukemogenesis in Ph-positive malignancies is a multi-step process, the first step of which may not necessarily involve acquisition of the Ph.

Transformation of chronic lymphocytic leukemia to small non-cleaved cell lymphoma: a cytogenetic, immunological, and molecular study.

A patient with chronic lymphocytic leukemia (CLL) transforming into a small non-cleaved cell lymphoma (SNCL) with the occurrence of a t(8;22) is described. The SNCL and the CLL were both found to have a germline lambda light chain gene configuration and the same heavy chain and kappa light chain gene rearrangements. The SNCL was CD10 (CALLA) negative and appeared to be CD5 negative. It is concluded that the SNCL is derived from the CLL and that activation of the c-myc oncogene may have played a role in this transformation.

Morphology of chronic lymphocytic leukemia and its relationship to survival.

The morphology of lymphocytes in blood and bone marrow from patients with chronic lymphocytic leukemia was studied; blood lymphocyte morphology was related to survival. Three primary morphologic groups emerged. Group 1 was characterized by small to medium-sized lymphocytes with narrow rims of cytoplasm and coarsely clumped nuclear chromatin. In group II the predominant lymphocytes were large with abundant cytoplasm. Group III was characterized by a heterogeneous population of lymphocytes with characteristics of both groups I and II. Clinical features of the patients were studied, and B and T typing of the lymphocytes was done. The median survival in group I was 26+ months; in group II 46+ months; and in group III 50+ months. Our data are at variance with previous reports and suggest that survival in patients with large lymphocytes is longer than in those with small lymphocytes.

Clinical characteristics of post-transplant lymphoproliferative disorders.

PURPOSE: To study the histopathologic findings, clinical course, and therapeutic outcome of patients who developed a lymphoproliferative disorder after undergoing solid organ transplantation. PATIENTS AND METHODS: A series of 26 patients who developed a lymphoproliferative disorder after solid organ transplant during a 27-year period were studied. RESULTS: The 26 patients ranged in age from 6 to 68 years (median 42 years). The lymphoproliferative disorder was diagnosed from 1 to 211 months (median 80 months) after transplantation. The type of transplant was kidney (n = 21), heart or heart-lung (n = 4), or liver (n = 1). Most patients received azathioprine and prednisone, in addition to antilymphocyte globulin or cyclosporine, for post-transplant immunosuppression. Eight patients had lymphoma that could be classified according to the International Working Formulation (IWF-F, IWF-G, IWF-H). Sixteen patients had polymorphic lymphoma, and 2 patients were classified as having polymorphic lymphoid hyperplasia. Patients were staged by the Ann Arbor staging system. Nine patients had stage I disease, 4 stage II, 6 stage III, and 7 stage IV. Central nervous system, lung, or marrow involvement was present in 27%, 23%, and 14% of patients, respectively. In the 17 patients studied, immunophenotype was monoclonal B-cell (n = 12), malignant T-cell (n = 2), or polyclonal B-cell (n = 3). The initial therapeutic approach was generally a reduction in immunosuppression, but, thereafter, the approach to therapy varied. In patients with localized disease, surgical excision and/or involved field radiotherapy were utilized as applicable. For patients with more extensive disease, other approaches such as high-dose acyclovir, combination chemotherapy, or alpha interferon were utilized. Overall, 15 of 26 patients (58%) responded to systemic therapy or were rendered disease-free either by surgery or radiation, including 8 (31%) with a complete remission (CR). Only 3 of 9 patients responded to chemotherapy, whereas 4 of 13 patients responded to acyclovir (including 3 patients who experienced CR). Remission duration ranged from 8 to 122 months (median 32+ months). Twenty-one of 26 patients (81%) have died. Survival ranged from less than 1 to 122 months (median 14 months). CONCLUSION: The outcome of patients with post-solid organ transplant lymphoproliferative disorders is poor, and the optimal approach to therapy is not clear. Newer therapeutic approaches are thus needed to improve the outcome of these patients.

Chronic lymphoproliferative disorder with unusual clinical, morphologic, ultrastructural and membrane surface marker characteristics.

Four of 105 patients with chronic lymphocytic leukemia (CLL) manifested clinical, morphologic, ultrastructural and membrane surface marker characteristics that differed from those found in patients with typical CLL of demonstrated B-lymphocyte origin. These four patients presented with moderate increases in absolute lymphocyte counts, absolute neutropenia, polyclonal hypergammaglobulinemia and hepatosplenomegaly without lymphadenopathy. Two of them were unusually young, 19 and 25 years old, at the time of diagnosis. The proliferating lymphocytes carried receptors for sheep erythrocytes, a T-lymphocyte marker. In the three patients tested, the lymphocytes also carried Fc receptors. Ultrastructurally the lymphocytes contained cytoplasmic inclusion bodies consisting of parallel tubular arrays. The parallel tubular arrays corresponded to prominent cytoplasmic azurophilic granules on light microscopy. Parallel tubular arrays were found in less than 1 per cent of the lymphocytes in eight patients with typical B-lymphocyte CLL. The process in these four patients may be a distinctive chronic lymphoproliferative disorder originating in T lymphocytes with Fc receptors found in small numbers in the blood of normal persons.

B and T cell lymphomas. Analysis of blood and lymph nodes in 87 patients.

B and T cell populations were studied in blood and neoplastic tissues from 64 untreated and 23 treated patients with non-Hodgkin's lymphoma. This study was undertaken primarily to evaluate the relation of B and T cell markers in various lymphomas to the currently accepted morphologic classifications and to determine the utility of various tissues in defining the cell of origin of a lymphoma. When histologically involved blood, bone marrow, lymph nodes or body fluids were studied, a B or T cell origin of the lymphoma was identified in 26 of 28 (68 per cent) patients. A B cell origin was found in 17 adults classified as having nodular (N) or diffuse (D) poorly differentiated lymphocytic lymphoma (PDLL). One lymphoma of T cell origin was observed in an adult with poorly differentiated lymphocytic lymphoma-diffuse (PDLL-D). In contrast, all cases of PDLL-D in children were T cell in origin. The origin of American Burkitt's (stem cell) lymphoma in two children was the B cell. When histologically involved blood was studied, a B or T cell origin was demonstrated in 10 of 21 (48 percent) adults. Evidence of a monoclonal proliferation of B lymphocytes in the blood was found two adults with more than 7 per cent lymphoma cells in Wright-Giemsa stained blood smears. When neoplastic lymph nodes were studied, the diagnosis of a B cell lymphoma was made in 8 of 12 (67 per cent) adults. Study of surface markers on malignant cells in cerebrospinal or serosal fluids frequently revealed a B or T cell origin of the lymphoma. B and T lymphocyte numbers in the blood did not correlate with immunoglobulin or skin test abnormalities. Abnormalities in circulating B or T cell percentages at diagnosis were a poor prognostic sign in patients with PDLL-D.

Confirmation of the heterogeneity of posttransplant Epstein-Barr virus-associated B cell proliferations by immunoglobulin gene rearrangement analyses.

Immunoglobulin gene rearrangement analysis is a sensitive method for determining clonality of B cell proliferations. We have examined tissue obtained from five renal and one cardiac allograft recipient with Epstein-Barr virus-associated B cell proliferations for immunoglobulin gene rearrangements. Biopsies from two patients with lesions that were hyperplastic morphologically, polyclonal by cellular immunoglobulin staining, and had diploid karyotypes, had no detectable gene rearrangements and were, therefore, consistent with benign reactive processes. These patients are alive without evidence of disease 37 and 57 months after diagnosis. In a biopsy from one patient with a lesion that was malignant lymphoma morphologically, monoclonal by cellular immunoglobulin staining, and had clonal cytogenetic abnormalities, clonal gene rearrangements were detected in a majority of cells, confirming their neoplastic nature. In biopsies from an intermediate group of three patients with morphologically malignant proliferations that were composed predominantly of a polyclonal population of B cells, clonal gene rearrangements were also found, consistent with early malignant transformation in a subpopulation of cells. These findings confirm the heterogeneity of the posttransplant EBV-associated lymphoproliferative diseases and have significant implications for our understanding of the pathogenesis of EBV-induced infections and related lymphomas.

Epstein-Barr virus--determined clonality in posttransplant lymphoproliferative disease.

Analysis of the genomic termini of Epstein-Barr virus can provide valuable insight into the cofactor role of EBV in the development of B cell lymphomas and lymphoproliferative disease. We report EBV genomic findings in pathologic specimens from 10 patients who developed lymphoma or lymphoproliferative disease after renal or bone marrow transplantation. Endonuclease restriction patterns of EBV genomic termini are highly variable in size in both the episomal and linear configuration. This variability in fragment size permits direct assessment of tissue clonality in EBV-infected material. Hybridization with terminus-specific probes also reveals configuration of viral genome (circular and latent vs. linear and replicative). Nine of 10 patients had tumors with mono- or biclonal episomal markers, and 4 of 10 had evidence of linear or replicative virus. Analyses of virally determined markers were compared to immunoglobulin gene rearrangement studies, histologic immunophenotyping, cytogenetics, and clinical outcome. These 10 cases represent a spectrum of lymphoproliferative disorders ranging from benign polyclonal to malignant monoclonal disease. The molecular data lend credence to two important aspects of viral pathogenesis: (1) the finding of a homogeneous episomal population in the monoclonal tumors suggests that EBV infection is an early event in tumorigenesis that occurs before clonal expansion; and (2) therapeutic efficacy of acyclovir has been shown only in presence of polyclonal disease but may impact on intermediate stages where linear replicative virus can be found. Finally, the various assessments of tumor clonality were compared, and although heterogeneity was seen among patients and among diagnostic methods, analyses at the molecular level using virus and immunoglobulin gene specific probes were concurrent and provide the more sensitive means for detection of clonality.

Immunophenotyping of hematopoietic malignancies in paraffin sections.

Immunophenotyping of hematopoietic malignancies is usually accomplished in frozen sections or cell suspensions. To determine whether this procedure was also feasible in paraffin sections, we performed a double-blind immunoperoxidase study of 65 hematopoietic tumors whose phenotypes had been determined previously in fresh tissue. A selected antibody panel was used, including anti-LN2, UCHL-1, anti-cathepsin B, anti-Leu M1, anti-MB2, and anti-MT1. A correct phenotype was obtained on paraffin sections in 95% of cases. All 31 B-cell malignancies were properly classified, showing reactivity for LN2 and MB2. In 14 of 15 T-cell hematopoietic malignancies, all cells reacted with anti-MT1 and/or UCHL-1; the 1 case negative for these antigens was misdiagnosed as a B-cell tumor because of misinterpreted LN2 reactivity in benign histiocytes. Four of 5 true histiocytic neoplasms were positive for cathepsin B and LN2 but lacked other antigens; the fifth case was wrongly considered a B-cell proliferation because only bland histiocytes displayed cathepsin B. Only 1 of 7 Hodgkin's lymphomas was misdiagnosed (as a T-cell tumor); in the other 6 cases, Reed-Sternberg cells were reactive for LN2 and LEU M1. Five of 6 extramedullary myeloid leukemias also stained for LN2, MT1, and LEU M1. One showed LN2, MB2, and MT1; this case was classified as a B-cell neoplasm and indeed represented a pre-B-cell transformation of chronic myelogenous leukemia. These results show that the specified panel of antibodies may be useful for immunophenotyping of hematopoietic neoplasms when only paraffin sections are available for analysis. However, it cannot supplant traditional cell-marker studies of hematopoietic tumors because of its lesser accuracy.