KSHV-initiated notch activation leads to membrane-type-1 matrix metalloproteinase-dependent lymphatic endothelial-to-mesenchymal transition.

Kaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread.

Tanzanian malignant lymphomas: WHO classification, presentation, ploidy, proliferation and HIV/EBV association.

BACKGROUND: In Tanzania, the International Working Formulation [WF] rather than the WHO Classification is still being used in diagnosing malignant lymphomas (ML) and the biological characterization including the HIV/EBV association is sketchy, thus restraining comparison, prognostication and application of established therapeutic protocols. METHODS: Archival, diagnostic ML biopsies (N = 336), available sera (N = 35) screened by ELISA for HIV antibodies and corresponding clinical/histological reports at Muhimbili National Hospital (MNH) in Tanzania between 1996 and 2006 were retrieved and evaluated. A fraction (N = 174) were analyzed by histopathology and immunohistochemistry (IHC). Selected biopsies were characterized by flow-cytometry (FC) for DNA ploidy (N = 60) and some by in-situ hybridization (ISH) for EBV-encoded RNA (EBER, N = 37). RESULTS: A third (38.8%, 109/281) of the ML patients with available clinical information had extranodal disease presentation. A total of 158 out of 174 biopsies selected for immunophenotyping were confirmed to be ML which were mostly (84. 8%, 134/158) non-Hodgkin lymphoma (NHL). Most (83.6%, 112/134) of NHL were B-cell lymphomas (BCL) (CD20+), of which 50.9%, (57/112) were diffuse large B-cell (DLBCL). Out of the 158 confirmed MLs, 22 (13.9%) were T-cell [CD3+] lymphomas (TCL) and 24 (15.2%) were Hodgkin lymphomas (HL) [CD30+]. Furthermore, out of the 60 FC analyzed ML cases, 27 (M:F ratio 2:1) were DLBCL, a slight majority (55.6%, 15/27) with activated B-cell like (ABC) and 45% (12/27) with germinal center B-cell like (GCB) immunophenotype. Overall, 40% (24/60) ML were aneuploid mostly (63.0%, 17/27) the DLBCL and TCL (54.5%, 6/11). DNA index (DI) of FC-analyzed ML ranged from 1.103-2.407 (median = 1.51) and most (75.0%) aneuploid cases showed high (>40%) cell proliferation by Ki-67 reactivity. The majority (51.4%, 19/37) of EBER ISH analyzed lymphoma biopsies were positive. Of the serologically tested MLs, 40.0% (14/35) were HIV positive, mostly with high (> or =40.0%) Ki-67 reactivity. CONCLUSIONS: According to the 2001 WHO Classification, most subtypes are represented in Tanzanian ML. Extranodal presentation was common among MNH lymphoma patients who also showed high aneuploidy, tumor proliferation (KI-67) and EBER positivity. DLBCL was frequent and phenotype heterogeneity appeared similar to observations in Western countries suggesting applicability of established intervention approaches. HIV was apparently associated with high ML cell proliferation but extended studies are needed to clarify this.

Nucleophosmin phosphorylation by v-cyclin-CDK6 controls KSHV latency.

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

Autoimmunogenicity of the helix-loop-helix DNA-binding domain.

Nonimmunogenic character of native DNA, and its high immunogenicity when presented in complex with the DNA-binding proteins indicate that the latter might contain molecular triggers of anti-DNA response. To find if this is the case, we have evaluated the autoimmunogenic potential of the main DNA-binding domain of HIV-1 reverse transcriptase that belongs to the canonical helix-loop-helix type. BALB/c mice were immunized with a peptide representing the domain, alone or in complex with the fragmented human DNA in the presence of an adjuvant. Mice were assessed for specific antibodies, autoantibodies against a panel of self-antigens; glomerular immunoglobulin deposition; and for the signs of autoimmune disease, such as proteinuria, and changes in the blood components. Immunization with the adjuvanted peptide-DNA complex induced autoantibodies against double-stranded DNA, histones, heterochromatin, and kidney proteins; glomerular IgG and IgA deposition; proteinuria; thrombocytopenia, and anemia. Altogether, this identifies the helix-loop-helix DNA-binding domain as one of the molecular triggers of autoimmunity to DNA and DNA-associated proteins. The experiments cast new light on the role of the DNA-binding retroviral proteins in the induction of autoimmunity, and on the origins of autoimmune complications in the microbial infections in general. It also implies that choosing the DNA-binding proteins as vaccine candidates should be done with precaution.

Human herpesvirus-8 (HHV-8) sero-detection and HIV association in Kaposi's sarcoma (KS), non-KS tumors and non-neoplastic conditions.

BACKGROUND: The association of the human herpesvirus-8/Kaposi's sarcoma (KS)-associated herpesvirus (HHV-8/KSHV) serology with various malignancies in Tanzania is not currently well established while previous studies were based on either PCR or immunofluorescence assays [IFA] but not with a sensitive enzyme-linked immunosorbent assay (ELISA). Selected archival diagnostic biopsies (n = 184) and sera from indigenous patients with KS (n = 120), non-KS tumors (n = 24) and non-neoplastic lesions (n = 40) at Muhimbili National Hospital (MNH), Tanzania, were evaluated by diagnostic histopathology, immunohistology [anti-HHV-8 latency-associated nuclear antigen (LANA)] and serology for HIV (ELISA) and HHV-8 (IFA and ELISA). RESULTS: About 66.3% (n = 122) cases including AIDS-associated Kaposi's sarcoma (AKS) (n = 93), reactive conditions (n = 28) and only one non-KS tumour were HIV positive. Endemic KS (EKS) patients were mostly males (96.3%, 26/27) who were less (69.9%, 65/93) predominant in AIDS-associated (AKS). A high (89%) percentage of patients with anti-HHV-8 antibodies was found in the cohort including the HIV positive (92%) cases, males (81.2%), KS patients (93%), non-KS tumors (92%), and reactive conditions (75%). All HHV-8 seronegative KS cases were nodular stage whereas both sera and corresponding biopsies from early stage KS were HHV-8+. Assay sensitivity, positive predictive value (PPV) and specificity were 98.6%, 93.5% and 16.7% for IFA and 93.5%, 98.6% and 50.0% for ELISA respectively. CONCLUSION: HHV-8 seroprevalence at MNH appears high as expected among AKS cases and males but also in non-KS patients. ELISA showed a combination of high HHV-8 sensitivity as well as higher PPV and specificity than IFA which however, showed higher sensitivity. The apparent stage-dependent, inverted serum HHV-8 immunoreactivity supports a notion of viral immune-segregation during KS development. Routine HHV-8 screening should be considered particularly in patients at risk of KS and for selection of blood/organ donations.

Viral oncogene-induced DNA damage response is activated in Kaposi sarcoma tumorigenesis.

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Kaposi's sarcoma herpesvirus load in biopsies of cutaneous and oral Kaposi's sarcoma lesions.

OBJECTIVES: To evaluate human herpesvirus 8/Kaposi's sarcoma associated herpesvirus (HHV-8/KSHV) viral load in diagnostic, (formalin fixed, paraffinised) biopsies and patient serum during tumour progression of oral and cutaneous AIDS-related Kaposi's sarcoma (AKS), and endemic Kaposi's sarcoma (EKS) by a sensitive and specific quantitative real time polymerase chain reaction (qRT-PCR) assay. STUDY DESIGN: Eighty six biopsies of both AKS (oral and cutaneous AKS, 68) and EKS (cutaneous EKS, 18) were evaluated by qRT-PCR and immunohistochemistry (IHC). The viral load in human tumour tissue and serum of some individual patients were compared. RESULTS: Higher viral load as well as frequency of latency-associated nuclear antigen (LANA)+ tumour spindle cells (SC) and number of LANA granules per SC was found in oral AKS compared to cutaneous AKS. Although few cases were available, serum viral load appeared to decrease compared to tumour tissue during KS progression. CONCLUSIONS: The higher viral load in oral rather than cutaneous AKS is consistent with the well recognised reservoir function of the oral mucosa. Decrease of serum HHV-8 load during KS progression may indicate decreased virus release and/or increased virus clearance.

Oral Kaposi's sarcoma in Tanzania: presentation, immunopathology and human herpesvirus-8 association.

Oral Kaposi's sarcoma (OKS) from Tanzanian patients (78) at Muhimbili National Hospital/Muhimbili University College of Health Sciences corresponding to approximately 10% of KS registered during 1990-2005, were diagnosed (ELISA) as HIV-infected (OAKS) (74/78) and endemic KS (4/78). Females were 69.2% (54/78) with median age 31 and males 30.8% (24/78) with median age 38. More males (50%) had systemic KS than females (37%) and 4-times more multicentric OKS. All tested (34) oral KS patients sera had HHV-8 antibodies. Available (31/78) blood showed very low CD4+ T-lymphocyte counts. Most OKS (61.5%) had nodular histology. Immunostaining showed adult male nodular OAKS to have a significantly higher frequency of viral LANA+, endothelial CD34+ tumour spindle-cells (SC) and more Ki-67+ (median =24.1%) proliferating cells compared to females (17.2%). Juvenile nodular OAKS had more LANA+ and Ki-67+ cells than corresponding adult cases. Significantly more LANA+ and Ki-67+ cells were found in nodular OAKS compared to cutaneous HIV/AIDS Kaposi's sarcoma (CAKS). A positive correlation (60%) was found between the proliferation index (Ki-67+ cell frequency) and LANA+/CD34+ SC. OKS in Tanzania is since 1990, mostly seen in females, associated with HIV/AIDS and advanced (nodular) histopathology. Males have more systemic tumour burden while more females develop primary OAKS. HHV-8+ cells were more frequent in nodular male than female and in juvenile than adult nodular OAKS than cAKS. Higher tumoral HHV-8 content appeared to be correlated to proliferation index.

KSHV/HHV-8 and HIV infection in Kaposi's sarcoma development.

Kaposi's sarcoma (KS) is a highly and abnormally vascularized tumor-like lesion affecting the skin, lymphnodes and viscera, which develops from early inflammatory stages of patch/plaque to late, nodular tumors composed predominant of spindle cells (SC). These SC are infected with the Kaposi's sarcoma-associated herpesvirus or human herpesvirus-8 (KSHV/HHV-8). KS is promoted during HIV infection by various angiogenic and pro-inflammatory factors including HIV-Tat. The latency associated nuclear antigen type 1 (LANA-1) protein is well expressed in SC, highly immunogenic and considered important in the generation and maintenance of HHV-8 associated malignancies. Various studies favour an endothelial origin of the KS SC, expressing "mixed" lymphatic and vascular endothelial cell markers, possibly representing hybrid phenotypes of endothelial cells (EC). A significant number of SC during KS development are apparently not HHV8 infected, which heterogeneity in viral permissiveness may indicate that non-infected SC may continuously be recruited in to the lesion from progenitor cells and locally triggered to develop permissiveness to HHV8 infection. In the present study various aspects of KS pathogenesis are discussed, focusing on the histopathological as well as cytogenetic and molecular genetic changes in KS.

Hepatitis C virus core protein transforms murine fibroblasts by promoting genomic instability.

The oncogenic potential of hepatitis C virus (HCV) core protein has been demonstrated, but the precise mechanism of cell transformation triggered by HCV core is still unclear. This study shows that constitutive expression of HCV core protein (core) in NIH 3T3 murine fibroblasts triggers malignant transformation. At the preneoplastic stage, clones that expressed HCV core constitutively demonstrated genomic instability seen as disruption of the mitotic spindle cell checkpoint leading to increased ploidy. Transformation was completed by the loss of DNA and resistance to apoptosis induced by serum starvation. Simultaneously, cells acquired a capacity for anchorage independent growth and absence of contact inhibition. Inoculation of these transformed cells into severe combined immune deficiency (SCID) mice led to formation of solid core-expressing tumors. Transformation and tumorigenicity of core-expressing cell lines coincided with a 5- to 10-fold repression of endogenous p53 transactivation. Thus, long-term HCV core expression alone is sufficient for complete transformation of immortal fibroblasts that can then induce tumors in a susceptible host. This data suggests that malignant transformation by HCV core may occur through primary stress, induction of genomic instability, and further HCV core-induced rescue of surviving mutated cells.

CGH of microdissected Kaposi's sarcoma lesions reveals recurrent loss of chromosome Y in early and additional chromosomal changes in late tumour stages.

BACKGROUND: It is still unclear if Kaposi's sarcoma (KS) is a monoclonal cell proliferation or a polyclonal, hyperplastic, reactive process. Reports on KS cytogenetics are few and restricted to late stage disease and cell lines. METHOD: We analysed 27 KS, early and late, AIDS related (AKS) and endemic (EKS) by laser microdissection, global DNA amplification and comparative genomic hybridization (CGH). RESULT: Loss of Y chromosome was detected in 20/23 male KS, which was the only recurrent chromosomal aberration in all nine male early (patch) KS. Only one patch EKS showed in addition to the Y loss a loss of Xq. Late (nodular) AKS and EKS showed recurrent copy number changes in chromosomes 16, 17, 21, X and Y, as well as other random changes. The loss of chromosome 16, 17 and Y was confirmed by interphase fluorescence in situ hybridization (FISH) on paraffin sections. EKS showed a higher number of chromosomal abnormalities than AKS, indicating that rapid growth of AKS is less dependent on genetic changes than is EKS, possibly because of the immunosuppressed host environment in AKS. CONCLUSION: Clonal loss of chromosome Y was detected in all early male KS, while additional chromosomal aberrations appeared during development to late KS. This increase in chromosomal abnormalities during tumour growth indicates genetic instability and the selection of survival cell clones establishing late, aggressive sarcoma growth. Our data support the view that KS (in males) develops into a clonal tumour yet initially is a hyperplastic reactive cell proliferation.

AIDS and non AIDS-related malignant lymphoma in Tanzania.

BACKGROUND: Malignant lymphoma (ML) in HIV patients, are second in frequency to Kaposi's sarcoma (AKS) as AIDS-defining tumors. In Africa the frequency of AIDS-related lymphoma (ARL) is rare and the findings are controversial. Kaposi's sarcoma (KS) lesions are now causally associated with KSHV/HHV-8 but whether African ARL shows this association is not clear. METHOD: Cancer registry data was reviewed for retrospective cases. Both retrospective and prospective lymphoma cases were classified according to the revised European-American (REAL) classification. Immunephenotyping was performed on both frozen and fixed paraffin sections. Viral DNA was assessed by polymerase chain reaction (PCR) of formalin fixed or frozen biopsies. In situ hybridization (ISH) was used to determine the presence of EBV encoded RNA (EBER). OBJECTIVES: To determine the frequency and type of AIDS and non-AIDS related malignant lymphoma in Tanzania and a possible co-association with KSHV/HHV-8 and EBV. RESULTS: An overall increasing tendency for ML in Tanzania was observed during 1991-94 and a clear increase from 1993. The tumors were classified as Burkitt's (6), diffuse large cell (10), precursor-B lymphoblastic (1) and Hodgkin's disease (5) from HIV positive and negative patients. Ten (40%) high grade ML and three Hodgkin's lymphoma from HIV patients had HHV-8 DNA. These findings were not related to age, sex or type of lymphoma. There was no association of HHV-8 with the lymphoma cells. Epstein-Barr virus (EBV) was demonstrable in most (13/18; 72%) of the tested tumors and seven (31.8%) had both HHV-8 and EBV. CONCLUSIONS: This study suggests an overall increased frequency of ML patients infected with HHV-8 in Tanzania particularly in HIV patients which may result from the well established high HHV-8 prevalence in the general population, but HHV-8 was not associated with ARL pathogenesis as reflected by lack of tumor cell infection. As opposed to EBV, measures targeting HHV-8 for control of ML may therefore not be appropriate.

Hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha are expressed in kaposi sarcoma and modulated by insulin-like growth factor-I.

PURPOSE: Neoangiogenesis is essential for tumor development. Hypoxia-inducible factor (HIF), a transcriptional factor composed of two subunits (alpha and beta), plays a key role in this process, activating proangiogenic factors such as vascular endothelial growth factor (VEGF). The HIF alpha subunits are critically regulated by oxygen and are also modulated by growth factors. Kaposi sarcoma (KS) is a highly vascular tumor that releases large amounts of VEGF and for which we have recently described an essential role for the insulin-like growth factor (IGF) system. We therefore investigated the expression of HIF alpha subunits in biopsies from KS tumors and their modulation by IGF-I in KSIMM, a KS cell line. RESULTS: Both HIF-1alpha and HIF-2alpha were expressed in KS biopsies in all tumoral stages. HIF-1alpha immunopositivity increased through the tumor development with highest expression in the late nodular stages. In KSIMM cells, IGF-I induced accumulation of both HIF alpha subunits. The induction suggests a translation mechanism as documented by cycloheximide chase experiment coupled with constant RNA levels as evaluated by quantitative real-time PCR. IGF-I-induced HIF alpha accumulation was followed by an increase in HIF function as assessed both by reporter gene assay and by induction of endogenous target gene expression (VEGF-A). Specific blockade of IGF-I receptor with alphaIR3 antibody or with picropodophyllin, a specific IGF-IR tyrosine kinase inhibitor, diminishes the basal and IGF-I-dependent induction of both HIF alpha congeners. CONCLUSION: These novel findings show the coupling between the IGF and HIF signaling in KS and suggest a coordinated contribution by these pathways to the characteristic vascular phenotype of this tumor.

Lymphatic and vascular origin of Kaposi's sarcoma spindle cells during tumor development.

The histogenesis of Kaposi's sarcoma (KS) tumor spindle cells (SC) remains controversial but several immunohistochemical studies favor a lymphatic origin. Twenty KS surgical biopsies were analyzed for the coexpression of LANA, CD34, LYVE-1, D2-40, VEGFR-2, VEGFR3 by using double or triple immunostaining. Most of the SC in both early and late KS expressed the lymphatic markers LYVE-1, D2-40 and VEGFR-3 and the blood vascular endothelial/endothelial precursor cell markers CD34 and endothelial stem cell marker VEGFR-2. All the LANA+ SC in early and late KS were LYVE-1+, but only 75% of these LANA+ cells were CD34(+). The CD34(+)/LANA+ cells increased from early- (68.8%) to late-stage KS (82.2%). However, approximately 18% of the LANA+ SC in early KS were CD34(-) but were LYVE-1+, suggesting that resident lymphatic endothelial cells (LEC) are targeted for primary infection by human herpesvirus-8. This LANA+/LYVE-1+/CD34(-) (resident LEC) cell population clearly decreased during the development of KS from early (18.7%) to late KS (2.9%). Thus, in late stages of KS, most SC were LANA+/CD34(+)/LYVE-1+. However, in both early- and late-stage KS, approximately 18% of the SC were CD34(+)/LANA-/LYVE-1 -- and could represent newly recruited endothelial precursor cells, which become infected in the lesion and eventually undergo a phenotype switch expressing LEC markers. Our study apparently indicates that KS represents a unique variant of tumor growth with continues recruitment of tumor precursor cells as well as proliferation and decreased apoptosis of SC.

Coreceptor usage of primary HIV type 1 isolates obtained from different lymph node subsets.

Biological characteristics of virus quantitatively rescued from different cell types present in lymph nodes of HIV-1-infected individuals in various stages of their disease were determined, not including patients with AIDS defining illness. Viruses were obtained by cocultivation with donor monocyte-derived macrophages and T-lymphocytes and their biological phenotype compared to viruses obtained from the peripheral blood mononuclear cells of the same patient. The biological phenotype was determined on established cell lines (U937-2, CEM, and MT-2) and on the U87.CD4 coreceptor indicator cell lines and variable region 3 (V3) of the envelope was subjected to direct sequencing. All isolates obtained from lymph node subsets used CCR5 as coreceptor. Furthermore, these viruses were also sensitive to inhibition by beta-chemokines as analyzed for viruses of one patient. All 12 V3 regions showed a unique sequence indicating compartmentalization within each patient. The biological phenotype of CCR5-dependent (R5) HIV-1 isolates obtained from PBMC resembles the phenotype of viruses isolated from different lymph node cell subsets.

HHV-8/KSHV during the development of Kaposi's sarcoma: evaluation by polymerase chain reaction and immunohistochemistry.

The human gamma-herpes virus-8 (HHV-8) was first described in AIDS-related Kaposi's sarcoma (KS) tumour samples. In this study, we report comparative studies on paraffin-embedded biopsies of AIDS-related KS (AKS) and endemic KS (EKS) with regard to HHV-8 content as evaluated using polymerase chain reaction (PCR) and immunohistochemistry. DNA was extracted either using Chelex-100 or using Qia-gene kit and was evaluated with the help of a semiquantitative PCR assay. The PCR detection of HHV-8 was more sensitive to the Chelex method than to Qia-gene. The threshold for PCR test sensitivity with the help of serial dilution of DNA was at the level of five plasmid ORF-26 regions, and DNA from 25 body cavity-based lymphoma-1 cells. The results expressed as virus load/actin unit showed progressively higher HHV-8 levels in late (nodular) cases, compared to those in early (patch/plaque) stages. Evaluation of HHV-8 DNA levels in tumour tissues, thus, indicates a correlation between virus load and KS stage. Double immunostaining of spindle cells (SC) in KS biopsies for CD34 and HHV-8/latency-associated nuclear antigen (LANA) showed an increase in double-positive SC in the lesions of nodular AKS and EKS cases, compared to that in plaque and patch stages. However, 10-15% of CD34+/LANA- SC cells were observed during the development from patch to nodular cases of AKS and EKS. Our results indicate that PCR analysis is a simple and sensitive diagnostic method for HHV-8 evaluation in KS tissues, processed for conventional histopathology.

Epstein-Barr virus LMP1 status in relation to apoptosis, p53 expression and leucocyte infiltration in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection. EBV-encoded LMP1, expressed in most of NPC, has been suggested to have an important role in the pathogenesis and development of NPC and its expression correlates with poor prognosis. MATERIALS AND METHODS: Eighty-seven NPC biopsies were analyzed by immunohistochemistry for expression of markers of cell proliferation, apoptosis, infiltrating T lymphocytes and macrophages in relation to the LMP1 status. RESULTS: Our findings indicate that the p53 accumulation in NPC was significantly correlated to LMP1 and MMP9 overexpression in NPC cells. The frequency of apoptotic cells in NPC, as analyzed by TUNEL labeling, correlated to Fas-L and caspase-3 expression, and inversely to LMP1, p53 and MMP 9 expression. CD8+ T cell infiltration was predominately seen in nests of cancer cells with a high level of EBV-LMP1 expression, but these CD8+ T cells showed low expression of CD25 and TIA-1, indicating that they were not activated. CONCLUSION: Our observation suggests that the heavy infiltration by lymphocytes in LMP1-positive NPC tumors does not appear to counteract tumor growth by cytoxicity as indicated by the low apoptotic index. Thus, LMP1 seems to enhance survival- and proliferation-related signals in NPC. In analogy with other tumors, both the infiltrating T cells and the accumulated p53 may be inactive.