Yoshito Eizuru

Surgical Maneuvers Enhance Molecular Detection of Circulating Tumor Cells During Gastric Cancer Surgery

ObjectiveTo 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 DataSeveral 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. MethodsBlood 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. ResultsCEA-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. ConclusionsA 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.

Epstein-Barr virus associated gastric carcinoma: Epidemiological and clinicopathological features

In this paper, the roles of Epstein‐Barr virus (EBV) in gastric carcinogenesis are discussed, reviewing mainly epidemiological and clinicopathological studies. About 10% of gastric carcinomas harbor clonal EBV. LMP1, an important EBV oncoprotein, is only rarely expressed in EBV‐associated gastric carcinoma (EBV‐GC) while EBV‐encoded small RNA is expressed in almost every EBV‐GC cell, suggesting its importance for developing and maintaining this carcinoma. In addition, the hypermethylation‐driven suppressor gene downregulation, frequently observed in EBV‐GC, appears to give a selective advantage for carcinoma cells. EBV reactivation is suspected to precede EBV‐GC development since antibodies against EBV‐related antigens, including EBV capsid antigen (VCA), are elevated in prediagnostic sera. Interestingly, the average anti‐VCA immunoglobulin G antibody titer in EBV‐GC patients was significantly higher among men than among women, whereas EBV‐negative GC cases did not show such a sex difference. A higher frequency of human leucocyte antigen‐DR11 in EBV‐GCs suggests that major histocompatibility complex‐restricted EBV nuclear antigen 1 epitope recognition may enhance EBV reactivation. EBV infection of gastric cells by lymphocytes with reactivated EBV is suspected to be the first step of EBV‐GC development. Male predominance of EBV‐GC suggests the involvement of lifestyles and occupational factors common among men. The predominance of EBV with XhoI+ and BamHI type i polymorphisms in EBV‐GC in Latin America suggests a possibility of some EBV oncogene expressions being affected by EBV polymorphism. The lack of such predominance in Asian countries, however, indicates an interaction between EBV polymorphism and the host response. In conclusion, further studies are necessary to examine the interaction between EBV infection, its polymorphisms, environmental factors, and genetic backgrounds. (Cancer Sci 2008; 99: 195 –201)

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 104 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.

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.

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.

Detection and Clinical Significance of Lymph Node Micrometastasis Determined by Reverse Transcription-Polymerase Chain Reaction in Patients with Esophageal Carcinoma

We investigated micrometastasis in lymph nodes by detecting carcinoembryonic antigen (CEA) mRNA. A total of 400 lymph nodes obtained from 21 patients with esophageal carcinoma were examined by CEA-specific reverse transcription-polymerase chain reaction (RT-PCR). Serial sections of positive lymph nodes were reexamined histologically and immunohistologically. Twenty-seven lymph nodes of 11 patients were diagnosed as being positive by conventional histologic examination. CEA-mRNA positivity was found in 18 of 21 patients. Among 373 histologically negative nodes, 79 (21.2%) were positive for CEA mRNA. Of these, micrometastasis was detected in 2 by histological reexamination and in 11 by immunohistochemical staining using cytokeratin antibody. Two of 6 RT-PCR-positive patients (33.3%) had recurrent disease. Four of 11 patients (36.4%) whose nodal involvement was discovered by routine histological examination also had recurrent cancer. CEA-specific RT-PCR detected micrometastasis in lymph nodes at a higher rate than histological or immunohistochemical analysis of serial sections. Since the incidence of CEA-mRNA positivity is high in the lymph nodes of esophageal cancer patients except for those with early cancer, these patients should be treated with adjuvant therapy.

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.

Molecular epidemiology of Epstein‐Barr virus (EBV) in EBV‐related malignancies

The prevalent strain of Epstein‐Barr virus (EBV) in EBV‐related malignancies and in healthy adults in Southern Japan was examined by means of polymerase chain reaction (PCR) and/or restriction fragment length polymorphism (RFLP) analysis. In EBV‐related gastric cancers, 51/73 cases were subtype A, 4 were subtype B and the EBNA‐2 region was not amplified in 18 cases. Sixty‐three were wild‐type F, and only one was variant “f”. Sixty‐one cases had type C and 2 type D. EBNA‐2 subtype A was found in 10/12 EBV‐related T/NK‐cell lymphomas, and 11 samples harbored the wild‐type F. Neither subtype B nor the “f” variant was detected. Type C EBV was found in 8 cases and type D in 3 specimens. Two Japanese nasopharyngeal carcinomas (NPC) harbored subtype A with wild‐type F and type C. Throat washings from healthy adults harbored wild‐type F virus in 60/153 cases, and 25 of these samples were EBNA‐2 subtype A. Type C viruses were detected in 92% of cases and type D in 7.4%. Therefore, the prevalent strain in EBV‐related malignancies in Southern Japan was the same as in the healthy population in this geographical region. Int. J. Cancer 72:72–76, 1997.

The comparison of the prognosis between Epstein-Barr virus (EBV)-positive gastric carcinomas and EBV-negative ones.

The relationship between the degree of lymphocytic infiltration into the tumor and the prognosis has not been completely evaluated between Epstein-Barr virus (EBV)-positive and -negative gastric carcinoma (GC). Although the average numbers and the grades of the infiltrating CD8+T cells, natural killer cells, dendritic cells, Ki67-positive cells were significantly greater in EBV-positive GCs than in -negative GCs, there was no significant survival improvement in EBV-positive group. These findings suggest that the infiltration of lymphocytes in the EBV-positive GC does not necessarily meant better prognosis and that the EBV status is not a significant prognostic factor in the patients with gastric cancer.

Association of a distinctive strain of Epstein‐Barr virus with gastric cancer

Epstein‐Barr virus (EBV) has been linked to gastric carcinoma (GC) with worldwide geographical variations attributable to types and variants of EBV. Here, we compare EBV strains between EBVaGC and healthy donors in Latin America, a high frequency area for EBVaGC. Tumor samples from 73 EBVaGC cases and throat washings from 329 healthy adults were examined for types 1 and 2 EBV and polymorphism at BamHI‐F and BamHI‐W1/I1 boundary regions and XhoI restriction site in LMP1 gene. Type 1 and prototype F of BamHI‐ F polymorphism accounted 59 (81%) and 69 (95%) of EBVaGC cases and 257 (78%) and 267 (81%) of healthy donors, respectively. Types I and “i” of BamHI W1/I1 polymorphism accounted 2 (3%) and 62 (85%) of EBVaGC and 85 (26%) and 170 (52%) of healthy donors, respectively (p<0.001). XhoI+ and − polymorphism accounted 60 (82%) and 4 (5%) of EBVaGC and 142 (43%) and 92 (28%) of healthy donors, respectively (p<0.001). Cosegregation analysis demonstrated that most of the 62 type “i” EBVaGC cases harbor XhoI+ strain (81%). However, among 143 type “i” healthy adults, both XhoI polymorphism were present in relatively similar frequencies (XhoI+ 58% and XhoI− 42%) (OR 9.0; 95% CI 1.2–69). Our findings are against to the proposed hypothesis that EBV strains are geographically but not disease‐restricted. We conclude that most of the EBVaGC cases harbor a distinctive EBV strain (type “i”/XhoI +), but in healthy donors, this strain was as common as other strains. This finding is contrary to the proposed hypothesis that EBV strains are geographically but not disease‐restricted and identified a healthy population group that share the same strain that predominate in EBVaGC cases.