Real-time PCR assay for rapid detection of GSTM1 polymorphism in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC) is a common public health problem in Thailand. Glutathione S-transferase M1 gene deletion (GSTM1 null genotype) carriers have been reported to be at increased risk and therefore this parameter is a potential marker for screening of NPC high-risk individuals. However, the conventional polymerase chain reaction (C-PCR) assay commonly used for GSTM1 null genotype detection is not suitable for mass screening since it is inconvenient, time consuming and unsafe due to the use of a toxic chemical. Currently, real-time PCR (R-PCR) assay is recommended for quicker and safer detection of various genetic polymorphisms. The aim of this study was to develop a SYBR green I R-PCR assay combined with melting curve analysis for GSTM1 polymorphism detection in Thai NPC patients. The results were compared to those from the C-PCR assay using DNA samples from peripheral blood leukocytes of 120 Thai NPC patients. The frequencies of GSTM1 polymorphism detected by the R-PCR and the C-PCR were the same. Forty-eight individuals that were GSTM1+ in the R-PCR assay showed 2 peaks with melting points of 82.5 and 87.5 that correlated with the appearance of 2 DNA bands in the C-PCR assay (i.e., one for GSTM1 at 215 base pairs (bp) and one for ?-globin at 268 bp). By contrast, 72 individuals that were GSTM1?- in the R-PCR assay showed 1 peak with a melting point of 87.5C that correlated with the appearance of 1 DNA band for -globin at 268 bp in the C-PCR assay. The R-PCR assay using SYBR Green I and melting curve analysis for GSTM1 polymorphism detection was as reliable as C-PCR assay but was quicker and safer and more amenable to large scale screening in Thai NPC cases.

Detection of GSTM1 polymorphism in patient with nasopharyngeal carcinoma by real-time PCR.

Objective: To investigate whether the real-time polymerase chain reaction (R-PCR) assay with SYBR green I and melting curve analysis could be used for glutathione S-transferase M1 gene (GSTM1) polymorphism detection in Thai nasopharyngeal carcinoma (NPC) patients by comparing the results of this assay with the conventional PCR (C-PCR) assay. Methods: DNA samples from peripheral blood leukocytes of 60 Thai NPC patients were investigated in this study. GSTM1 polymorphism [GSTM1 normal genotype (GSTM1+) and GSTM1 null genotype (GSTM1-)] were examined by using the R-PCR assay with SYBR green I and melting curve analysis and the C-PCR assay. Results: The results of GSTM1 polymorphism detection by the R-PCR assay were in concordance with the C-PCR assay (k = 1.0). Twenty-six individuals with GSTM1+ in the R-PCR assay showed 2 peaks of melting point at 82.5oC and 87.5oC that correlated with the appearance of 2 DNA bands of GSTM1 [215 base pair (bp)] and b-globin (268 bp) in the C-PCR assay, respectively. In addition, thirty-four individuals with GSTM1- in the R-PCR assay showed only 1 peak of melting point at 87.5oC that correlated with the appearance of 1 DNA band of b-globin (268 bp) in the C-PCR assay. Moreover, we found that the R-PCR assay was a faster and safer method for detection of GSTM1 polymorphism than the C-PCR assay. Conclusion: The present study suggests that the R-PCR assay with SYBR Green I and melting curve analysis may be a useful screening tool for more convenient, rapid, reliable, and safer detection of GSTM1 polymorphism in Thai NPC as compared to the C-PCR assay.

Selected risk factors, human papillomavirus infection and the p53 codon 72 polymorphism in patients with squamous intraepithelial lesions in northeastern Thailand.

Risk factors for cervical squamous intraepithelial lesions (SIL) including human papillomavirus (HPV) infection and the p53 codon 72 polymorphism were investigated in a case-control study with 103 cases and 105 controls in Northeastern Thailand. Increased risk for SIL was observed for age at menarche (odds ratio (OR) = 2.2; p< 0.005), age at the first sexual intercourse (OR=2.4; p< 0.05), number of sexual partners (OR=2.7; p< 0.005) and partners' smoking history (OR=2.3-3.2; p< 0.01). Prevalence of malignant type of HPV infection in the control and SIL groups was 18.1% and 60.2%, respectively. HPV infection significantly increased risk for SIL 6.8-fold (p< 0.001). HPV-16 infection was the commonest (31 out of 62 carriers) in SIL patients and highly associated with risk. The p53 codon 72 polymorphism was not identified as a genetic risk for SIL in this study, as demonstrated in Thai cervical cancer. Therefore, to prevent cervical neoplasia or HPV infection, inclusion of knowledge on sexual behavior and effects of smoking into public health programs is important and, at the same time, a nation-wide screening scheme for cervical abnormalities including HPV-typing is a high priority in Thailand.

Glutathione S-transferase M1 gene polymorphism in Thai nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a serious health problem in Thailand. It is caused by the combined effects of Epstein-Barr virus (EBV), carcinogens and genetic susceptibility. The glutathione S-transferase M1 gene (GSTM1) encodes a phase II enzyme responsible for detoxifying carcinogenic electrophiles. Polymorphic null forms of the gene GSTM1 lack enzyme activity and have been associated with susceptibility to several cancers including NPC. To examine the association between GSTM1 polymorphism and NPC susceptibility in Thais, GSTM1 genotypes (normal and null genotypes) in 78 NPC patients and 145 age-matched healthy controls were determined using PCR assays. Overall, no statistically significant differences were observed in the frequency of GSTM1 genotypes between cases and controls, nor among NPC patients compared on the basis of sex and clinical stage of disease. Carriers with the GSTM1 null genotype had a 2.9-fold increased risk for NPC of WHO type III when compared to those with GSTM1 normal genotype (P < 0.05 with OR =2.9, 95% CI = 1.2-6.8). When cases and controls were categorized into 3 age groups (>40, (>45 and (>50 years), the frequencies of GSTM1 null genotype in cases the (>45 and (>50 age groups were significantly different from controls (P< 0.05). In addition, carriers of the GSTM1 null genotype in age groups (>45 and (>50 years had a 2-fold and 3-fold increased risk for NPC when compared to those with GSTM1 normal genotype (OR = 2.2, 95% CI = 1.1-4.7 and OR = 3.0, 95% CI = 1.2-7.5). We suggest that GSTM1 polymorphism may be associated with NPC susceptibility in Thais, especially for GSTM1 null genotype carriers of age higher than 45 years. The GSTM1 null genotype may be a useful genetic marker for predicting Thai NPC and for screening of early stages of Thai NPC.

Co-risk factors for HPV infection in Northeastern Thai women with cervical carcinoma.

HPV infection is the main cause of cervical cancer; however, factors that promote and maintain HPV infection are still unclear. This study was designed to search for factors responsible for the HPV infection in Northeastern Thai women. A total of 190 volunteers with a normal histopathologic appearance of cervix as controls (n=100) and with squamous cell cervical carcinoma (SCCA) (n=90) were the subjects. Variables of risk factors including sexual behaviors, history of reproduction, history of sexually transmitted diseases and smoking were conducted with self-report and direct interview. Number of sexual partners and smoking history increased the likelihood of high-risk HPV infection. Multiple sexual partners showed significantly higher 3.94-fold risk for HPV infection (95% CI = 1.82-8.82, p-value<0.001). Smoking history of partner increased the risk for HPV infection 3.03-fold (95%CI=1.42-6.58, p-value< 0.002). After OR were adjusted, significant difference was still observed in the number of sexual partners (p-value <0.0001) and smoking history of the partner (p-value<0.005). To decrease the incidence of cervical cancer, we should prevent HPV dissemination and be on the alert for having multiple sexual partners and a partner's smoking habit, which must be included in our public health planning.

Epstein-Barr virus-associated nodal malignant lymphoma in Thailand.

Specific subtypes of malignant lymphoma are highly associated with Epstein-Barr virus (EBV) infection. In the present study, the authors evaluated EBV-encoded RNA (EBER) expression by in situ hybridization in 300 cases of malignant lymphomas diagnosed by lymph node biopsy, with 100 cases of reactive lymphoid hyperplasia in lymph nodes as controls, for comparison. There were 100 consecutive cases of classical Hodgkin's lymphoma (cHL), 100 consecutive cases of non-Hodgkin's lymphoma, B cell (NHL-B), and 100 consecutive cases of non-Hodgkin's lymphoma, T cell (NHL-T). EBER expression was detected in 46% of reactive lymphoid hyperplasia cases, but the positively stained cells in those cases constituted less than 5 percent of the total cell populations. When using the presence of EBER in 5 percent or more of the cell population and/or the presence of EBER in the Hodgkin's Reed-Sternberg's cells as indicators of positivity, 64% of cHL, 13% of NHL-B, and 51% of NHL-T were found to be positive. The study indicates a strong association of cHL and NHL-T with EBV infection, the link apparently being weaker for NHL-B except for the subtypes of Burkitt's lymphoma and diffuse large B cell lymphoma.

Epstein-Barr virus-associated peripheral T-cell lymphoma with gastrointestinal tract involvement.

Peripheral T-cell lymphoma (PTCL) is a group of diseases which are common in Asia and areas of South and Central America. They are highly associated with the Epstein-Barr virus (EBV) infection. In the present study the authors evaluated patients with gastrointestinal involvement of PTCL with respect to clinical findings and outcome, pathologic features, and molecular analysis for EBV infection and the clonality of tumor cells. From January 1997 through December 2000, 7 patients with gastrointestinal tract involvement of PTCL were identified. The frequency of gastrointestinal tract involvement in the various types of PTCL was 5.4 per cent (7 of 129 cases). The pertinent clinical features were prolonged fever, weight loss, anemia, hepatosplenomegaly, lymphadenopathy, multiorgan involvement, and gastrointestinal bleeding. Laboratory results showed a significantly high serum level of alkaline phosphatase and lactate dehydrogenase, and abnormal coagulograms. Five patients died within 4 months after onset of illness, while two were in complete remission after chemotherapy. The tumor cell morphology was classified into three categories: small-sized cells, mixed medium- and large-sized cells, and large-sized cells. The antigenic phenotypes of the tumor cells were LCA+, CD3+, CD15-, CD16-, CD30-, CD45R0+, CD57-, CD68-, EMA-, betaF1-, granzyme B+, TIA-1+, and p53+. The expression of CD4, CD8, CD56 and CD20 was variable. EBV-RNA expression by in situ hybridization (EBER-ISH) study was positive and T-cell receptor (TCR) beta and/or gamma gene rearrangements were detected in all patients. DNA sequence analysis showed high identity to the human TCR germline gene. PTCL with gastrointestinal tract involvement was associated with EBV infection. The tumor cells were mature T cells with some NK-cell antigenic expression and all demonstrated TCR gene rearrangements.

The p53 codon 72 polymorphism and risk of oral cancer in Southern Thailand.

The codon 72 polymorphism of the p53 tumor suppressor gene has been investigated extensively for its association with various cancers around the world. However, its influence has not been elucidated in the Thai population. Therefore, a case-control study with 97 patients and 97 matched controls was conducted to elucidate the association between the polymorphic p53 and oral cancer risk in a Southern Thai population. The frequencies of the Arg/Arg, Arg/Pro, and Pro/Pro genotypes were 36%, 35%, and 29%, respectively in the controls and 33%, 45% and 22%, respectively in the patients. This study shows that there was no significant association between the p53 codon 72 polymorphism and oral cancer risk. There was also no link with respect to smoking or drinking habits. However, our data suggest that for individuals who were younger than 65 years old, the Pro/Pro genotype may offer some protection against oral cancer (OR = 0.13, 95%CI 0.04-1.10). This is the first report on p53 polymorphism and oral cancer in Thailand.

Significance of plasma IgA and IgG antibodies to Epstein-Barr virus early and viral capsid antigens in Thai nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is an important causal factor of human nasopharyngeal carcinoma (NPC). High levels of serum IgA and IgG antibodies to EBV early and viral capsid antigens (IgA/EA, IgA/VCA, IgG/EA and IgG/VCA) have been reported in NPC patients. Since specific serum IgA/EA, IgA/VCA and IgG/EA are claimed to be useful serological markers for NPC. In order to evaluate whether plasma IgA/EA, IgA/VCA, IgG/EA and IgG/VCA antibody levels are useful markers for diagnosis and prognosis of Thai NPC, we examined the prevalence of these antibodies in 79 NPC patients, and 127 age-matched controls (47 healthy subjects (HS), 32 cases of other disease (OD) and 48 cases of other cancer (OC)) by using an indirect immunofluorescence assay. The prevalence of plasma IgA/EA, IgA/VCA, and IgG/EA in NPC patients (55.7, 68.4 and 68.4%) was significantly higher than in the HS (0.0, 0.0 and 20.5%,), OD (0.0, 0.0 and 3.1%) and OC (0.0, 0.0 and 20.8%) groups (p<0.05). The prevalence of plasma IgG/VCA in NPC patients (93.7%) was significantly different from those for the OD and OC groups (71.9 and 43.8%) but not for the HS group (89.4%). In NPC patients, the geometric mean titers (GMT) of plasma IgA/EA, IgA/VCA and IgG/EA were increased with an advanced clinical stage of disease but not IgG/VCA. In contrast, GMT of IgG/VCA was increased with aggressive type of disease (histological type) but not IgA/EA, IgA/VCA, and IgG/VCA. The results of our study suggest that plasma IgA/EA, IgA/VCA and IgG/EA antibodies may be useful markers for diagnosis and assessing prognosis of Thai NPC.

Epstein-barr virus-associated non-Hodgkin's lymphoma of B-cell origin, Hodgkin's disease, acute leukemia, and systemic lupus erythematosus: a serologic and molecular analysis.

Parallel studies of (a) patients with Epstein-Barr virus (EBV)-associated peripheral T-cell proliferative disease/lymphomas and (b) a group of patients with a prolonged fever from other causes were conducted at Songklanagarind University Hospital from 1997 through 2000. (Reports on EBV-associated peripheral T-cell and NK-cell proliferative disease/lymphomas have been published elsewhere) In this study, the authors identified 58 patients; 14 were non-Hodgkin's lymphoma of B-cell origin (NHL-B), 8 were Hodgkin's disease, 6 were acute leukemia, 9 were systemic lupus erythematosus (SLE), and 21 were patients with other diseases. Serologic tests for the EBV infection, the study of EBV genome in circulating non-T-cells (CD3-cells) and T-cells (CD3+ cells), and the EBV-RNA study in the tumor cells were performed. EBV internal repeat-1 region (IR-1) in peripheral blood CD3+ cells was detected in 10 of 14 patients (71.5%) with NHL-B, 3 of 8 patients (37.5%) with Hodgkin's disease, 1 of 6 patients (16.7%) with acute leukemia, 4 of 9 patients (44.5%) with SLE, and was not detected in any of the 21 patients with other diseases. Anti-viral capsid antigen-IgG was significantly elevated in hematologic malignancy patients with EBV IR-1 genome in the peripheral blood CD3+ cells when compared to hematologic malignancy patients with a negative result, whereas there was no significant difference in anti-EBV nuclear antigen among these two groups. EBV-RNA expression in tumor cells by in situ hybridization was detected in 4 of 13 patients (31%) with NHL-B (all showed EBV IR-1 genome in peripheral blood CD3+ cells), and 3 of 5 patients (60%) with Hodgkin's disease (only two showed EBV IR-1 genome in peripheral blood CD3+ cells). These data support the theory that chronic EBV infection is often found in association with cases of NHL-B, Hodgkin's disease, acute leukemia, and SLE.

Malaria species and Southeast Asian ovalocytosis defined by a 27-bp deletion in the erythrocyte band 3 gene.

To evaluate the resistance of SAO against species specific malaria infection, relationships between parasite species and the 27-bp deletion in the band 3 gene were studied in malaria endemic Sumba Island, eastern Indonesia. Thick blood films were prepared from patients with malaria symptoms (n=129) and healthy controls (n=231). Species of Plasmodium was identified by microscopic observation. The 27-bp deletion was screened by the PCR method. Among 231 healthy controls, 29 (12.6%) had the 27-bp deletion, whereas 14 (10.9%) among 129 patients confirmed with malaria infection harbored the 27-bp deletion. No significant difference was observed in the prevalence of the 27-bp deletion between controls and patients (p>0.8). There was no significant difference in the frequency of the 27-bp deletion between P. vivax and P. falciparum infected subjects at 5% level by Fisher's exact test. The present result showing no correlation between the presence of the 27-bp deletion and infected parasite species is consistent with the post-invasion resistance hypothesis that may involve not a single malaria species.