Frequent expression of thymidine phosphorylase in Epstein-Barr virus-associated gastric carcinoma of diffuse type.

UNLABELLED: The aim of the present study was to elucidate the etiological roles of Epstein-Barr virus (EBV) in the development of EBV-associated GC (EBV-GC), EBV-GCs and non EBV-GCs were compared with regard to the expression of thymidine phosphorylase (TP), which is known to have angiogenic activity in various tumor tissues. PATIENTS AND METHODS: TP expression was examined by immunohistochemistry assay among 156 gastric carcinoma cases (21 EBV-GC cases and 135 non EBV-GC cases). RESULTS: The frequency of tumors with TP expression was nearly twice as high in EBV-GCs (71%) than in non EBV-GCs (37%) (p=0.005). However, such an association was only observed in Lauren's diffuse-type tumors. CONCLUSION: Our finding suggests that the mechanism involved in TP expression of gastric carcinoma appears to be different in intestinal- and diffuse-type tumors.

Human papillomavirus is frequently detected in gefitinib-responsive lung adenocarcinomas.

A number of studies have reported the presence of human papillomavirus (HPV) in lung carcinoma. Interestingly, its detection rate appears to differ histologically and geographically. The present study examined 30 adenocarcinomas and 27 squamous cell carcinomas of the lung in a southern area of Japan, and detected high-risk HPV genome in 9 (30%) adenocarcinomas and 2 (7%) squamous cell carcinomas, using PCR with SPF10 primers and INNO-LiPA HPV genotyping assay. The difference of HPV detection rates in adenocarcinomas and squamous cell carcinomas was statistically significant (P=0.044, Fisher's exact test). HPV-16 was the most prevalent HPV genotype, and was detected in 27% (8/30) of adenocarcinomas and in 7% (2/27) of squamous cell carcinomas. High-risk-HPV positive carcinomas had decreased proportions of pRb (P=0.107) and significantly increased proportions of p16INK4a expressing cells (P=0.031) when compared to HPV-negative lung carcinomas. All HPV-16-positive cases were considered to have an integrated form of HPV-16 but its viral load was low (geometric mean = 0.02 copy per cell). In 20 additional adenocarcinomas treated with gefitinib, a tyrosine kinase inhibitor specific for epidermal growth factor receptor, the presence of HPV was examined. Note that East Asian ethnicity is a predictive factor of gefitinib response. High-risk HPV genome was found in 75% (6/8) of adenocarcinomas with complete or partial response to gefitinib but was not found in the remaining 12, which did not respond to gefitinib. In conclusion, the present study suggests that high-risk HPV may be more strongly related to adenocarcinomas, particularly gefitinib-responsive adenocarcinomas, when compared to squamous cell carcinomas. However, its low viral load makes it difficult to determine the etiological significance of these findings.

E-cadherin and beta-catenin expression in Epstein-Barr virus-associated gastric carcinoma and their prognostic significance.

AIM: To examine the role of E-cadherin and beta-catenin in carcinogenesis and to assess their prognostic implication in Epstein-Barr virus-associated gastric carcinomas (EBV-GCs). METHODS: We compared the frequency of E-cadherin and beta-catenin expression in 59 EBV-GCs and 120 non-EBV-GCs, and examined the association between patients' prognosis and the expressions of these proteins. RESULTS: Neither the cellular-membranous nor the cytoplasmic E-cadherin expression showed any difference between EBV-GCs and non-EBV-GCs. On the other hand, loss of membranous expression of beta-catenin occurred more frequently in non-EBV-GCs than EBV-GCs [odds ratio = 0.41; 95% confidence interval (CI), 0.19-0.90]. Furthermore, the nuclear and/or cytoplosmic expression of beta-catenin was seen more frequently in EBV-GCs than non-EBV-GCs (odds ratio = 2.23; 95% CI, 0.97-5.09), and was observed in a larger proportion of carcinoma cells of EBV-GCs than non-EBV-GCs (P = 0.024). Survival analysis for non-EBV-GC revealed that lymph node metastasis was significantly associated with poor prognosis (P < 0.001). Among EBV-GCs, the depth of invasion (P = 0.005), lymph node metastasis (P = 0.004) and an intestinal type by Lauren classification (hazard ratio = 9.47; 95% CI, 2.67-33.6) were significantly associated with poor prognosis. On the other hand, nuclear and/or cytoplasmic expression of beta-catenin was associated with a better prognosis in patients with EBV-GC (hazard ratio = 0.32; 95% CI, 0.11-0.93). CONCLUSION: We observed more frequent preservation of beta-catenin in cell membrane and accumulation in nuclei and/or cytoplasm in EBV-GCs than in non-EBV-GCs. Factors involved in the prognosis of EBV-GCs and non-EBV-GCs are different in the two conditions.

Human papillomavirus in esophageal squamous cell carcinoma in Colombia and Chile.

AIM: To examine the presence of human papillomavirus (HPV) in esophageal squamous cell carcinoma (ESCC) specimens collected from Colombia and Chile located in the northern and southern ends of the continent, respectively. METHODS: We examined 47 and 26 formalin-fixed and paraffin-embedded ESCC specimens from Colombia and Chile, respectively. HPV was detected using GP5+/GP6+ primer pair for PCR, and confirmed by Southern blot analysis. Sequencing analysis of L1 region fragment was used to identify HPV genotype. In addition, P16(INK4A) protein immunostaining of all the specimens was conducted. RESULTS: HPV was detected in 21 ESCC specimens (29%). Sequencing analysis of L1 region fragment identified HPV-16 genome in 6 Colombian cases (13%) and in 5 Chilean cases (19%). HPV-18 was detected in 10 cases (21%) in Colombia but not in any Chilean case. Since Chilean ESCC cases had a higher prevalence of HPV-16 (without statistical significance), but a significantly lower prevalence of HPV-18 than in Colombian cases (P = 0.011) even though the two countries have similar ESCC incidence rates, the frequency of HPV-related ESCC may not be strongly affected by risk factors affecting the incidence of ESCC. HPV-16 genome was more frequently detected in p16 positive carcinomas, although the difference was not statistically significant. HPV-18 detection rate did not show any association with p16 expression. Well-differentiated tumors tended to have either HPV-16 or HPV-18 but the association was not statistically significant. HPV genotypes other than HPV-16 or 18 were not detected in either country. CONCLUSION: HPV-16 and HPV-18 genotypes can be found in ESCC specimens collected from two South American countries. Further studies on the relationship between HPV-16 presence and p16 expression in ESCC would aid understanding of the mechanism underlying the presence of HPV in ESCC.

Risk factors of gastric cancer specific for tumor location and histology in Cali, Colombia.

AIM: To examine histology- and tumor-location specific risk factors of gastric cancer (GC). METHODS: This was a case-control study. The study subjects were 216 GC patients newly diagnosed during the period 2000-2002 and 431 controls selected from non-cancer patients matching in age, gender, and hospital. We obtained information on lifestyles, dietary habits, and others by a questionnaire. RESULTS: The subjects who were not eldest among his/her siblings were at a slightly elevated GC risk (OR 1.3; 95% CI 0.8-2.0). Salting meals before tasting was related to an increased GC risk (OR 3.5; 95% CI 1.6- 7.3). Frequent consumptions of fruits (OR 0.3; 95% CI 0.1-1.0) and vegetables (OR 0.3; 95% CI 0.1-1.0) were related to decreased GC risks. On the other hand, frying foods (OR 1.9; 95% CI 1.0-3.6) and cooking with coal (OR 1.8; 95% CI 1.3-2.6) were related to increased GC risks. Neither Laurenos histological classification (intestinal and diffuse types) nor tumor location significantly affected those associations except birth order. The subjects who were not eldest among his/her siblings had an increased risk of GCs in the distal and middle thirds, and their ORs were 1.7 (95% CI 1.0-2.8) and 1.9 (95% CI 0.8-4.3), respectively. The corresponding OR in the upper third stomach was 0.3 (95% CI 0.1-0.9). The differences of those three ORs were statistically significant (P = 0.010). CONCLUSION: The present study shows that birth order, salt intake, consumption of fruits and vegetables, the type of cooking, and cigarette smoking are related to GC risk. In histology and tumor-location specific analyses, non-eldest person among their siblings is related to an increased GC risk in the distal and middle thirds of the stomach, and is related to a decreased GC risk in the cardia.

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

Epstein-Barr virus (EBV)-encoded small RNA can be detected in about 1-17 % of gastric carcinomas. To elucidate lifestyles and other factors related to such an EBV-associated gastric carcinoma (EBV-GC), we conducted a case-control study in Cali, Colombia. The study subjects were 368 patients with gastric carcinoma newly diagnosed during the period between September 2000 and June 2003, including 42 EBV-GC cases. We obtained information on lifestyles, dietary habits, and occupational exposure by a questionnaire. The frequency of EBV-GC was related to birth order of patients (P for trend =0.025). More precisely, EBV-GC was much less frequent among the patients who were the eldest child in a family (P=0.007). Those findings were contrary to what was reported by the study conducted in Japan, where EBV-GC was more frequently observed among eldest brothers/sisters. A possible explanation for the apparently conflicting results is that EBV-GC risk is related to the age at first EBV infection but its relationship is not monotonic. In addition to the relationship with birth order, the present study showed that high salt intake and metal dust exposure may be related to EBV-GC as reported by the Japanese study although these associations observed in the present study were not statistically significant. No significant association was observed in other factors, including dietary habits. Further studies seem warranted to elucidate the difference between Japan and Colombia with respect to the environmental factors related to EBV-GC cases.

Epstein-Barr virus-associated gastric carcinoma: Evidence of age-dependence among a Mexican population.

AIM: To investigate features of Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) among a Mexican population. METHODS: Cases of primary gastric adenocarcinoma were retrieved from the files of the Departments of Pathology at the Instituto Nacional de Cancerologia and the Instituto Nacional de la Nutricion in Mexico City. The anatomic site of the gastric neoplasia was identified, and carcinomas were histologically classified as intestinal and diffuse types and subclassified as proposed by the Japanese Research Society for Gastric Cancer. EBV-encoded small non-polyadenylated RNA-1 (EBER-1) in situ hybridization was conducted to determine the presence of EBV in neoplastic cells. RESULTS: We studied 330 consecutive, non-selected, primary gastric carcinomas. Among these, there were 173 male and 157 female patients (male/female ratio 1.1/1). EBER-1 was detected in 24 (7.3%) cases (male/female ratio: 1.2/1). The mean age for the entire group was 58.1 years (range: 20-88 years), whereas the mean age for patients harboring EBER-1-positive gastric carcinomas was 65.3 years (range: 50-84 years). Age and histological type showed statistically significant differences, when EBER-1-positive and -negative gastric carcinomas were compared. EBER-1 was detected in hyperplastic- and dysplastic-gastric mucosa surrounding two EBER-1-negative carcinomas, respectively. CONCLUSION: Among Latin-American countries, Mexico has the lowest frequency of EBVaGC. Indeed, the Mexican population >50 years of age was selectively affected. Ethnic variations are responsible for the epidemiologic behavior of EBVaGC among the worldwide population.

Epstein-Barr virus-associated gastric carcinoma in Papua New Guinea.

Using in situ hybridization assay, we examined Epstein-Barr virus (EBV) encoded RNA (EBER) expression in 66 cases of oral cancer, 40 esophageal cancer cases, 150 stomach cancer cases, and 46 colorectal cancer cases diagnosed in the Pathology Department of Port Moresby General Hospital, University of Papua New Guinea during the period between 1986-2002. There were no malignancies with positive EBER expression except for the following two male stomach cancer cases: a male case with a gastric carcinoma in pylorus whose age was unknown; and a male case aged 55 years without information on location of tumor. Both cases were histologically classified as non-solid poorly differentiated adenocarcinoma of the Japanese histological classification. The frequency of EBV-associated gastric carcinomas was 1.3% (2/150), and was the lowest ever reported in the world. We examined genotypes of two EBV strains detected from gastric carcinomas. Four different regions of EBV genome were examined by PCR-RFLP, coupled with Southern blot hybridization. The EBV genotype of the first case were type A, wild-type F at BamHI-F region, type D of BamHI-I region and the kept type of the XhoI cleavage site in LMP1. The second case had EBV whose genotypes were type A, wild-type F at BamHI-F region, and the kept type of the XhoI cleavage site in LMP1. The BamHI-I region of this case could not be analyzed.

Loss of p16/CDKN2A protein in Epstein-Barr virus-associated gastric carcinoma.

We examined the expression of p16, the CDKN2A gene product, in EBV-associated gastric carcinomas (EBV-GCs). EBV-GCs were identified by detecting EBV-encoded small RNA (EBER) using an in situ hybridization assay of paraffin-embedded tissue. Two non-EBV-GC cases for each EBV-GC case were selected, matched for age, sex, tumor location, and depth of invasion. After excluding cases without sufficient tissue samples for immunohistochemical analysis, 54 EBV-GC and 117 non-EBV-GC cases were available for the present study. The loss of p16 expression was more frequently observed in EBV-GCs (89%) than non-EBV-GC cases (32%; p < 0.001). Among non-EBV-GC cases, the loss of p16 expression was more frequent in female cases (57%) than male cases (29%) (p = 0.042). Expression of p16 was not related to the location of tumor, clinical stage of tumor, age, or prognosis of the patients. In conclusion, the present study suggests that the loss of p16-related cell cycle regulation may be associated with the development of EBV-GC.

Histology-specific gender, age and tumor-location distributions of Epstein-Barr virus-associated gastric carcinoma in Japan.

We examined 1,918 Japanese gastric cancer cases diagnosed during the period 1976-1995 to clarify histology-specific gender, age and tumor-location distributions of Epstein-Barr virus-associated gastric carcinoma (EBV-GC). EBV-GCs accounted for 4.5% and 6.1% of 1,088 intestinal-type and 830 diffuse-type gastric carcinomas, respectively. Both intestinal- and diffuse-type EBV-GCs showed male predominance, but the observed gender difference was statistically significant only in diffuse-type carcinomas (P<0.001). An age-dependent decrease of the EBV-GC proportion was observed in intestinal-type carcinomas (P=0.002), but not in diffuse-type carcinomas. In intestinal-type tumors, the estimated incidence of EBV-GCs reached its peak around age 70. Diffuse-type EBV-GCs appeared to have a much older peak incidence, if any. Both intestinal- and diffuse-type EBV-GCs were least prevalent in the stomach antrum. This study, examining the largest number of EBV-GCs in current literature, showed different patterns of age-dependence in intestinal- and diffuse-type EBV-GCs, suggesting that pathogenic pathways of EBV-GCs may be different in these 2 histological types.

Epstein-Barr virus-associated gastric carcinoma in Cali, Colombia.

To estimate the incidence of Epstein-Barr virus-associated gastric carcinoma (EBV-GC) in Colombia and to clarify its clinicopathological features, we examined 178 consecutive gastric carcinoma cases, diagnosed during the period from 1996 to 1998, at Hospital Universitario del Valle in Cali, Colombia. The mean age of the cases was 60 years in males and 58 years in females. Using in situ hybridization assay of EBV-encoded small RNA-1 in paraffin-embedded tissue samples, we identified 23 cases of EBV-GC (13%). After excluding remnant carcinoma, which was found to be EBV-negative in this series, there were 19 (18%) male and 4 (6%) female EBV-GC cases, and the male predominance was statistically significant (P=0.004). The proportion of EBV-GCs decreased with age (P for trend = 0.022). Using sex- and age-specific proportions of EBV-GCs estimated by logistic models and gastric cancer incidence in Cali, which was obtained from tumor registry during the period 1987-1991, we estimated sex- and age-specific incidence of EBV-GCs. The incidence of EBV-GCs (per 100,000 person-years) was 4.1 and 1.4 among men and women, respectively, after age adjustment using the standard world population. Pathological features of EBV-GCs were also examined. EBV-GCs accounted for 33% (8/24) of carcinomas located in the stomach cardia, 14% (6/43) of carcinomas in the middle-part of the stomach, and 7% (6/81) of carcinomas in the antrum. The difference by tumor location was statistically significant (P=0.009). Histology-specific analysis using Lauren classification revealed that the proportion of EBV-GCs was not different in intestinal- and diffuse-type carcinomas (13% in both types). When the classification scheme of the Japanese Research Society for Gastric Cancer was used, EBV-GCs were identified more frequently in moderately differentiated tubular adenocarcinoma, and solid poorly differentiated adenocarcinoma when compared to other histological types. No lymphoepitelioma-like histology was found in the present series. The frequency of EBV-GC was slightly higher in advanced tumors, which involved serosa. Further analysis of clinico-pathological features of EBV-GC using a larger number of cases would give invaluable insights into its etiology.

Prognostic significance of Epstein-Barr virus involvement in gastric carcinoma in Japan.

We examined the prognosis of 64 EBV-associated gastric carcinoma (EBV-GC) cases and 128 EBV-negative gastric carcinoma cases. EBV-GCs were identified by detecting EBV-encoded small RNA (EBER) using in situ hybridization assay of paraffin-embedded tissue. For each EBV-GC case, 2 EBER-negative cases (EBV-negative cases) were selected, matching the EBV-GC case with respect to age, sex, tumor location, and depth of invasion. The average follow-up period was 70.9 months (SD=61.1) in EBV-GCs and 63.8 months (SD=59.7) in EBV-negative cases. Tumor-advanced stage determined by TNM classification of UICC, tumor location, and p53 over-expression were statistically significant prognostic factors. On the other hand, EBER expression was not related to the survival of patients. However, further analysis specific for intestinal and diffuse types of Lauren classification revealed that the association of EBER expression with prognosis was different in the two histological types. EBER expression was related to poor prognosis in intestinal-type carcinoma [hazard ratio (HR) =2.5, 95% confidence intervals (CI) =1.3-4.8] after adjusting for stage, p53 over-expression, and tumor location, whereas the diffuse-type EBV-GC had better prognosis (HR=0.4, 95% CI=0.2-0.9) even when lymphoepithelioma-like carcinomas were excluded. To examine the interactive prognostic effects between EBER expression and p53 over-expression, the study subjects were divided into 4 groups on the basis of EBER expression and p53 over-expression. In intestinal-type tumors, the cases having both EBER expression and p53 over-expression showed the poorest prognosis (HR=10.0, 95% CI=3.3-30.4), and the cases with either EBER expression or p53 over-expression had an intermediate prognosis. In diffuse-type tumor, only EBER was an important prognostic factor. These results give additional evidence implicating EBV in the natural history of EBV-GCs.

The Epstein-Barr virus-associated gastric carcinoma in Southern and Northern China.

In the present study, we examined the proportions of Epstein-Barr virus-associated gastric carcinomas (EBV-GCs) in Guangzhou, southern China and Shenyang, northern China, two areas differing markedly in nasopharyngeal carcinoma (NPC) incidence. Using in situ hybridization assay, the presence of EBV-encoded small RNA (EBER) was examined in 198, and 180 gastric cancer cases in Guangzhou and Shenyang, respectively. The proportion of EBV-GC in Guangzhou (9%) was significantly higher than that in Shenyang (6%), and the odds ratio (OR) for Guangzhou, after adjusting for the effects of age, sex, and tumor subsite, was 2.7 (95% CI = 1.1-6.2) when Shenyang was taken as reference. There was a male predominance of EBV-GC, and the OR for male was 3.0 (95% CI = 1.2-7.3) when female was taken as reference. We observed a weak and negative age dependence in the proportion of EBV-GC (p-values for trend = 0.077). The EBV-GC was most commonly observed in the middle part of stomach in both series. The frequency of EBV-GCs was higher in cases with p53 overexpression than in cases without p53 expression (OR = 2.4, 95% CI = 1.0-5.8). Among p53-positive cases, the frequency of EBV-GC decreased as the proportion of p53-positive carcinoma cells increased (p for trend = 0.021). In conclusion, the present study suggested that the frequency of EBV-GC in Guangzhou, southern China, where NPC is the most common in the world, may be higher than that in other parts of China.

Epstein-Barr virus-associated gastric carcinoma in southern India: A comparison with a large-scale Japanese series.

Epidemiological and clinicopathological features of Epstein-Barr virus (EBV) associated gastric carcinoma was compared in India and Japan, two countries differing markedly in gastric cancer incidence. Using in situ hybridization assay, the presence of EBV-encoded small RNA (EBER) was examined in 215, and 2,011 gastric cancer cases in Kerala, India, and Japan, respectively. Ten cases (5%), all males, in the Indian series were EBER-positive. This frequency was similar to that in the Japanese series (6.2%). As was the case with Japanese series, the EBV-associated gastric carcinoma in the Indian series was observed most frequently in the middle part of the stomach (1 in antrum, 4 in middle part, 2 in cardia, and 3 unknown), and, histologically, the diffuse type Lauren's classification (8 cases) was more common than the intestinal type (2 cases). Virus subtyping by PCR-RFLP revealed that all of the 10 EBV strains isolated from the EBER-positive Indian cases were subtype A, and wild-type F for Bam HI F region. In Bam HI I region, 8 cases were type C and the remaining 2 cases were type D. In either series, there was no significant difference in the frequency of tumors with p53 overexpression between EBER-positive and -negative cases. However, the proportion of cells with p53 overexpression in EBER-negative tumors was significantly higher than that in EBER-positive tumors regardless of histological type in both series. In conclusion, the frequency and major clinicopathological features of EBV-associated gastric carcinoma in south India were similar to those observed in Japanese series although gastric cancer incidence in these two countries differs markedly.

Amplification and overexpression of c-met gene in Epstein-Barr virus-associated gastric carcinomas.

To reveal the role of oncogenes in Epstein-Barr virus (EBV)-positive gastric carcinomas, the amplification and overexpression of the c-met gene were examined by a competitive polymerase chain reaction and immunohistochemistry, respectively. The proportion of c-met amplification and overexpression in EBV-positive and -negative carcinomas did not differ significantly. The amplification and overexpression of the c-met gene in EBV-negative gastric carcinomas were significantly associated with upper location, deeper invasion and lymphatic invasion, while in EBV-positive gastric carcinomas a significant correlation with c-met activation was observed only in deeper invasion. However, none of the observed associations of c-met amplification or overexpression with clinicopathological features in the EBV-positive and -negative carcinomas differed significantly in their strength or direction. These results suggest that the amplification and overexpression of c-met gene do not play a different role in the progression and metastasis of EBV-positive and EBV-negative gastric carcinomas.