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.

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.

Detection of virus-specific RNA in herpes simplex virus type 1-transformed hamster cell lines.

In situ hybridization experiments with the use of recombinant herpes simplex virus type 1 DNA as probes have detected virus-specific RNA in herpes simplex virus type 1-transformed Syrian hamster cell lines. The relatively most abundant virus transcripts hybridized to the herpes simplex virus type 1 EcoRI-F fragment at map position 0.32-0.42.

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 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.

Human T lymphotropic virus-type I Tax induction of CD21/Epstein-Barr virus receptor expression on T cells and its significance in leukemogenesis of adult T cell leukemia.

CD21, which is expressed on B cells, is also expressed on human T lymphotropic virus-type I (HTLV-I)-infected T cell lines. CD21 also serves as a receptor of Epstein-Barr virus (EBV). We evaluated the mechanism of CD21 induction on HTLV-I-infected T cells and its clinical significance in the leukemogenesis of adult T cell leukemia (ATL). CD21 induction was detected at very low levels in T cell lines (Jurkat and CEM cells), and in non- or low-Tax-producing HTLV-I-infected T cell lines (Oh13T, S1T, and Su9T01 cells). In contrast, marked induction of CD21 was detected in high-Tax-producing HTLV-I-infected T cell lines (K3T, F6T, and MT-2). A Jurkat T cell clone stably transfected with tax-expressing cDNA expressed a significant amount of CD21 on the cell surface. These results strongly suggest that HTLV-I Tax induces CD21 on T cells. On two-color analysis, CD21 expression was detected in CD4+ T cells of the primary ATL cells from a subset of patients, suggesting that EBV infection may be associated with the leukemogenesis of ATL, at least in part. However, no genome of EBV was detected in the genomic DNA of six HTLV-I-infected T cell lines or the primary ATL cells separated from all patients, indicating the irrelevance of EBV infection to ATL leukemogenesis.

Comparison of human cytomegalovirus DNA polymerase activity for ganciclovir-resistant and -sensitive clinical strains.

Previously, we found that a ganciclovir (GCV)-resistant clinical human cytomegalovirus (HCMV) isolate had an amino acid substitution at codon 501 (Leu --> Phe) in the delta-region C of the DNA polymerase gene. DNA polymerases have now been (partially) purified from both the GCV-resistant and sensitive parental strains and the activity of DNA polymerase and 3'-5' exonuclease compared. With respect to DNA polymerase activity, the Michaelis constant (Km) and maximum velocity (Vmax) of the GCV-resistant strain for the DNA template were lower than those of the GCV-sensitive strain. With respect to 3'-5' exonuclease activity, the Km and Vmax of the GCV-resistant strain for the DNA substrate in the presence of ammonium sulfate were lower than those of the GCV-sensitive strain, while being similar in the absence of ammonium sulfate. Although the polymerase activity of the two strains showed almost the same sensitivity for the different polymerase inhibitors, the 3'-5' exonuclease activity of the GCV-resistant strain was more resistant to these inhibitors than that of the GCV-sensitive strain.

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.

Genetic analysis of a ganciclovir-resistant human cytomegalovirus mutant.

We isolated a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) from a laboratory strain, AD169, and analysed the mutant. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of GCV for the mutant strain was five times higher than that of the wild-type strain. The mutant strain showed similar sensitivity to phosphonoacetic acid and cidofovir as the wild-type strain. These data suggest mutation in the UL97 gene encoding for the phosphotransferase that phosphorylates GCV. Molecular analysis of the mutant strain revealed that a single base substitution of adenine by cytosine occurred at the 1796 nucleotide position of the UL97 gene region, resulting in the substitution of lysine by threonine at codon 599 in the UL97 gene product. Marker transfer experiment confirmed that this mutation conferred HCMV resistance to GCV. The mutation at codon 599 was easily identified by means of RsaI digestion of the selected PCR product.

Anti-herpesvirus activity profile of 4'-thioarabinofuranosyl purine and uracil nucleosides and activity of 1-beta-D-2'-fluoro-4'-thioarabinofuranosyl guanine and 2,6-diaminopurine against clinical isolates of human cytomegalovirus.

Newly synthesized 4'-thio- and 2'-fluoro-4'-thioarabinofuranosyl purine and pyrimidine nucleosides were compared with the corresponding 4'-oxo type arabinosyl nucleosides for anti-herpesvirus and anti-cell proliferative potencies. 4'-Thioarabinosyl- and 2'-fluoro-4'-thioarabinofuranosyl 5-substituted uracils had selective antiviral activities, but were not superior to 4'-oxo nucleosides, except for the activity of 5-ethyl-uracil 4'-thio nucleosides against herpes simplex virus. Furthermore, 4'-thio substituted derivatives of sorivudine (BV-araU) and related compounds, and 2'-fluoro-5-methyl-arabinosyluracil exhibited reduced activity against varicella-zoster virus compared with the parent compounds. The 4'-thioarabinosyluracils, except for 5-methyluracil derivatives, were inactive against human cytomegalovirus (HCMV). 4'-Thioarabinofuranosyl guanine and diaminopurine had the most potent anti-HCMV and anti-proliferative activities, whereas arabinosyl guanine and diaminopurine had only marginal antiviral activity. 2'-Fluoro-4'-thioarabinofuranosyl derivatives of guanine (4'-thio-FaraG) and 2,6-diaminopurine (4'-thio-FaraDAP), however, had particularly high activity against all herpesviruses tested with anti-proliferative activity equipotent to that of arabinosyl guanine and diaminopurine. 4'-Thio- and 2'-fluoro-4'-thioarabinofuranosyladenines exhibited biological activities similar to that of arabinosyladenine. Both 4'-thio-FaraG and 4'-thio-FaraDAP had a 6-fold lower ED50 than ganciclovir against clinical isolates of HCMV. A ganciclovir-resistant isolate, obtained from a patient who had received long-term ganciclovir-treatment, was susceptible to 4'-thio-FaraG and 4'-thio-FaraDAP.

Human herpesvirus-6 genomes in histiocytic necrotizing lymphadenitis (Kikuchi's disease) and other forms of lymphadenitis.

The cervical lymph nodes of 27 patients with histiocytic necrotizing lymphadenitis (HNL) were examined, as were those of 9 patients with tuberculous lymphadenitis (Tb), 10 with reactive paracortical hyperplasia (RPH), and 10 with nonspecific lymphadenitis (NSL). Southern blot analysis, the polymerase chain reaction (PCR), and in situ hybridization were use to locate the human herpesvirus-6 (HHV-6) genome. Southern blot analysis showed that all cases were negative for HHV-6 genomes, although all but one HNL case expressed HHV-6 genome using PCR. On in situ hybridization all 10 HNL cases, 6 of the 10 RPH cases, 6 of the 10 NSL cases, and 2 of the 9 Tb cases showed HHV-6 DNA. These results indicate that the presence of HHV-6 genome is not specifically related to HNL, and that this virus could hibernate in a latent form in the cervical lymph nodes. In addition, we examined three different primers (A, B, and C) for PCR amplification of HHV-6 genomes.

Analysis of human herpes virus-6 genomes in lymphoid malignancy in Japan.

Ninety cases of malignant lymphoma and 56 cases of reactive lymphadenopathy were studied using Southern blot analysis and the polymerase chain reaction to identify human herpes virus-6 (HHV-6) DNA. This was detected in cases of lymphoid malignancy at a rate which ranged from 50.0% to 68.8%. There were no differences in rates for different types of lymphoid malignancies. Herpes virus-6 DNA was detected by PCR in lymphoid malignancies less frequently than in reactive lymphadenopathies. It was not detected in lymphoid malignancies using Southern blotting. These results suggest that HHV-6 DNA was not related to lymphoid malignancy and was only a latent infection of non-neoplastic cells in tumour tissue.

Detection of human T-cell lymphotropic virus type 1 infection by the polymerase chain reaction using dried blood specimens on filter papers.

A simple method for detection of proviral DNA sequences of human T-cell lymphotropic virus type 1 (HTLV-1) was developed using dried blood specimens on filter papers. The whole blood was blotted onto the Guthrie paper. After the blood has dried, the blotted paper was punched out into small discs. The discs were then boiled to prepare the template for PCR (filter paper-PCR method). The filter paper-PCR method detected even a single HTLV-1-infected cell in three discs. The sensitivity of the filter paper-PCR method was equivalent to that of the method in which DNA was extracted with phenol and used as the template for PCR (DNA extraction-PCR method). In addition, DNA in the blotted filter paper was still utilizable as the template after the storage at 25 degrees C for at least 7 wk. A total of 53 clinical specimens from 30 seropositive and 23 seronegative individuals who were screened by particle agglutination (PA) test were analysed for HTLV-1 DNA by both PCR methods. Of 30 PA-positive specimens, 28 were also positive for HTLV-1 antibody by Western blot (WB) analysis, but two were indeterminate. The twenty eight WB-positive and one of the two indeterminate specimens were positive for HTLV-1 proviral DNA by both PCR methods. Of 23 PA-negative specimens, 22 were negative for HTLV-1 proviral DNA by both PCR methods. However, one PA-negative specimen was positive by both PCR methods. This patient was a 16-mth-old infant who was born to an HTLV-1 carrier mother and fed thereafter without her breast milk. In comparison to DNA extraction-PCR method, the sensitivity and specificity of the filter paper-PCR method was 100%, respectively.

Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections.

Non-isotopic in situ hybridization with a novel phenytoin (PHE)-labeled probe was developed. The mixture of cloned cytomegalovirus (CMV) DNA fragments was labeled by random primer technique using PHE-11(spacer)-dUTP, instead of dTTP. The tissue sections were treated with 0.2 N HCl and with proteinase K (1 microgram/ml), and then heated at 70 degrees C in the presence of 50 or 75% formamide. The sections were hybridized with PHE-labeled probe at 37 degrees C overnight. The hybridization signal was visualized by alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate (BCIP)/4-nitroblue tetazolium (NBT) system. Strong hybridization signals were detected in sections of the small intestine and the placenta, even when denatured in the presence of 50% formamide. In the case of small intestine, CMV DNA was also detected in the endothelial cells of the mucosa where apparent infected cell was not observed histologically. In the sections of the submaxillary gland, the lung, the adrenal gland and the ovary, hybridization signal was not detected when denatured in the presence of 50% formamide, but detected after denaturation with 75% formamide. Thus, in situ hybridization with the novel PHE-labeled probe is applicable to conventional formalin-fixed, paraffin-embedded tissue sections.

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.

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.

Progressive encephalopathy associated with cytomegalovirus infection without immune deficiency.

Slowly progressive encephalopathy caused by cytomegalovirus is an unusual disorder, and its pathogenesis remains unknown except for cases associated with the acquired immune deficiency syndrome and organ transplantation. We report a case who showed clinical features of progressive encephalopathy. Cytomegalovirus was repeatedly isolated from urine, and cytomegalovirus-infected cells were detected in bone marrow. Serial computed tomographic head scan revealed periventricular calcification and its progression to the thalamus, cerebellum, and brain stem. On autopsy, there were multiple calcifications and diffuse glial proliferation in the gray and white matter. Perivascular inflammation was only minimal. There was no evidence of immune deficiency. This case suggests that progressive encephalopathy can be caused by cytomegalovirus infection without immune deficiency. This type of cytomegalovirus infection may be unusual, but its serious outcome should remind us to detect it accurately.

Cytomegalovirus, human herpesvirus 6 and human T cell leukemia virus infection in renal transplanted patients in the northeast of Brazil.

The seroprevalence of antibodies against cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human T cell leukemia virus type 1 (HTLV-1), determined by ELISA and by fluorescence in 54 renal transplanted patients, was 96.2%, 88.8% and 11.1%, respectively. These values are relatively high when compared with the results obtained for healthy individuals of the same age groups from Recife, Northeastern Brazil. Active CMV infection was detected by the presence of IgM antibodies and/or virus isolation in 13 (24%) patients. Kidney rejection and renal dysfunction were observed in 11 of these 13 patients, whereas 3 of 6 HTLV-1 antibody-positive individuals presented these complications. All HTLV-1 positive patients were also positive to IgG CMV and HHV-6 antibodies. The importance of the three viruses in this clinical condition is suggested by the high seropositivity rates compared with the healthy population. The group may also represent a potential source of HTLV-1 infection in this non-endemic area.