Determinants of Epstein-Barr virus-positive gastric cancer: an international pooled analysis.

BACKGROUND: Meta-analyses of the published literature indicate that about 9% of gastric cancers contain Epstein-Barr virus (EBV), with consistent and significant differences by sex and anatomic subsite. This study aimed to identify additional determinants of EBV positivity and their joint effects. METHODS: From 15 international populations with consistent laboratory testing for EBV, we pooled individual-level data for 5081 gastric cancer cases including information on age, sex, subsite, histologic type, diagnostic stage, geographic region, and period of diagnosis. First, we combined population-specific EBV prevalence estimates using random effects meta-analysis. We then aggregated individual-level data to estimate odds ratios of EBV positivity in relation to all variables, accounting for within-population clustering. RESULTS: In unadjusted analyses, EBV positivity was significantly higher in males, young subjects, non-antral subsites, diffuse-type histology, and in studies from the Americas. Multivariable analyses confirmed significant associations with histology and region. Sex interacted with age (P=0.003) and subsite (P=0.002) such that male predominance decreased with age for both subsites. The positivity of EBV was not significantly associated with either stage or time period. CONCLUSION: Aggregating individual-level data provides additional information over meta-analyses. Distinguishing histologic and geographic features as well as interactions among age, sex, and subsite further support classification of EBV-associated gastric cancer as a distinct aetiologic entity.

Human papillomavirus detected in female breast carcinomas in Japan.

To investigate the aetiological role of human papillomavirus (HPV) in breast cancer, we examined the presence, genotype, viral load, and physical status of HPV in 124 Japanese female patients with breast carcinoma. Human papillomavirus presence was examined by PCR using SPF10 primers, and primer sets targeting the E6 region of HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). In 11 normal epithelium specimens adjacent to 11 HPV-16-positive carcinomas, 7 were HPV-16-positive. However, none of the normal breast tissue specimens adjacent to HPV-negative breast carcinomas were HPV-positive. The real-time PCR analysis suggested the presence of integrated form of viral DNA in all HPV-16-positive samples, and estimated viral load was low with a geometric mean of 5.4 copies per 10(4) cells. In conclusion, although HPV DNA was detected in 26 (21%) breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered integrated into the host genome, their low viral loads suggest it is unlikely that integrated HPV is aetiologically involved in the development of Japanese breast carcinomas that we examined.

Human papillomavirus-16 is integrated in lung carcinomas: a study in Chile.

The human papillomavirus (HPV) was detected in 20 (29%) out of 69 lung carcinomas (LCs) in Chile, by PCR and Southern blot, and was more frequently detected in squamous cell carcinoma (SQC) than in adenocarcinomas (46 vs 9%, P=0.001). HPV-16, positive in 11 cases, was the most frequently detected HPV genotype determined by DNA sequencing. HPV-16 E2/E6 ratio, estimated from real-time PCR analysis, was much lower than the unity, suggesting that at least a partial HPV-16 genome was integrated in all but one HPV-16-positive SQCs. The remaining one case was suspected to have only episomal HPV-16. Although the viral load was low in most of the LCs, a case showed the HPV-16 copy number as high as 8479 per nanogram DNA, which was even a few times higher than the minimum viral load of seven cervical carcinomas (observed viral load: 3356-609 392 per nanogram DNA). The expression of the HPV-16/18 E6 protein was found in only two HPV-16-positive SQCs (13%) but not in the case with the highest viral load. Although the viral load was in general very low and HPV E6 expression is none or weak, further studies seem warranted to examine aetiological involvement of high-risk HPV in lung carcinogenesis.

Nasal natural killer/T-cell lymphoma and its association with type "i"/XhoI loss strain Epstein-Barr virus in Chile.

BACKGROUND: Nasal T/natural killer (NK)-cell lymphoma is an aggressive type of non-Hodking's lymphoma associated with Epstein-Barr virus (EBV) and striking geographical variations worldwide. AIM: To characterise nasal NK/T-cell lymphoma associated with genotypes of EBV in Chile, a Latin American country, where multiple strains of EBV, including two new recombinant strains, in healthy individuals were recently found. METHODS: Cases with diagnosis of primary nasal lymphoma were selected for histological and immunohistochemical analysis (CD3, CD3e, CD4, CD8, CD79a, CD56, CD57 and TIA-1) and in-situ hybridisation, serology and genotyping analysis for EBV. RESULTS: Out of 22 cases, 9 (41%) cases fulfilled the World Health Organization criteria for nasal NK/T-cell lymphoma; of these 7 (78%) cases were positive for EBV. Genotyping analysis revealed 6 cases of type 1 EBV and wildtype F at the BamHI-F region, 4 cases type "i" EBV at the BamHI-W1/I1 region; XhoI wild type was found in 2 and XhoI loss in 4 cases, respectively. Cosegregation analysis of the BamHI-W1/I1 region and XhoI restriction site showed the new recombinant strain type "i"/XhoI loss in 3 cases and type "i"/XhoI wild-type strain in 1 case. Most patients were treated with combined anthracycline-containing regimens. Half of the cases attained complete remission. CONCLUSION: Although nasal NK/T-cell lymphomas from Chile share similar clinicopathological features, high association with EBV and unfavourable prognosis with those described elsewhere, genotype analysis shows that the new recombinant type "i"/XhoI loss strain might contribute to explain the intermediate incidence of nasal NK/T-cell lymphomas in Latin America.

Novel real-time monitoring system for human cytomegalovirus-infected cells in vitro that uses a green fluorescent protein-PML-expressing cell line.

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

Transcripts of the human herpesvirus 8 genome in skin lesions and peripheral blood mononuclear cells of a patient with classic Kaposi's sarcoma.

We report a 75-year-old Japanese woman with classic Kaposi's sarcoma. PCR amplified human herpesvirus 8 (HHV-8) DNA sequences from her skin lesions and peripheral blood mononuclear cells (PBMC), but not her plasma, saliva or urine. An antibody test against HHV-8 lytic antigens was positive. Immunohistochemical staining detected latent antigen. There was no evidence of HHV-8 infection in her husband, sister or daughter. Genes coding for HHV-8-encoded viral interleukin-6, viral macrophage inflammatory protein I, viral G protein-coupled receptor, viral cyclin D and viral Bcl-2 were expressed to the same degree in both her skin lesion and PBMC. Latency-associated T0.7 mRNA and HHV-8-encoded viral tegument protein genes were expressed in her PBMC at levels lower than in the skin lesions. Based on the gene expression profile, we concluded that lytic HHV-8 infection was present in her skin lesions and PBMC.

Epstein-barr virus detection in tumors of upper gastrointestinal tract. An in situ hybridization study in Pakistan.

To examine the potential role of Epstein-Barr virus (EBV) in the carcinogenesis of upper gastrointestinal tract, we conducted an in situ hybridization assay for EBV-encoded small RNA (EBER) expression in the tumors of 56 oral and 50 esophageal squamous cell carcinoma (SCC) cases, and 52 stomach adenocarcinoma cases diagnosed in the King Edward Medical College and Allama Iqbal Medical College Lahore, Pakistan between 1996-2002. There were no malignancies with positive EBER expression in oral and esophageal SCC. Only one out of the 52 gastric adenocarcinoma cases (1.9%) was positive for EBER expression, and this frequency was relatively low as compared to cases reported worldwide. The case was a 42 year-old male patient and histologically classified as moderately differentiated tubular adenocarcinoma. In conclusion, the frequency of EBV-associated gastric carcinoma was relatively low in Pakistan. The present study could not confirm the involvement of EBV in the carcinogenesis of oral and esophageal SCC.

Epstein-Barr virus-associated gastric carcinoma in Lima, Peru.

We examined 254 gastric carcinomas (GCs) diagnosed in four hospitals in Lima, Peru, and its suburban area during the period between 1994-2001. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) was identified by the in situ hybridization (ISH) technique to detect EBV-encoded small RNA (EBER) in gastric tissue. EBVaGCs, where EBER ISH staining was observed in all carcinoma cells, accounted for 3.9% (10/254) of gastric adenocarcinomas, the lowest frequency ever reported in Latin American countries. EBVaGC incidence rates in Peru, which we estimated on the basis of the present study and cancer incidence in Lima, were 0.8 per 100,000 among men and 0.5 per 100,000 among women. These estimates are much lower than those reported in our previous studies in Colombia (4.1 and 1.4 per 100,000 among men and women, respectively), a neighboring country, and in Japan (6.4 and 1.1 per 100,000 among men among women, respectively). Interestingly, EBVaGC in Peru showed no evident male predominance, as opposed to the findings reported in a majority of studies. Other clinicopathological features of EBVaGC in Peru were similar to those found in literature: EBVaGC showed no age dependence, a predominance in the non-antrum part of the stomach, and high frequencies in histological subtypes of moderately differentiated tubular adenocarcinoma and solid poorly differentiated adenocarcinoma. There was a case of well-differentiated adenocarcinoma showing a partial EBER-1-positive staining. In this carcinoma, the tumor in the body (middle third of the stomach) was EBER-1 positive but the tumor in the stomach antrum showed no noticeable EBER-1 ISH staining. We suspect this was a case of synchronous double carcinomas. Further studies are needed to identify the cause of the low frequency and lack of male predominance of EBVaGC in Peru.

Environmental factors related to Epstein-Barr virus-associated gastric cancer in Japan.

Epstein-Barr virus (EBV)-encoded small RNA can be detected in about 1-17% of gastric carcinomas. To elucidate the lifestyles and other factors related to the EBV-associated gastric carcinoma (EBV-GC), we interviewed 43 EBV-GC cases and 162 non EBV-GC cases in Kagoshima Prefecture, Japan from 1996-2001. We mainly focused on lifestyles predominant among men because of its male predominance. Although the prevalence of smokers in EBV-GC cases was higher than among non EBV-GC cases, the difference was not significant (P = 0.131). Frequent drinking of coffee and high-temperature drinks, as well as frequent intake of salty and spicy foods, were more prevalent among EBV-GC cases, but only frequent intake of salty food showed a significant difference between EBV-GC and non EBV-GC cases (P = 0.026). In addition, EBV-GC cases tended to be exposed to wood dust and/or iron filings (P = 0.068) and tar (P = 0.097). These findings, together with a high frequency of EBV-GC among remnant cancers after partial gastrectomy, suggest an association between mechanical injuries to the stomach membrane and the high frequency of EBV-GC. The present study also showed that EBV-GC cases tended to be elder brothers/sisters (P for trend = 0.029) suggesting that age at primary infection with EBV may be older in EBV-GC cases than non EBV-GC cases.

Epstein-Barr virus in gastric carcinoma is associated with location in the cardia and with a diffuse histology: a study in one area of Chile.

Epstein-Barr virus (EBV) has been associated with the most common form of stomach neoplasms, the gastric carcinoma (GC). The presence of EBV-encoded small RNAtype-1 (EBER-1), a marker for EBV infection was analyzed by in situ hybridization (ISH) in 185 formalin-fixed and paraffin-embedded cases of GC from a high risk region. We found 31 (16.8%) EBV-positive cases with no relationship to age. Although male predominance (19% in males and 12.5% in females) was observed, the gender difference did not achieve statistical significance. Odds ratio (OR) for cardia location was 5.4 (95% CI 1.7-17.3) when antrum was used as referent category and the effects of gender and age were taken into account. The proportion of EBV-positive cases in diffuse histology was higher than intestinal type (OR = 4.8, 95% CI = 2.0-11.1). Our findings are contrary to a previously accepted hypothesis, that high-risk countries for GC have low rates of EBV-associated GC. In addition, our findings regarding location, histology and weak male predominance are different from what has been described in Asian and European countries, but similar to those described in Mexico and Mexican descendants living in the U.S. suggesting unique characteristics of EBV-associated GC in Latin-America.

Herpes simplex virus pneumonia after cardiac surgery: report of a case.

A rare case of a 61-year-old man who developed herpes simplex virus (HSV) pneumonia after cardiac surgery is presented. He was immunocompetent before the operation and had no history of a mucocutaneous herpesvirus infection. This potentially fatal complication was successfully managed with acyclovir treatment after establishing the diagnosis with bronchoalveolar lavage. A depression of the patient's cell-mediated immunity after cardiopulmonary bypass may have been a causative factor. An unusual type of pneumonia such the HSV pneumonia seen in the present case should therefore be considered in patients with severe hypoxemia accompanied with unexplained pulmonary infiltrates after cardiac surgery using cardiopulmonary bypass which does not improve with conventional treatment.

Epstein-Barr virus-associated gastric carcinoma in Japanese Brazilians and non-Japanese Brazilians in São Paulo.

The proportion of Epstein-Barr virus-associated gastric carcinoma (EBV-GC) was examined in 149 Japanese-Brazilian and 151 non-Japanese-Brazilian gastric-carcinoma cases using in situ hybridization (ISH) assay to detect EBV-encoded small RNA (EBER), and the results were compared with our referent Japanese data. We found that 4.7% of cases in Japanese Brazilians were EBER-positive. This frequency was slightly lower than that of the referent Japanese, among whom 6.2% of 2038 gastric-carcinoma cases were EBER-positive. On the other hand, the non-Japanese-Brazilian series showed a significantly higher proportion of EBV-GC (11.2%) than the referent group did (P = 0.01). Although EBV-GC was predominant in males among non-Japanese Brazilians (M / F = 3.6, P = 0.047), as was the case in Japanese (M / F = 2.7), Japanese Brazilians did not show such a male predominance. The sex-ratio difference between the Japanese Brazilians and Japanese was statistically significant (P = 0.005). In conclusion, the present study in Japanese Brazilians and Japanese yielded no evidence suggesting any change in the frequency of EBV-GC caused by migration, except the absence of male predominance, which was observed both in Japanese and non-Japanese Brazilians.

Epstein-Barr virus involvement is mainly restricted to lymphoepithelial type of gastric carcinoma among various epithelial neoplasms.

To demonstrate the association of Epstein-Barr virus (EBV) with primary epithelial neoplasms in the south part of Kyushu, Japan, 761 carcinomas consisting of 75 lung, 61 breast, 107 esophagus, 102 colon, 58 pancreas, 45 thyroid, and 313 gastric cancers were examined by EBER-1 in situ hybridization. EBER-1 was detected in 23 cases (7.3%) out of 313 gastric carcinomas, while none of the other carcinomas was positive for EBER-1. Twenty-eight (9.4%) out of 313 gastric carcinomas were differentiated poorly to moderately carcinomas with prominent lymphoid cell infiltration, similar to so-called lymphoepithelioma-like carcinoma, and 19 cases (67.9%) were positive for EBER-1. Although two (2.6%) and 11(10.3%) out of 75 lung and 107 esophagus carcinomas were so-called lymphoepithelioma-like carcinomas, respectively, but EBER-1 was not detected in other epithelial neoplasms that originated from the lung, esophagus, breast, colon, pancreas, and thyroid in the south of Kyushu, Japan. As a result, EBV was associated with only some gastric carcinomas but not with other epithelial neoplasms originating from the lung, esophagus, breast, colon, pancreas, and thyroid in southern Japan.

The significance of cytomegalovirus infection over the clinical course of adult T-cell leukemia/lymphoma.

The significance of cytomegalovirus (CMV) infections developed over the clinical course of adult T-cell leukemia/lymphoma (ATLL) were evaluated in relation to the patient survival rate, ATL activity and immunocompetent cells. ATLL patients with CMV infections on admission exhibited a poor survival rate, while patients with CMV infections at any time after admission survived longer than those not infected with this virus. ATLL patients who exhibited a numbers of CMV infection on admission showed higher ATL activity and had lower numbers of CD8-positive and CD56-positive cells than those who developed CMV infections at any time after admission. Therefore, it appears likely that patients with CMV infections on admission were in an immunosuppressive state due to aggressive ATL activity.

Human leukocyte antigens related to Epstein-Barr virus-associated gastric carcinoma in Japanese patients.

To assess the association between specific types of human leukocyte antigen and the risk of Epstein-Barr virus (EBV)-associated gastric carcinoma, serological typing for major histocompatibility complex class I and class II antigens was performed for 110 EBV-positive and 155 EBV-negative gastric carcinoma cases. In class I analysis, the frequency of B59 in the EBV-positive cases was higher than for the EBV-negative cases (odds ratio (OR) 3.06; 95% confidence interval (CI) 1.02-9.23). For class II antigens, DQ3 and DR9 frequencies in the EBV-positive cases were higher (OR 1.94; 95% CI 1.16-3.24 and OR 1.93; 95% CI 1.11-3.37, respectively), whereas DR11 frequency was lower than found in the EBV-negative cases (OR 0.10; 95% CI 0.01-0.79). After adjusting for multiple comparisons, only DR11 frequency remained significantly lower in the EBV-positive cases (P = 0.04), and the association of DQ3 was marginally significant (P = 0.05). These results suggest that the presence of DR11-restricted cytotoxic T cells (CTLs) related to EBV-associated gastric carcinoma, or a deficiency of DR11 and a high frequency of DQ3 may be genetic markers for a population at greater risk of EBV-associated gastric carcinoma. However, further extensive studies to more cases and DNA typing are needed because our findings in this study are exploratory.

Surgical maneuvers enhance molecular detection of circulating tumor cells during gastric cancer surgery.

OBJECTIVE: To evaluate the relation between the presence of cancer cells in blood according to the time course during a surgical procedure and liver metastases in patients with gastric cancer. SUMMARY BACKGROUND DATA: Several studies have reported on the detection of circulating cancer cells in blood by reverse transcriptase-polymerase chain reaction (RT-PCR). However, few reports have examined the relation between molecular detection of circulating cancer cells according to the time course during a surgical procedure and blood-borne metastases. METHODS: Blood samples from 57 patients with gastric cancer were obtained from the portal vein, peripheral artery, and superior vena cava before and after tumor dissection. After total RNA was extracted from each blood sample, carcinoembryonic antigen (CEA)-specific RT-PCR was performed. RESULTS: CEA-mRNA was detected in the blood of 21 (36.8%) of the 57 patients. CEA-mRNA was not detected in the blood obtained from 15 healthy volunteers and 15 patients with benign disease. The positive rate increased in proportion to the depth of tumor. The incidence of positive CEA-mRNA did not differ among the various sites of blood sampling. The appearance of circulating cancer cells was related to the surgical maneuver. A significant relation was found between the detection of CEA-mRNA and blood-borne metastases. CONCLUSIONS: A high incidence of positive CEA-mRNA was found in the blood during gastric cancer surgery. Surgical maneuvers are a possible cause of hematogenous metastasis. The authors found that patients with positive CEA-mRNA had a high risk of blood-borne metastasis even after curative resection.

Isolation and analysis of an aciclovir-resistant murine cytomegalovirus mutant.

An aciclovir (ACV)-resistant murine cytomegalovirus (MCMV) was isolated from the Smith strain and the mutant was analysed. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC(50)) of ACV for the mutant strain was approximately 30 times higher than that for the wild-type strain. The mutant strain was equally sensitive to ganciclovir (GCV), but slightly resistant to cidofovir (CDV) and foscarnet (PFA) when compared with the wild-type. Molecular analysis of the mutant strain revealed that a single base mutation of cytosine (C) to guanine (G) occurred at the 2476th nucleotide position in the DNA polymerase gene region, resulting in an amino acid substitution of proline (Pro) with alanine (Ala) at codon 826. The marker transfer experiment confirmed that this mutation conferred ACV resistance to MCMV. This mutation at codon 826 was easily identified by means of Hae III digestion of the selected PCR product and electrophoresis.

Clinicopathological features of Epstein-Barr virus-associated nasal T/NK cell lymphomas in southern Japan.

OBJECTIVE: Nasal T/natural killer cell lymphomas (NTCL) are highly prevalent among Oriental populations. However the characteristic immunophenotype of NTCL is still controversial and it can be difficult to make a firm histologic diagnosis of malignancy. Therefore, 14 cases of NTCL in patients from southern Japan were evaluated for clinicopathological features and immunophenotypic status. Furthermore, the genetic variations in the latent membrane protein 1 (LMP-1) gene of Epstein-Barr virus (EBV)-related NTCL were evaluated. METHODS: Biopsy specimens were fixed in formalin and embedded in paraffin before examination using in situ hybridization for EBV-encoded RNA-1 (EBER-1) as well as immunohistochemical staining for CD3, CD3epsilon, CD4, CD8, CD43, CD45RA, and CD45RO. To detect genetic variations, single-strand conformation polymorphism (SSCP) analysis and DNA sequencing were applied. RESULTS: Clinically, 14 cases were divided into two groups. The first group consisting of eight patients showed good prognosis. The other group consisting of six patients showed poor prognosis. The vast majority of neoplastic cells in NTCL were EBER-1 positive. These cells did not express CD4, CD8, or CD45RA, but often expressed CD43 and CD45RO. In addition, they were negative for CD3 when stained with a mouse monoclonal antibody but stained for CD3epsilon when a rabbit polyclonal was used. The 3'-terminal of LMP-1 gene of seven cases were amplified and all of them have 30 base pair (bp) deletion. CONCLUSION: NTCL are a heterogeneous mix of cell types although EBV-associated NTCL in patients from southern Japan appear to originate from natural killer cells rather than T cells, and also prognosis is variable and not always poor. The ability to make a firm diagnosis can be enhanced through the combined use of in situ hybridization and immunohistochemistry. High prevalence of the 30-bp deletions of the LMP-1 gene in EBV-related NTCL may also reflect the prevalence of the deletion variant in the normal population in Japan.

Differentiation of Herpes simplex virus types 1 and 2 in sera of patients with HSV central nervous system infections by type-specific enzyme-linked immunosorbent assay.

OBJECTIVE: To differentiate by enzyme-linked immunosorbent assay (ELISA) using type-specific glycoprotein G herpes simplex virus (HSV) type 1 and type 2 in serum collected from patients with HSV central nervous system (CNS)infections. METHODS: HSV 1 and 2 typing in convalescent sera of 17 patients with HSV acute encephalitis, myelitis, or meningitis was determined by the type-specific IgG ELISA kit (Gull Laboratory, Inc.). HSV CNS infections were diagnosed by polymerase chain reaction (PCR) or conventional serologic tests from acute to convalescent stages. RESULTS: In 13 of 17 patients, HSV type 1 and HSV type 2 antibodies were positive; 11 patients with HSV type 1 and 2 patients with HSV type 2 were found. All 10 patients with encephalitis showed equivocal or positive results for HSVtype 1. In two of three cases of myelitis, HSV type 1 was demonstrated. Each case of myelitis and meningitis reacted to both types 1 and 2. CONCLUSION: These data suggest that the kit is useful for type differentiation of HSV CNS infections from convalescent sera, and can play a supplementary role in HSV typing by PCR or previous serologic tests.