Non-neoplastic Lymphocytes Research Articles

Lack of evidence for an association of Epstein–Barr virus infection with breast carcinoma – authors' response

In her letter, recently published in Breast Cancer Research, Maria Victoria Preciado points out that some published evidence suggests a possible association of breast cancer with Epstein–Barr virus (EBV) [1]. EBV DNA has been detected by PCR in up to 50% of cases (for a review see [2]), and expression of the EBV-encoded nuclear antigen EBNA1 has been demonstrated in approximately 40% of cases by immunohistochemistry using the mAb designated 2B4 [3]. In combination, these findings seem to implicate EBV in the pathogenesis of breast carcinoma. Interestingly, however, most investigators agree that the EBV-encoded RNAs (EBERs), a hallmark of latent EBV infection, are not detectable in breast carcinoma tumour cells [2]. In two independent studies, we have analysed breast carcinomas for evidence of EBV infection [4,5]. In whole tissue sections, EBV DNA was detectable by quantitative PCR in up to 21% of cases at low copy number [5]. However, this does not allow conclusions regarding the cellular source of the viral DNA. We therefore attempted to detect viral DNA directly in the neoplastic cells using a sensitive DNA in situ hybridisation assay [4] or using quantitative PCR of microdissected tumour cells [5]. These studies yielded negative results [4,5]. In support of these results, in situ hybridisation for the detection of the EBERs was also negative in all cases while occasional EBV-positive non-neoplastic lymphocytes were detected by this approach [4]. The latter finding explains the detection of EBV DNA in whole tumour sections by PCR. There remains the issue of reactivity of breast carcinoma cells with the EBNA1-specific mAb 2B4. We have previously reported that this mAb yields nuclear labelling not only of EBV-infected cells, but also of some EBV-negative cells, notably epithelial cells [6]. We therefore recommended the use of another EBNA1-specific mAb, 1H4, when studying epithelial tumours [6]. In one of our recent studies, 31% of breast cancers, including cases that had tested negative for EBV genomes by PCR of microdissected tumour cells, showed nuclear labelling of tumour cells with the 2B4 mAb [5]. However, no labelling of tumour cells was observed with both mAbs in the other study [4]. The discrepancy between these results may relate to technical differences (e.g. fixation of tissues or antigen retrieval). We thus conclude that EBV is not present in tumour cells of breast carcinomas. The reactivity of tumour cell nuclei with the EBNA1-specific mAb 2B4 is likely to be the result of cross-reactivity with an unrelated epitope. There is currently no evidence that breast carcinoma is an EBV-associated malignancy.

Epstein-Barr virus encoded latent membrane protein 1 (LMP1) and TNF receptor associated factors (TRAF): colocalisation of LMP1 and TRAF1 in primary EBV infection and in EBV associated Hodgkin lymphoma

Aims: Epstein-Barr virus (EBV) immortalises B cells in vitro and is associated with several malignancies. Most phenotypic effects of EBV are mediated by latent membrane protein 1 (LMP1), which interacts...

The XRCC1 codon 399 Gln allele is associated with adenine to guanine p53 mutations in non-small cell lung cancer

The X-ray repair cross-complementing group 1 ( XRCC1) gene plays a critical role in the repair of DNA single-strand breaks. A polymorphism at codon 399 of the XRCC1 gene (Arg to Gln) is associated with increased DNA adduct binding and an increase in sister chromatid exchanges after exposure to tobacco carcinogens and may be linked with an increased risk of lung cancer. To further define the interaction between tobacco carcinogens, XRCC1-mediated DNA repair and DNA damage, we examined the role of the XRCC1 codon 399 polymorphism in mutation of the p53 gene in non-small cell lung cancer (NSCLC). Tumor and non-neoplastic (lung or lymphocyte) samples were collected from 116 cigarette smokers with NSCLC. p53 mutations were detected by direct sequencing and/or the GeneChip ® p53 assay in 63 of 116 (54%) tumors. XRCC1 polymorphisms were identified by PCR/RFLP analysis. The distribution of XRCC1 codon 399 genotypes was (Arg/Arg [74 of 116, 64%], Arg/Gln [29 of 116, 25%], and Gln/Gln [13 of 116, 11%]). The prevalence of p53 mutations was similar among subjects with all three XRCC1 genotypes (Arg/Arg [39 of 74, 53%], Arg/Gln [18 of 29, 62%], and Gln/Gln [6 of 13, 46%]). However, the prevalence of specific p53 mutations varied among different XRCC1 genotypes. AT to GC transitions were significantly ( P=0.01) more common among subjects with the Gln/Arg or Gln/Gln genotype (5 of 42, 12%) than in subjects with the Arg/Arg genotype (1 of 74, 1.4%). In summary, the XRCC1 Gln allele is associated with AT to GC mutations in p53 in NSCLC. The XRCC1 gene may play a role in the repair of cigarette smoking-induced DNA damage.

Comparison of T-lymphocyte proliferation in canine epitheliotropic lymphosarcoma and benign lymphocytic dermatoses.

The lesions of patch/plaque cutaneous T-cell lymphosarcoma (CTCL) are similar to many benign inflammatory dermatoses, both histopathologically and clinically. In humans, attempts to develop a simple, reliable immunophenotypic technique to differentiate between the malignant and benign dermatoses have been unsuccessful. The purpose of our study was to determine, using a proliferating cell nuclear antigen (PCNA)/CD3 double immunolabelling technique of paraffin-embedded sections, if the rate of T-cell proliferation in canine CTCL was greater than that of other diseases with non-neoplastic lymphocyte epitheliotropism. We selected cases of patch/plaque (PP) and tumour stage CTCL, erythema multiforme (EM) and cutaneous lupus erythematosus (CLE). We did not find a significant difference in the rate of T-cell proliferation in the epithelia nor the superficial dermis between PP-CTCL and EM. Whereas epithelial T-cell proliferation is significantly higher in PP-CTCL than CLE, it is not diagnostically useful because of overlap in the labelling indices.

Clinicopathological consultation. Lymphoepithelial carcinoma of the larynx hypopharynx, and trachea.

Lymphoepithelial carcinoma of the larynx, hypopharynx, and trachea is a rare neoplasm composed of large, poorly differentiated, nonkeratinized cells intermingled with small nonneoplastic lymphocytes and plasma cells. It is histologically similar to its more common counterpart occurring in the nasopharynx. In contrast to nasopharyngeal carcinoma, most cases have not been associated with Epstein-Barr virus (EBV), although rare cases have been reported to be EBV-positive. The diagnosis often requires immunohistochemistry or electron microscopy for confirmation. The neoplasm seems to behave in a fashion reminiscent of nasopharyngeal carcinoma. Lymph node metastasis occurs in the majority of patients, and eventual visceral dissemination occurs in one fourth. Radiotherapy is the main treatment for the primary tumor and regional metastases, but chemotherapy is indicated for more advanced disease. The initial stage is the primary determinant of prognosis. Death from disease occurs in about one third of patients.

Epstein‐Barr virus infection in the neoplastic and nonneoplastic cells of lymphoid malignancies

BACKGROUND The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied. METHODS Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein–Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases. RESULTS Although EBER-1-positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T-cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1. EBV genomic DNA was detected in 8 of the 39 examined cases. CONCLUSIONS T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes. Cancer 1996;77:2339-47.

Epstein-Barr virus infection in the neoplastic and nonneoplastic cells of lymphoid malignancies

The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied. Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein-Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases. Although EBER-1-positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T-cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1, EBV genomic DNA was detected in 8 of the 39 examined cases. T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes.

Contribution of Epstein‐Barr virus to development of malignant lymphoma of the thyroid

Epstein-Barr virus (EBV)-related mRNA, their products and apoptosis were investigated in 32 cases of malignant lymphoma of the thyroid (MLT) and 30 cases of Hashimoto's thyroiditis (HT) by in situ hybridization, immunohistochemistry and nick end labeling method on routinely processed tissue sections. In MLT, EBV-encoded small RNA (EBER) were detected in three cases, consisting of a follicular, predominantly large cell type (FL), a diffuse, large cell type (DL) and a large cell, immunoblastic type (IBL). In EBER-positive cases, IBL that was positive for T cell marker, exhibited neither BamHl H Left Frame 1 (BHLF1) transcript, EBV-encoded latent membrane protein (LMP) nor BamHl Z Left Frame 1 (BZLF1) gene product (ZEBRA), whereas both BHLF1 and ZEBRA were found in a small portion of the tumor cells in the FL and DL that expressed B cell marker and LMP. Apoptotic cells were observed in only a few lymphocytes in HT, and in a few non-neoplastic lymphocytes and various numbers of lymphoma cells in MLT. The apoptotic cell ratio of MLT tended to be higher in lower grade lymphomas. These results suggest that EBV may participate in the malignant transformation from HT to MLT.

Epstein‐Barr virus in B‐cell non‐Hodgkin's lymphomas: Unexpected infection patterns and different infection incidence in low‐ and high‐grade types

Two hundred and eight cases of B-cell non-Hodgkin's lymphoma (B-NHL) occurring in Europeans without any signs of HIV infection were investigated for their association with an Epstein-Barr virus (EBV) infection. The polymerase chain reaction (PCR) was applied for EBV-DNA detection, in situ hybridization (ISH) for the cellular localization of EBV-encoded small nuclear RNAs (EBER) and immediate-early RNAs (BHLF), and immunohistology (IH) for the detection of EBV-encoded latent membrane protein (LMP) and EBV nuclear antigen 2 (EBNA2) expression. PCR and EBER-ISH produced congruent results in those cases with amplifiable DNA. EBV was present overall in 26 per cent (54/208) of the B-NHL cases. Through EBER-ISH, the virus could be localized merely in rare non-neoplastic bystander lymphocytes in 27 and additionally in tumour cells of 27 cases. Unexpectedly, the proportion of EBV-infected tumour cells present in the different cases varied between 1 and 100 per cent. All but three of the cases with infected tumour cells were of high-grade malignancy. Correlation with the morphological and immunological tumour phenotype revealed that all cases with more than 80 per cent EBER-positive tumour cells were either B-anaplastic large cell lymphomas (B-ALCL), sporadic Burkitt's lymphomas, or B-NHLs with partial or full plasmacellular differentiation. LMP was consistently absent from Burkitt's lymphomas and constantly expressed in B-ALCLs with EBER-positive tumour cells, while in all other instances it varied greatly and was rarer than EBER expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary Cutaneous Immunocytoma A B-Cell Lymphoma that can Easily Be Mistaken for Cutaneous Lymphoid Hyperlasia

It has been estimated that immunocytomas comprise roughly 2% of all cutaneous lymphomas. We studied five patients with primary cutaneous immunocytomas who presented with cutaneous nodules or plaques. Many of the infiltrates were "top-heavy" and polymorphous with admixed eosinophils, macrophages, lymphoid follicles, and non-neoplastic lymphocytes. Other potentially confusing findings were one case each of spongiotic dermatitis and leukocytoclastic vasculitis. The neoplastic cells were often situated at the peripheries of nodules and ranged from those with nuclei that resembled small lymphocytes to others that resembled immunoblasts. Most had eccentrically placed nuclei and fan-shaped cytoplasm. Monotypic kappa-light chain was found in all five cases, accompanied by gamma-heavy chain in two cases, and mu-heavy chain in one. In situ hybridization detected only kappa-mRNA in the four cases that yielded technically satisfactory results. The neoplastic cells did not express the B-cell antigen CD20; T-cells formed the centers of many nodules. Inappropriate staining for CD43 was evident in the neoplastic cells of one case. Because of reports of immunocytomas complicating acrodermatitis chronica atrophicans, we stained sections with an antiserum to Borrelia burgdorferi, which did not detect that organism. In situ hybridization did not detect EBER-1 RNA of the Epstein-Barr virus, which can be present in immunocytomas in immunocompromised patients. One patient died of disease after failing chemotherapy; another is alive with disseminated disease, and three are in remission following excision of lesions alone in two patients and chemotherapy in one patient who had relapsed following both excision and radiation therapy.

Lack of restricted T-cell receptor beta-chain variable region (V beta) usage of reactive T-lymphocytes in Hodgkin's disease.

T-cell response against tumour-associated antigens is mediated by the TCR complex. To determine a possibly restricted TCR-V beta repertoire in reactive T-lymphocytes in Hodgkin's disease (HD), 20 cases (of which 10 were EBV-positive cases) were investigated using 14 monoclonal antibodies (MoAbs) recognizing 11 different TCR-V beta region family products and Northern blot analysis with cDNA probes specific for mRNA transcripts of 11 V beta families that were not detectable by MoAbs. Four V beta families (V beta 5, V beta 6, V beta 8, V beta 19) were investigated using both immunohistochemistry (IHC) with anti-V beta MoAbs and Northern blot analysis. Immunohistochemical and Northern blot findings were correlated with the detection of the Epstein-Barr virus (EBV) genome in Hodgkin's and Reed-Sternberg cells (H-RS). The non-neoplastic lymphocytes in HD were predominantly of T-phenotype (CD3+). Most of these cells were TCR-alpha beta+ (beta F1+) and only a few T-cells were reactive for TCR-delta 1 antibody (TCR-gamma delta+). In the majority of cases helper/inducer T-cells (CD4+) outnumbered suppressor/cytotoxic T-cells (CD8+). Labelling of these samples with the panel of 14 anti-V beta MoAbs showed that only a small percentage (0.2-5.5%) of beta F1+ lymphocytes were positive with each of these MoAbs. The proportion of these cells was comparable to that seen in normal tissues. Most TCR V beta+ cells were randomly distributed, but in virtually all cases occasional V beta+ cells pertaining to the various V beta families were seen in close contact to H-RS cells. Using total RNA extracted from malignant and normal tissues, no visible band was detected with the various V beta probes. As determined in the present study, the percentage of T-cells expressing a given V beta family must be > or = 10% to be detected with Northern blot. Thus, the percentage of V beta+ cells expressing V beta families which were explored only with Northern blot were within the same range as those of the 11 different TCR-V beta region families assessed with IHC, i.e. 1-10% of lymphoid cells. The results of the present study show that in HD there is no restricted T-cell V beta repertoire usage regardless of the detection of EBV. In addition, since the various V beta families are represented in T-cell subpopulations forming rosettes around H-RS cells, we conclude that the T-cells attracted by H-RS cells constitute a polyclonal population.

Association of the subtype 2 of the Epstein-Barr virus with T-cell non- Hodgkin's lymphoma of the midline granuloma type [see comments

Lethal midline granuloma (LMG) is associated with Epstein-Barr virus (EBV). The latter has at least two subtypes with different biological properties. The subtypes can be identified by their genomic configuration. Using EBV-RNA (EBER) in situ hybridization and EBV polymerase chain reaction (PCR), we have looked for the presence of EBV in six LMGs and six non-Hodgkin's lymphomas (NHLs) located in the nasopharyngeal region, and determined the subtype of EBV. Six of six LMGs were positive by PCR and EBER in situ hybridization, whereas NHLs were either negative or, in three of six cases, showed few EBER- positive cells considered to be nonneoplastic lymphocytes. The subtype 2 was found in LMG lesions of three of six patients; the remaining three of six patients with LMG had the generally occurring subtype 1. The results indicate that the association of EBV with NHL may depend more on tumor type than on its localization. The occurrence of the rare subtype 2 in LMG may relate to a covert immune defect.

Association of the Subtype 2 of the Epstein-Barr Virus With T-Cell Non-Hodgkin's Lymphoma of the Midline Granuloma Type

Lethal midline granuloma (LMG) is associated with Epstein-Barr virus (EBV). The latter has at least two subtypes with different biological properties. The subtypes can be identified by their genomic configuration. Using EBV-RNA (EBER) in situ hybridization and EBV polymerase chain reaction (PCR), we have looked for the presence of EBV in six LMGs and six non-Hodgkin's lymphomas (NHLs) located in the nasopharyngeal region, and determined the subtype of EBV. Six of six LMGs were positive by PCR and EBER in situ hybridization, whereas NHLs were either negative or, in three of six cases, showed few EBER-positive cells considered to be nonneoplastic lymphocytes. The subtype 2 was found in LMG lesions of three of six patients; the remaining three of six patients with LMG had the generally occurring subtype 1. The results indicate that the association of EBV with NHL may depend more on tumor type than on its localization. The occurrence of the rare subtype 2 in LMG may relate to a covert immune defect.

Frequency of Epstein-Barr viral DNA in "Western" sinonasal and Waldeyer's ring non-Hodgkin's lymphomas.

Sinonasal non-Hodgkin's lymphomas (SNHL) of B- or T-cell immunophenotype have been associated with the Epstein-Barr virus (EBV) in Asian patients. We investigated eight sinonasal and 10 Waldeyer's ring NHL from Western patients for evidence of EBV genomes using a sensitive in situ hybridization technique. EBV DNA was detected in each of three sinonasal NHL with a T-cell immunophenotype and two of five cases with a B-cell immunophenotype. Two of 10 B-cell Waldeyer's ring NHL were positive for EBV genomes. In each positive case, EBV genomes were evenly distributed among the neoplastic cells, whereas no evidence of EBV in associated nonneoplastic lymphocytes or epithelium was seen. The results indicate that B-cell and T-cell sinonasal NHL are associated with EBV in Western as well as in Asian patients, and that EBV may have a role in oncogenesis in NHL of the upper aerodigestive tract. The strong association of EBV with nasopharyngeal carcinoma suggests that the anatomic site is important in the development of EBV-related neoplasms.

Regulation of bcl-2 proto-oncogene expression during normal human lymphocyte proliferation.

The bcl-2 and c-myc proto-oncogenes are brought into juxtaposition with the immunoglobulin heavy chain locus in particular B-cell lymphomas, resulting in high levels of constitutive accumulation of their messenger RNAs. Precisely how the products of the bcl-2 and c-myc genes contribute to tumorigenesis is unknown, but observations that c-myc expression is rapidly induced in nonneoplastic lymphocytes upon stimulation of proliferation raise the possibility that this proto-oncogene is involved in the control of normal cellular growth. In addition to c-myc, the bcl-2 proto-oncogene also was expressed in normal human B and T lymphocytes after stimulation with appropriate mitogens. Comparison of the regulation of the expression of these proto-oncogenes demonstrated marked differences and provided evidence that, in contrast to c-myc, levels of bcl-2 messenger RNA are regulated primarily through transcriptional mechanisms.

Phytohemagglutinin activation of the transcription of the bovine leukemia virus genome requires de novo protein synthesis.

Addition of supramitogenic doses of phytohemagglutinin (PHA) to short-term cultures of neoplastic or nonneoplastic lymphocytes infected with bovine leukemia virus increased the synthesis of the major core virion antigen (p25) by 5- to 10-fold. Such stimulation was not due to the mitogenic effect of PHA or to a generalized increase in cellular RNA or protein synthesis but rather to enhanced transcription of the viral genome by a PHA-induced protein.

Acid Phosphatase and Alpha-Naphthyl Acetate Esterase in Neoplastic and Non-Neoplastic Lymphocytes. A Statistical Analysis

Acid phosphatase and alpha-naphthyl acetate esterase reaction patterns were evaluated in lymphocytes from patients with a variety of neoplastic and non-neoplastic conditions: leukemia, 59; NHL, 53; and reactive follicular hyperplasia, 23. Fifteen individuals with normal peripheral blood were also studied. For both enzymes, statistical analysis showed a strong correlation between a globular reaction pattern and T lymphocytic origin in both non-neoplastic lymph nodes and normal peripheral blood specimens (P less than 0.0001). A similarly strong correlation was found between a granular acid phosphatase pattern and T lymphocytic origin in cell isolated from non-neoplastic lymph nodes (P less than 0.0001) but not in those obtained from normal peripheral blood where this pattern was observed with equal frequency in B, T, and null lymphocytes (P = 0.415). A granular alpha-naphthyl acetate esterase pattern was correlated with non-T lymphocytes from normal peripheral blood (P less than 0.0001), but was observed with equal frequency in B, T, and null lymphocytes fron non-neoplastic lymph nodes (P = 0.76). In the eight T cell neoplasias studied, a globular pattern was evident in the majority of cells for both enzymes. In the majority of the B cell neoplasias, however, a granular pattern was observed for both enzymes.

Acid phosphatase and alpha-naphthyl acetate esterase in neoplastic and non-neoplastic lymphocytes. A statistical analysis.

Acid phosphatase and alpha-naphthyl acetate esterase reaction patterns were evaluated in lymphocytes from patients with a variety of neoplastic and non-neoplastic conditions: leukemia, 59; NHL, 53; and reactive follicular hyperplasia, 23. Fifteen individuals with normal peripheral blood were also studied. For both enzymes, statistical analysis showed a strong correlation between a globular reaction pattern and T lymphocytic origin in both non-neoplastic lymph nodes and normal peripheral blood specimens (P less than 0.0001). A similarly strong correlation was found between a granular acid phosphatase pattern and T lymphocytic origin in cell isolated from non-neoplastic lymph nodes (P less than 0.0001) but not in those obtained from normal peripheral blood where this pattern was observed with equal frequency in B, T, and "null" lymphocytes (P = 0.415). A granular alpha-naphthyl acetate esterase pattern was correlated with non-T lymphocytes from normal peripheral blood (P less than 0.0001), but was observed with equal frequency in B, T, and "null" lymphocytes fron non-neoplastic lymph nodes (P = 0.76). In the eight T cell neoplasias studied, a globular pattern was evident in the majority of cells for both enzymes. In the majority of the B cell neoplasias, however, a granular pattern was observed for both enzymes.

Human Malignant Lymphomas in Vitro

A series of 55 biopsies from different types of malignant lymphomas were characterized in short-term culture experiments and during prolonged growth in vitro. The majority of the lymphocytic lymphomas and half of the histiocytic lymphomas expressed surface immunoglobulin, either in monoclonal or polyclonal form, indicating B-lymphocyte derivation. No lysozyme production was noted in either type of lymphoma, giving further support to the notion that histiocytic lymphomas are not truly histiocytic. Production of beta2-microglobulin was higher in histiocytic than in lymphocytic lymphoma and Hodgkin's disease but did not significantly differ from the production observed in non-neoplastic lymph node disorders. Incorporation of 3H-thymidine varied greatly within each category of lymphoma; the highest mean labelling index was noted in histiocytic lymphoma, possibly reflecting the generally more malignant course in such cases. Epstein-Barr virus-associated nuclear antigen was observed in one case of Hodgkin's disease. Attempts to establish permanent tumor cell lines were successful only from two explants of lymphocytic lymphoma and one pleural effusion from histiocytic lymphoma. The two cell lines derived from lymphocytic lymphomas both exhibited B-lymphocyte characteristics. The histiocytic lymphoma line lacked lymphocyte markers, produced lysozyme and was found to be rich in cytoplasmic esterases. These features are consistent with a "true" histiocytic derivation of this line. Lymphoblastoid cell lines representing non-neoplastic EBV-carrying lymphocytes contaminating the biopsies were derived from 19 biopsies, with the highest frequency noted in cultures of biopsies from Hodgkin's disease. The tumor lines were all EBV-genome negative.