Differential expression of thymus and activation regulated chemokine and its receptor CCR4 in nodal and cutaneous anaplastic large-cell lymphomas and Hodgkin's disease.

Recent studies demonstrated that Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkin's disease (HD) express thymus and activation-regulated chemokine (TARC), whereas reactive lymphocytes surrounding H/RS cells express its ligand, CC-chemokine receptor 4 (CCR4). Because in vitro studies showed that CCR4 expression is a marker for lymphocytes bearing a T-helper 2 (Th2) phenotype, it was suggested that expression of TARC is a new immune escape mechanism in HD. To find out whether this mechanism might also be operative in CD30+ malignant lymphomas other than HD, TARC and CCR4 expression was investigated by immunohistochemistry on paraffin and frozen-tissue sections of 39 nodal CD30+ anaplastic large cell lymphomas (ALCL), including 27 ALK-negative and 12 ALK-positive ALCL, 25 primary cutaneous CD30+ ALCL, including 11 patients with lymphomatoid papulosis, and 31 cases of HD. TARC was expressed by the neoplastic cells in 12/27 (44%) nodal ALK-negative ALCL and all cases of classic HD, but not in nodal ALK-positive ALCL (0/12) and only rarely in primary cutaneous CD30+ ALCL (3/25). In contrast, CCR4 was expressed by the neoplastic cells in 9/9 cutaneous CD30+ ALCL, and in 9/15 (60%) nodal ALK-negative ALCL, but only in 1/4 (25%) nodal ALK-positive ALCL and not by the H/RS cells in HD (0/8). Apart from three cases of HD showing 10 to 15% CCR4-positive lymphocytes surrounding TARC-positive H/RS cells, CCR4-positive reactive T cells were few (<5%) in all other cases studied. Our results demonstrate a differential expression of TARC and CCR4 in different types of CD30+ malignant lymphomas. The small number of CCR4-positive reactive T cells in most cases studied argues against an important role of TARC expression in the evasion of antitumor responses.

Differential expression of cell cycle and apoptosis related proteins in colorectal mucosa, primary colon tumours, and liver metastases.

AIMS: Tumour cell growth results from a disturbance in the balance between the rate of proliferation and cell death. In this study, proteins involved in the regulation of cell cycle arrest and apoptosis were studied as possible factors responsible for uncontrolled cell growth in colorectal cancer. METHODS: The expression of proteins involved in these processes was investigated in 48 metastases from patients with colorectal cancer and compared with eight normal colon mucosa samples and 14 primary tumours. Both primary tumours and metastases were obtained from eight patients. The expression of thymidylate synthase (TS), p53, retinoblastoma protein (Rb), Fas receptor, Fas ligand, bcl-2, mcl-1, bax, and bcl-x was measured using immunohistochemistry. Proliferation was determined by Ki67 staining, whereas apoptosis was assessed by M30 immunostaining, which recognises cleaved cytokeratin 18. RESULTS: In the limited number of cases in which paired comparisons were possible, the expression of TS and Ki67 was significantly higher in metastases than in the matched primary tumour samples (p = 0.014 and 0.016, respectively), whereas Rb expression was lower in metastases than in primary tumours (p = 0.024). Fas receptor expression was high in normal mucosa but absent in primary tumours and metastases, whereas the opposite was seen for p53. The expression of bax, mcl-1, and bcl-x in normal mucosa was more apical than that seen in malignant cells, where a more diffuse expression pattern was seen (p < 0.04). Apoptosis was more abundant in primary tumours than in metastases. CONCLUSIONS: These results demonstrate that proliferation and apoptosis are disturbed during colorectal cancer progression, and this is accompanied by loss of Rb and Fas expression, the accumulation of p53 and TS, and changes in the expression patterns of bax, mcl-1, and bcl-xl.

Rb, mcl-1 and p53 expression correlate with clinical outcome in patients with liver metastases from colorectal cancer.

BACKGROUND: Thymidylate synthase (TS) has been associated with clinical outcome in disseminated colorectal cancer. However, many patients with low TS expression still fail to respond to treatment. Therefore, we studied the cell cycle proteins, Rb, E2F2, Ki67, p21 and p53 and the apoptotic proteins, mcl-1, hax, bcl-xl, bcl-2, Fas receptor, Fas ligand, caspase-3, M30 and PARP as potential predictive factors. PATIENTS AND METHODS: In biopsy specimens of liver metastases from 31 colorectal cancer patients, protein expression was retrospectively determined by immunohistochemistry and related to response to hepatic arterial or intravenous (i.v.) 5-fluorouracil (5-FU) treatment, time to tumour progression (TTP) and overall survival. RESULTS: Expression of both p53 and Rb correlated with survival benefit after 5-FU treatment. A median survival time of 79 weeks was found in patients with high levels of p53 or Rb compared to 36 and 44 weeks for patients expressing low levels of p53 (P = 0.027) or Rb (P = 0.030), respectively. Multivariate analysis showed that p53 was the best predictor of survival independent of sex, age or prior treatment. Following 5-FU hepatic arterial infusion, patients with a high TS expression had a shorter survival time than those with a low expression (P = 0.025). The anti-apoptotic protein mcl-1 was the only factor, which correlated with response to 5-FU treatment. Thirty-five percent of patients with a diffuse mcl-1 expression responded whereas ninety percent of patients with a peri-nuclear expression responded (P = 0.041). CONCLUSIONS: These results indicate that besides TS, also Rb, p53 and mcl-1 are correlated with clinical outcome in patients with liver metastases from colorectal cancer.

5-Fluorouracil induced Fas upregulation associated with apoptosis in liver metastases of colorectal cancer patients.

BACKGROUND: In vitro, thymidylate synthase (TS) inhibition by 5-fluorouracil (5-FU) induces thymineless apoptosis possibly via Fas receptor Fas ligand interactions and cell-cycle arrest. In colorectal cancer patients we evaluated whether 5-FU administration also resulted in apoptosis and cell-cycle arrest and which proteins might be involved. PATIENTS AND METHODS: Biopsy specimens were taken from 36 patients 2, 22 or 46 hours after administration of 500 mg/m2 5-FU, and from 12 control patients who did not receive 5-FU. In frozen tissue-sections from liver metastases immunohistochemistry was performed with antibodies directed against p53, p21, E2F2, Rb, Ki67 and TS (cell-cycle related) and bax, BCL-2, BCL-x, mcl-1, PARP, caspase-3, Fas receptor and Fas ligand (apoptosis related). Apoptosis was determined by M30 immunostaining, which recognises a cleavage product of cytokeratin 18. RESULTS: Fas receptor expression was 50% higher (P = 0.036) 46 hours after 5-FU administration compared to the control group. This was associated with a 12% increase (P < 0.02) in M30 positive tumour cells and with elevation of caspase-3 and PARP expression. The expression of Ki67 and E2F2 was 30% lower after 46 hours compared to the control group, whereas TS was 56% lower after 2 hours and 32% higher again after 46 hours. No differences in the expression of the other proteins were found. CONCLUSIONS: These results suggest that 5-FU decreases proliferation status and induces apoptosis possibly via the Fas pathway. Since Fas mediated cell killing is important for cytotoxic T cells this indicates that clinical studies combining immunotherapy for activation of T cells and chemotherapy using 5-FU might be very effective.

Expression of killer cell inhibitory receptors is restricted to true NK cell lymphomas and a subset of intestinal enteropathy-type T cell lymphomas with a cytotoxic phenotype.

BACKGROUND/AIMS: Killer inhibitory receptors (KIR) have a modulating effect on the cytotoxic functions of natural killer (NK) cells and T cells. Because lymphoma cells often have the same receptors as their non-neoplastic counterparts, this study investigated the expression of KIR on well defined groups of NK and T cell lymphomas, with and without a cytotoxic phenotype, from different sites of origin. METHODS: Nine CD56+/CD3- NK cell lymphomas, 29 CD3+/CD56- T cell lymphomas with a cytotoxic phenotype, and 19 T cell lymphomas without a cytotoxic phenotype were stained for KIR using monoclonal antibodies specific for CD94, CD158a, and CD158b. In addition, the expression of KIR was studied on normal lymphoid tissues. RESULTS: KIR expression was seen in five of nine true NK cell lymphomas including three of four nasal, one of four cutaneous, and one of one intestinal lymphoma nasal type. Double staining for CD56 and CD94 in normal lymphoid tissues revealed that KIR was predominantly expressed by CD56+ NK cells and sporadically on CD8+ T cells. Moreover, enteropathy-type T cell lymphomas with a cytotoxic phenotype showed KIR expression (three cases expressing CD94 and one case expressing CD158a). All nodal and extranodal nonintestinal T cell lymphomas with or without a cytotoxic phenotype lacked expression of KIR. CONCLUSIONS: These results show that KIR expression is restricted to CD56+/CD3- true NK cell lymphomas originating from the nose, gut, and skin, as well as in a subset of extranodal T cell lymphomas originating from the small intestine, which possessed a cytotoxic phenotype. Thus, the presence of KIR on NK/T cell lymphomas seems to mimic the distribution of KIR found on NK and T cells in normal lymphoid tissue.

Percentage of activated cytotoxic T-lymphocytes in anaplastic large cell lymphoma and Hodgkin's disease: an independent biological prognostic marker.

Recently, we demonstrated that the presence of high percentages of activated cytotoxic T-lymphocytes (CTLs) in biopsy specimens of both Hodgkin's disease (HD) and ALK negative anaplastic large cell lymphoma (ALCL) is associated with a poor prognosis. To test whether this biological prognostic factor is more important in predicting clinical outcome than histological diagnosis or clinical factors, we compared the prognostic value of these parameters in an expanded group of classical HD and ALK negative ALCL. Tumor biopsies of classical HD (n = 83) and ALK negative systemic nodal ALCL (n = 43) were investigated for the presence of activated CTLs by immunohistochemistry, using a monoclonal antibody directed against granzyme B. Percentages of activated CTLs were quantified using Q-PRODIT, and their prognostic value was compared to that of histological diagnosis and clinical parameters, including age and stage. Both in classical HD and ALK negative ALCL, a high percentage of activated CTLs (ie > or = 15%) identified a group of patients with poor overall and progression-free survival time, even when adjusted for stage. In multivariate analysis, percentage of activated CTLs remained a strong independent prognostic marker, and was more sensitive than histological diagnosis or clinical factors in predicting overall survival time. We conclude that a high percentage of activated CTLs in the reactive infiltrate of ALK negative ALCL and classical HD is a strong indicator for an unfavorable clinical outcome, regardless of histological diagnosis or clinical parameters. As such, this biological parameter may be an especially helpful tool to determine therapeutic strategies in cases in which the differentiation between ALK negative ALCL and HD remains difficult.

Direct immunosuppressive effects of EBV-encoded latent membrane protein 1.

In neoplastic cells of EBV-positive lymphoid malignancies latent membrane protein (LMP1) is expressed. Because no adequate cellular immune response can be detected against LMP1, we investigated whether LMP1 had a direct effect on T lymphocyte activation. In this study we show that nanogram amounts of purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cells. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first transmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane protein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-gamma release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides showed strong inhibition of T cell proliferation and NK cytotoxicity, while acetylated LALLFWL peptides had an even stronger effect. In addition, Ag-specific IFN-gamma release was severely inhibited. To exert immunosuppressive effects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell lines (LCL), and differential centrifugation in combination with Western blot analysis of the pellets indicated that LMP1 is probably secreted by LCL in the form of exosomes. The amount of secreted LMP1 in B cell cultures is well below the immunosuppressive level observed with rLMP1. Our results demonstrate direct immunosuppressive properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restricted to RNA tumor viruses, but can also be found in DNA tumor viruses.

All infiltrating T-lymphocytes in Hodgkin's disease express immunohistochemically detectable T-cell receptor zeta-chains in situ.

AIM: We studied the expression of TCR zeta-chain on tumour-infiltrating lymphocytes in EBV-positive and EBV-negative cases of Hodgkin's disease (HD), to assess whether downregulation of TCR zeta-chain on tumour-infiltrating lymphocytes might be a mechanism for immune escape of the neoplastic cells. METHODS AND RESULTS: By immunohistochemistry we investigated tissue of 27 cases of primary HD, both paraffin embedded and frozen, for the presence of T-cell receptor complex zeta-chain and other T-cell markers on the reactive cells. Strong membranous staining of TCR zeta-chain was present in all cases in frozen tissue. In contrast, in paraffin-embedded material substantial loss of TCR zeta-chain was detected in old (> 6 years) tissues. However, no differences in either the number of positive cells or their staining intensity were observed in EBV-positive and negative cases of HD as detected in frozen tissue. Storage of paraffin-embedded tissue leads to a rapid and substantial loss of TCR zeta-chain reactivity compared to frozen material of the same HD cases. Staining reactivity of other T-cell markers (CD3, CD4 and CD8) on paraffin-embedded material remained unaffected. Immunofluorescent double-staining confirmed colocalization and coexpression of TCR zeta-chain and CD3. CONCLUSIONS: In frozen biopsies of primary HD TCR zeta-chain was expressed on all reactive CD3-positive cells, both in EBV-positive and EBV-negative cases. This suggests that zeta-chain downregulation is not a likely mechanism whereby neoplastic cells of HD can escape immune surveillance.

The Mr 193,000 vault protein is up-regulated in multidrug-resistant cancer cell lines.

Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR). To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the Mr 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines. We find that, like MVP, p193 mRNA and protein levels are increased in various multidrug-resistant cell lines. Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells. Furthermore, protein analysis of postnuclear supernatants and co-immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193. Our observations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins.

Quantitative immunohistochemical analysis of cytokine profiles in Epstein-Barr virus-positive and -negative cases of Hodgkin's disease.

Hodgkin's disease (HD) is a malignant lymphoproliferative disease characterized by the presence of Hodgkin-Reed-Sternberg cells surrounded by a reactive infiltrate. In Epstein-Barr virus (EBV)-associated cases (40-60%), at least two EBV-encoded proteins [latent membrane protein 1 (LMP1) and LMP2] are expressed, which are potential targets for cytotoxic T-lymphocytes (CTLs). Although in EBV-positive cases significantly more activated (granzyme B-positive) CTLs and natural killer (NK) cells are present, the cytotoxic immune response is not sufficient for adequate killing of tumour cells. The production of immunomodulating cytokines within the tumour may be one of the mechanisms causing circumvention of the immune system. This study investigated by immunohistochemistry the presence of the immunosuppressive cytokine interleukin-10 (IL-10) and other Th1/Th2-associated cytokines [IL-2, IL-4, interferon-gamma (IFN-gamma)] in the neoplastic cells and reactive lymphocytes of nine EBV-positive and 18 EBV-negative cases of HD. The percentage of IL-10-expressing cells, both neoplastic and reactive, in EBV-positive cases was significantly higher (33.1% vs. 18.5% for the neoplastic cells and 21.6% and 12.2% for the reactive cells, p=0.003 and 0.04, respectively) than in EBV-negative cases. No difference in the percentage of IL-2-, IL-4- and IFN-gamma-expressing cells was observed. These results suggest that escape from local immune surveillance is not due to a shift from Th1 towards Th2, but may be caused by a direct effect of IL-10 on the cytotoxic cells.

A cytotoxic phenotype does not predict clinical outcome in anaplastic large cell lymphomas.

AIM: To investigate whether anaplastic large cell lymphomas (ALCL) expressing cytotoxic proteins have a relatively worse clinical outcome compared with ALCL lacking a cytotoxic phenotype. METHODS: 59 primary cases of ALCL originating from different sites were investigated by immunohistochemistry for the presence of the cytotoxic proteins T cell intracytoplasmic antigen (TIA-1) and granzyme B in the neoplastic cells. Since site of origin and expression of anaplastic lymphoma kinase (ALK) strongly influence prognosis, the presence of a cytotoxic phenotype was also investigated in relation to the primary site of origin (lymph node, gut, or skin) and ALK expression. The prognostic value was investigated by analysis of overall and relapse-free survival time, including Cox regression analysis. RESULTS: 39 of 59 ALCL (66%) appeared to have a cytotoxic phenotype as shown by expression of TIA-1 or granzyme B or both in the neoplastic cells. The presence of a cytotoxic phenotype did not have any influence on prognosis. Even when the survival data were corrected for site of origin and stage at presentation or were analysed separately for ALK positive and negative cases, no prognostic influence of a cytotoxic phenotype was observed. CONCLUSIONS: In primary biopsies of patients with ALCL, the presence of a cytotoxic phenotype is not related to clinical outcome of the disease.

Adverse effects of activated cytotoxic T lymphocytes on the clinical outcome of nodal anaplastic large cell lymphoma.

Systemic (nodal) anaplastic large cell lymphoma (ALCL) is a subgroup of T-cell non-Hodgkin's lymphomas with a relatively favorable clinical outcome. Part of systemic ALCLs harbor a genetic aberration (usually the t(2;5)(p23;q35) translocation) containing the anaplastic lymphoma kinase (ALK) gene at 2p23, which results in aberrant expression of the ALK protein. Recently, we have shown that the presence of high percentages of activated cytotoxic T lymphocytes (CTLs) in tumor biopsy specimens of Hodgkin's disease (HD) is associated with a poor prognosis. In the present study, we investigated the prognostic value of percentages of activated CTLs in combination with ALK expression in primary nodal ALCL. Primary nodal biopsies of 42 patients with ALCL were investigated for the percentage of activated CTLs (quantified using Q-PRODIT) and the expression of ALK by immunohistochemistry using monoclonal antibodies (MoAbs) directed against T-cell antigen granzyme B (GrB) and ALK, respectively. These parameters were evaluated for their predictive value regarding progression-free and overall survival time. The presence of a high percentage of activated CTLs (ie, >/=15%) was found to be an unfavorable prognostic marker. In combination with a lack of ALK expression, it was possible to identify a group of patients with a very poor prognosis. In this group, 13 of 16 patients died within 2 years as a result of the disease. Of the remaining 26 patients, only three (all ALK negative) died (P <.0001). Furthermore, the percentage of activated CTLs combined with ALK status appeared to be of stronger prognostic value than the International Prognostic Index (IPI). We conclude that a high percentage of activated CTLs present in biopsy material of patients with primary nodal ALCL is a strong indicator for an unfavorable clinical outcome. The combination of ALK expression and percentage of activated CTLs appears to be more sensitive than the IPI in identifying a group of patients with a highly unfavorable clinical outcome who may be eligible for alternative (high dose) therapy schemes.

Presence of Epstein-Barr virus latency type III at the single cell level in post-transplantation lymphoproliferative disorders and AIDS related lymphomas.

AIMS: To investigate the expression pattern of Epstein-Barr virus (EBV) latent genes at the single cell level in post-transplantation lymphoproliferative disorders and acquired immunodefiency syndrome (AIDS) related lymphomas, in relation to cellular morphology. METHODS: Nine post-transplantation lymphoproliferative disorders and three AIDS related lymphomas were subjected to immunohistochemistry using monoclonal antibodies specific for EBV nuclear antigen 1 (EBNA1) (2H4), EBNA2 (PE2 and the new rat anti-EBNA2 monoclonal antibodies 1E6, R3, and 3E9), and LMP1 (CS1-4 and S12). Double staining was performed combining R3 or 3E9 with S12. RESULTS: R3 and 3E9 anti-EBNA2 monoclonal antibodies were more sensitive than PE2, enabling the detection of more EBNA2 positive lymphoma cells. Both in post-transplantation lymphoproliferative disorders and AIDS related lymphomas, different expression patterns were detected at the single cell level. Smaller neoplastic cells were positive for EBNA2 but negative for LMP1. Larger and more blastic neoplastic cells, sometimes resembling Reed-Sternberg cells, were LMP1 positive but EBNA2 negative (EBV latency type II). Morphologically intermediate neoplastic cells coexpressing EBNA2 and LMP1 (EBV latency type III), were detected using R3 and 3E9, and formed a considerable part of the neoplastic population in four of nine post-transplantation lymphoproliferative disorders and two of three AIDS related lymphomas. All samples contained a subpopulation of small tumour cells positive exclusively for Epstein-Barr early RNA and EBNA1. The relation between cellular morphology and EBV expression patterns in this study was less pronounced in AIDS related lymphomas than in post-transplantation lymphoproliferative disorders, because the AIDS related lymphomas were less polymorphic than the post-transplantation lymphoproliferative disorders. CONCLUSIONS: In post-transplantation lymphoproliferative disorders and AIDS related lymphomas, EBV latency type III can be detected by immunohistochemistry in a subpopulation of tumour cells using sensitive monoclonal antibodies R3 and 3E9. Our data suggest that EBV infected tumour cells in these lymphomas undergo gradual changes in the expression of EBV latent genes, and that these changes are associated with changes in cellular morphology.

Expression of epstein-barr virus encoded nuclear antigen 1 in benign and malignant tissues harbouring EBV.

AIMS: To determine levels of expression of Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) in benign and malignant tissues harbouring EBV in relation to EBNA1 promoter usage. METHODS: Expression of EBNA1 was investigated by means of immunohistochemistry using a mixture of two EBNA1 specific monoclonal antibodies, 1H4-1 and 2B4-1. The presence of EBV was detected by EBER1/2 RNA in situ hybridisation. Detection of promoter specific EBNA1 transcripts was by RT-PCR analysis. RESULTS: EBNA1 positive cells were detected in all 20 EBV associated B cell lymphomas, 18 of which had arisen in immunocompromised patients; in eight of nine EBV associated T cell lymphomas; in 11 of 27 EBV positive cases of Hodgkin's disease; and in reactive lymphoid tissue harbouring EBV, including four cases of infectious mononucleosis. A diffuse EBNA1 staining pattern was observed in most of the EBV associated B cell lymphomas and was comparable with the EBER1/2 staining pattern. In the T cell lymphomas the number of EBNA1 positive cells was usually considerably less than the number of EBER1/2 positive ones. RT-PCR analysis revealed that in tumours with restricted EBNA1 expression-that is, T cell lymphomas and Hodgkin's disease lesions, EBNA1 transcripts were usually generated only by the F/Q promoter, whereas in B cell lymphomas EBNA1 transcripts were usually generated by both the C/W and F/Q promoters. CONCLUSIONS: EBNA1 is expressed in all types of tissue harbouring EBV, but the level of expression varies greatly. This may be the result of differential promoter usage.

Epstein-Barr virus and human T-cell lymphomas.

Epstein-Barr virus has been classically associated with certain B-lymphocytic benign and malignant proliferations. However, using molecular biological techniques it becomes clear that EBV is also associated with several T-NHL in non-immunocompromised patients. The distribution of EBV-associated T-NHL seems to be site-restricted, i.e. in about 100% of the nasal T-NHL and in 20% of the lung and gastrointestinal lymphomas and rarely in primary cutaneous T-cell lymphomas. Moreover, the expression of the LMP1 protein seems to be associated with a poor prognosis. In this section the role of EBV in the pathogenesis of T-NHL will be discussed.

Immunohistochemical demonstration of different latent membrane protein-1 epitopes of Epstein-Barr virus in lymphoproliferative diseases.

AIM: To compare the immunoreactivity of monoclonal antibodies S12 and CS1-4, which recognise different epitopes of the Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1), in EBV associated benign and malignant lymphoproliferative disorders and control tissues processed using different methods. RESULTS: Both monoclonal antibodies gave comparable results on frozen tissue sections and formalin fixed, paraffin wax embedded samples from cases with Hodgkin's disease and infectious mononucleosis. In all cases S12 stained more cells than CS1-4. For EBV associated B and T non-Hodgkin's lymphomas, frozen tissue sections yielded better LMP-1 staining results than formalin fixed material. Again, in all these cases S12 stained more cells and gave stronger results than CS1-4. For EBV negative tissues, both monoclonal antibodies showed cross-reactivity with melanocytic-like cells in the basal cell layer of the skin, synaptophysin-like staining in layers three and four of the cortex of the brain, and myelin-like staining in peripheral nerves and peripheral ganglion cells. Staining with S12 was always much stronger. Moreover, in contrast to CS1-4, S12 stained pancreatic islands in formalin fixed material but not in frozen tissue sections and sporadically stained solitary epithelial cells in the large bowel especially in formalin fixed tissue sections. CS1-4 also cross-reacted with myoepithelial cells around hair follicles and other adnexa of the skin. CONCLUSION: The results indicate that for optimal detection of LMP-1, S12 yields better results than CS1-4 and that tissue processing is very important especially when B and T non-Hodgkin's lymphomas are examined.