Establishment of cancer cell line originating from a patient with high-grade serous ovarian carcinoma.

Aim: Ovarian cancer is a serious malignancy with high prevalence and mortality. Methods: We isolated and characterized an ovarian high-grade serous cancer cell line (M4) from a tumor of a Vietnamese patient with ovarian carcinoma. Results: The M4 cancer cell line showed good proliferation and stability in culture. Morphologically, the M4 cells showed similar characteristics to tumor cells such as a polyhedral shape, large irregular nuclei, high nuclear/cytoplasmic ratio, high nuclear density and expressing cancer markers like CA125, p53 and Ki67 markers. Conclusion: We have successfully isolated and characterized the M4 cell line from a Vietnamese patient with ovarian carcinoma.

Expansion of EBNA1-specific effector T cells in posttransplantation lymphoproliferative disorders.

Immunosuppression resulting in impaired Epstein-Barr virus (EBV)-specific T-cell immunity is involved in the pathogenesis of EBV-positive post-transplantation lymphoproliferative disorder (EBV(+) PTLD). Restoration of EBV-specific T-cell immunity by adoptive immunotherapy can induce remission. EBV-nuclear antigen-1 (EBNA1) is unique in being expressed in all cases of EBV(+) PTLD. Recent data demonstrate that EBNA1 is not immunologically silent and can be exploited as a T-cell target. There are no data on EBNA1-specific T cells in PTLD. EBNA1-specific T cells capable of proliferation, interferon-γ release, and CD107a/b degranulation were assayed in 14 EBV(+) PTLD diagnostic blood samples and 19 healthy controls. EBNA1-specific CD4(+) T cells predominated and were expanded in 10 of 14 patients and 19 of 19 controls. Although human leukocyte antigen class I alleles influenced the magnitude of the response, EBNA1-specific CD8(+) effector T cells were successfully generated in 9 of 14 EBV(+) PTLD patients and 16 of 19 controls. The majority of PTLD patients had a polymorphism in an EBNA1 epitope, and T-cell recognition was greatly enhanced when EBNA1 peptides derived from the polymorphic epitope were used. These results indicate that EBNA1-specific T cells should be included in adoptive immunotherapy for PTLD. Furthermore, expansion protocols should use antigenic sequences from relevant EBV strains.

Expression profiling of Epstein-Barr virus-encoded microRNAs from paraffin-embedded formalin-fixed primary Epstein-Barr virus-positive B-cell lymphoma samples.

Epstein-Barr virus (EBV) is implicated in a range of B-cell malignancies and expresses unique microRNAs (EBV-miRNAs). Due to the requirements for high-quality RNA, studies profiling EBV-miRNA in EBV-positive lymphomas have been restricted to cell-lines or frozen samples. However, the most commonly available archived patient material is paraffin-embedded formalin-fixed (FFPE) tissue. This has impeded the widespread profiling of EBV-miRNA expression in clinical samples. The requirements for accurate EBV-miRNA real-time RT-PCR quantitation in FFPE tissues representing a broad-spectrum of EBV-positive lymphomas were determined systematically, including where the neoplastic cells are sparse relative to the non-malignant infiltrate. The level of cellular EBV-load correlated strongly with the sum of EBV-miRNA expression and the number of EBV-miRNAs detectable. As calibrators for cellular EBV-load, the sum EBV-miRNA was optimal to EBV-genome copy number and EBER2 expression level, with the added advantage of not requiring additional assays. EBV-miRNA was profiled reliably within archival FFPE tissue in 14/23 patients, but not in tissues with low abundance EBV. This method enabled specific and simultaneous detection of numerous EBV-miRNAs in FFPE lymphoma samples that contain EBV at high to medium levels, making it as a useful tool for studies of EBV-miRNA in the majority of diagnostic biopsies.

Development of a non-invasive method, multiplex methylation specific PCR (MMSP), for early diagnosis of nasopharyngeal carcinoma.

Increasing evidence demonstrated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis. Aiming at developing early diagnostic or prognostic tools for various tumors, we took an EBV-associated tumor, nasopharyngeal carcinoma (NPC), as a model and developed a powerful assay based on "multiplex methylation specific-PCR (MMSP)". The MMSP assay was designed to detect tumor-specific methylation status of several NPC-related genes and was capable of acquiring multiplex information simultaneously through a single PCR reaction with the tiny tumor DNA derived from the direct body fluid close to the primary tumor. In this study, we collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. A panel of markers including two EBV, and two cellular TSG markers were applied in this NPC-specific-MMSP assay. We optimized the working condition of MMSP so that it provides information equal to that from the corresponding separate PCRs. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies and significantly distinguished themselves from those of 20 noncancerous volunteers. Among the 69 samples (49 NPCs and 20 normal controls), the sensitivity of detecting NPC from NP swabs is 98%. The specificity is as high as 100%. In conclusion, being characterized by its noninvasiveness, high reproducibility and informativeness, MMSP assay is a reliable and potential diagnostic tool for NPC. It paves the way for the development of population screening and early diagnosis approaches for various tumor types.

Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly expresses EBNA3A with conserved CD8 T-cell epitopes.

Post-transplantation lymphoproliferative disorders (PTLD) arise in the immunosuppressed and are frequently Epstein-Barr virus (EBV) associated. The most common PTLD histological sub-type is diffuse large B-cell lymphoma (EBV+DLBCL-PTLD). Restoration of EBV-specific T-cell immunity can induce EBV+DLBCL-PTLD regression. The most frequent B-cell lymphoma in the immunocompetent is also DLBCL. 'EBV-positive DLBCL of the elderly' (EBV+DLBCL) is a rare but well-recognized DLBCL entity that occurs in the overtly immunocompetent, that has an adverse outcome relative to EBV-negative DLBCL. Unlike PTLD (which is classified as viral latency III), literature suggests EBV+DLBCL is typically latency II, i.e. expression is limited to the immuno-subdominant EBNA1, LMP1 and LMP2 EBV-proteins. If correct, this would be a major impediment for T-cell immunotherapeutic strategies. Unexpectedly we observed EBV+DLBCL-PTLD and EBV+DLBCL both shared features consistent with type III EBV-latency, including expression of the immuno-dominant EBNA3A protein. Extensive analysis showed frequent polymorphisms in EB-NA1 and LMP1 functionally defined CD8+ T-cell epitope encoding regions, whereas EBNA3A polymorphisms were very rare making this an attractive immunotherapy target. As with EBV+DLBCL-PTLD, the antigen presenting machinery within lymphomatous nodes was intact. EBV+DLBCL express EBNA3A suggesting it is amenable to immunotherapeutic strategies.

Epstein-Barr virus genetic variation in Vietnamese patients with nasopharyngeal carcinoma: full-length analysis of LMP1.

Genetic variation in tumor virus genes and its impact on function might contribute to the understanding of geographic differences in risks for virus-associated tumors. This is particularly true for the genes known to contribute to the biology of the tumor. It is has been proposed that Epstein-Barr virus (EBV) gene variation has a role in the high risk of nasopharyngeal carcinoma (NPC) in South-East Asia. NPC is among the five most common cancers in Vietnam. EBV-NPC cells always express EBV nuclear antigen 1 (EBNA1) and also frequently latent membrane protein 1 and 2 (LMP1 & LMP2). To investigate EBV gene variation in Vietnamese NPC patients we analyzed the full length of LMP1 gene including its promoter region, and the N-termini of both EBNA1 and LMP2A genes from five NPC biopsies. We detected two EBV variants V1 and V2 based on the LMP1 nucleotide sequence pattern compared with the prototype B95-8 and some available sequences including Chinese variants. The V1 variant shows strong similarity to a variant dominant in Southern China (China 1), while the V2 variant is similar to a Thai variant SEA 2 and partly identity with GD1 in the C-terminus. The promoter region and transmembrane domain of the SEA 2-like samples contained some specific differences compared with previously published variants. In contrast, analysis of EBNA1 N- and LMP2A N-termini only revealed minor changes. Our findings reinforces that the polymorphisms of whole LMP1 sequence should be considered in future EBV molecular epidemiology studies in different geographic populations.