Thai Cancer Journal

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    Animal Model for Using Allogeneic CD3/CD28 Cross-Linked Th1 Memory Cells Showing a Potent Active Immunotherapy of Leukemia/Lymphoma
    (Thai Cancer Journal, 2010-04-07) Wirote Lausoontornsiri
    We hypothesized that allogeneic CD3/CD28 cross-linked Th1 memory cells (allogeneic Th1 cells) would provide a therapeutic effect which would create systemic anti-tumor immunity and break tolerance to self antigens of the tumor. In the experiment, nude mice were inoculated with BCL1 leukemia into both hips. The mice were divided into 4 groups : control, ablation left side tumor alone, ablation and injection with allogeneic Th1 cell and injection allgeneic Th1 cell alone groups. Forty percent of mice treated with the combination therapy survived without evidence of tumor. The contralateral tumor area is displayed to separately show the response of the 40% mice that survived and the 60% that eventually succumbed to disease. Growth of the contralateral tumors was significantly suppressed (p \< 0.01) in the 60% of mice that eventually succumbed to disease. In this model, mice treated with intratumoral allogeneic Th1 cells alone survived a mean of 28 days, which was significantly longer than control mice which survived a mean of 19 days (p \< 0.0001; hazard ratio=4.291). Forty percent of mice treated with cryo+Th1 survived \> 90 days. Our results indicate that the ‘danger signals’ and immunimodulatory effects of these allogeneic Th1 cells are capable of eliciting the therapeutic anti-leukemia effects in the BCL1 tumor model. This is an evidence of allogeneic CD3/CD28 crosslinked Th1 cell after processing necrotic bodies in-situ, that is able to provide an effect of immunotherapy in this animal model. The findings will be a strong warrant to proceed into the phase1/2 clinical trial in the future.
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    Assessment of the Relationship Between RNA Integrity and Post-Chip QC Variables of Affymetrix GeneChip Human Genome U133 Plus 2.0 Array Experiments
    (Thai Cancer Journal, 2010-04-07) Anchalee Tantiwetrueangdet; Ravat Panvichian; Sansanee Wongwaisayawan; Koset Pinpradap; Surasak Leelaudomlipi
    RNA purity and integrity are important factors for reliability and reproducibility of downstream applications, especially for microarray analysis. However, it is difficult in practice to obtain perfect RNA from cancerous tissues and thus microarray experiments of gene expression are very expensive. So, it would be very useful to be able to predict, based on their RNA integrity, which RNA samples should be proceeded and will give interpretable results after hybridization. To assess the relationship between the RNA integrity and post-chip quality control variables of Affymetrix GeneChip Human Genome U133 Plus 2.0 Array experiments, total cellular RNA was isolate from 13 snap-frozen tissues including 7 breast tissues (2 tissues of normal breast, 2 tissues of primary breast cancer without metastasis and 3 tissues of primary breast cancer with metastasis) and 6 axillary lymph node tissues (3 tissues of normal axillary lymph node and 3 tissues of metastatic axillary lymph node). All samples were verified and confirmed by pathologist. RNA samples with RNA integrity number (RIN) between 5.5 and 7.9 were selected as starting material for conducting 13 chips of Affmetrix GeneChip Human U133 Plus 2.0 Array. Pearson correlation test was used to determine the correlation between RIN and post-chip quality control variables of Affymetrix GeneChip Human Genome U133 Plus 2.0 Array experiments. The assessed variables include the followings: background, noise (RawQ), scaling factor, percentage of present genes (%P), as well as 3’/5’ ratio of GAPDH (Sig3’/5’). We found that post-chip quality control variables after hybridizing RNA samples with RIN 5.8-7.9 to Affmetrix GeneChip Human Genome U133 Plus 2.0 Array were acceptable. We also observed a significant negative correlation between RIN and scale factor (r =-0.593, p=0.033, n=13) but no correlation between RIN and others variables. Based on this study, our data suggested that RNA samples with RIN not lower than 5.8 could be processed to conduct AFFmetrix GeneChip Human Genome U133 Plus 2.0 Array experiments.
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    Association between GSTM1 Gene Polymorphism and Oral Cavity Cancer in Suratthani Province
    (Thai Cancer Journal, 2010-04-07) Danai Tiwawech; Somjin Chindavijak; Sudjit Tongnun; Takafumi Ishida
    Oral cavity cancer (OCC) is a serious malignant disease in Thailand, particularly in the South, including Suratthani province, with a trend to increase in the number of its death yearly. However, patients with early stages of OCC are treatable. Hence, a mass screening for early stages of OCC patients without cancer-related symptoms is urgent. GSTM1 gene (GSTM1) that produced enzyme for detoxification of carcinogens has been reported to be polymorphic and associated with the risk of several cancer developments. The purpose of this study is to investigate the association between GSTM1 polymorphisms and the risk of OCC development in Suratthani province population. The frequency of GSTM1 genotypes [GSTM1 normal (GSTM1+) and GSTM1 null genotype (GSTM1-)] was detected in DNA extracted from peripheral white blood cells of 200 cases of OCC patients and 200 healthy controls using the polymerase chain reaction (PCR) assay. Overall, the frequency of GSTM1 genotypes between OCC and healthy control groups was significantly different (P \< 0.001). Individuals with GSTM1- had increased risk about 1.95-fold for OCC development as compared with those GSTM1+ [Odds ratio (OR) = 1.95, 95% confidence interval = 1.31-2.90]. In addition, GSTM1 polymorphism was found to be associated with an increased risk of OCC development in cigarette smokers, alcohol drinkers, and betel-nut chewers. In conclusion, the findings of this study suggest that GSTM1 polymorphism is associated with the risk of OCC development and the GSTM1- is the key factor for increasing the risk of OCC. Therefore, the detection of GSTM1 polymorphism is a useful tool in screening for the high-risk group that may be lead to identification of early stages of OCC in population who reside in Suratthani province.
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    Association Between GSTO1 Polymorphism and Clinicopathological Features of Patients with Breast Caner
    (Thai Cancer Journal, 2010-04-07) Wichai Purisa; Sunanta Chariyalertsak; Suleeporn Sangrajrang
    To our knowledge, only two reports concerning association between GSTO1 polymorphism and breast cancer risk have been revealed, but no association between GSTO1 polymorphism and clinicopathological features of patients with breast cancer have previously been reported. Therefore, in this study, the association of GSTO1 genotypes with a number of clinical parameter was investigated in 101 patients with breast cancer diagnosed and treated at the National Cancer Institute. DNA extracted from buffy coats of the cases was used to determine the genotypes by polymerase chain reaction-restriction-fragment length polymorphism. Our findings revealed that wild-type GSTO1 gene (A140/A140) significantly correlated with advanced-stage breast cancer (P=0.008). Form the literature, GSTO1 appears to be involved in drug and xenobiotic metabolism, it would be of great interest to investigate further whether resistance to radiation or chemotherapy occurs in carriers of wild-type GSTO1 gene, particularly those with advanced-stage breast cancer. Elucidating this relationship should lead to the improved clinical management of these patients.
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    Association between p53 codon 72 and GSTM1 polymorphisms and risk of hepatocellular carcinoma
    (Thai Cancer Journal, 2010-04-07) Danai Tiwawech; Wutthi Sumetchotimaytha; Aree Prastthipayong; Yaninee Jarratwisarutporn; Patcharin Homchaem; Takafumi Ishida
    Hepatocellular carcinoma (HCC) is a serious public health problem that caused vastly losses in Thailand annually. Recently, P53 codon 72 and GSTM1 polymorphisms have been found to associate with several cancers. The aim of this study is to investigate the association between P53 codon 72 and GSTM1 polymorphisms and risk of HCC development in Thais. The frequencies of P53 codon 72 genotypes (Arg/Arg, Arg/Pro and Pro/Pro genotypes) and GSTM1 genotypes (GSTM1+) and (GSTM1-) from 200 cases of HCC as well as 400 age-matched healthy control were analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR, respectively. In overall, the frequencies of P53 codon 72 polymorphism in HCC group were significantly different from those of healthy control. The Pro/Pro carriers had increased risk for HCC at 1.9-fold as compared to Arg/Arg carriers (OR=1.9; 95%CI=1.2-3.3). Similarly, the overall of frequencies of GSTM1 genotypes of GSTM1 polymorphism in HCC group were significantly different from those of healthy control. In addition, GSTM1- carriers had increased risk for HCC at 1.8-fold as compared to GSTM1+ carriers (OR=1.8; 95%CI=1.3-2.5). The results of this study suggest that P53 codon 72 and GSTM1 polymorphisms are associated with the increased risk of HCC and may be a useful tool for predicting and searching the high risk group of HCC in Thai population.
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    Association of GSTO2 Polymorphism and Survival for Colorectal Cancer
    (Thai Cancer Journal, 2010-04-07) Sunanta Chariyalertsak; Wichi Purisa; Nareerat Petpiroon; Tanett Pongtheerat
    Since reports on GSTO2 polymorphism are still limited, therefore, this study was conducted to investigate an association of genetic polymorphism of GSTO2 and survival for colorectal cancer. Formalin-fixed, paraffin-embedded colorectal cancerous tissues from 26 patients who were continuously followed-up, were used to extract DNA and the gene polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism. Our findings revealed that the patients carried wild-type GSTO2 gene (N142/N142) had poorer survival rate, when compared with those carried variant GSTO2 gene (adjusted hazard ration, 14.02, P=0.08). No association of GSTO2 genotypes with overall survival was noted (P=0.142). However, this study is only a preliminary report of which the number of cases is small. Further study in a larger population is required to achieve high statistical power and consequently clarify this finding.
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    Breaking Bad News about Cancer at Buddhachinaraj Hospital: Patients' Perceptions
    (Thai Cancer Journal, 2011-01-24) Pongsakorn Lengdee; Ulan Wilekha; Niwat Sriwijarn; Sakorn Samdao; Nutcha Jaroenputtravut
    This qualitative study, which aimed to describe patients' perceptions related to breaking bad news about cancer, was undertaken in the Chemotherapy Ward of Buddhachinaraj Hospital. Key informants were recruited by purposive sampling; 16 patients participated in the study. Data were collected between April-September 2008, using in-depth interviews, observations, focus-group discussions, and field notes, then analyzed using content analysis. The patients' experiences, while waiting for the final diagnosis, focused on thinking about the possibility of having a serious disease, the need to know the final diagnosis, and preparing themselves for bad news. When patients received bad news, the information source was their doctor and other related persons. The process of breaking bad news was not completed in one session. The study participants felt that receiving step-by-step information from doctors helped them to cope with the disease. Doctors' behaviors also affected patients' levels of satisfaction. It was found that, after being told of the diagnosis, patients felt both good and bad about the bad news and could adjust themselves to it, each according to their capacity. They appreciated nurses' helping them cope. Regarding patient expectations of the process, patients felt that they needed their doctor to be the person who told them about the diagnosis, and they expected that the doctor would have a good technique for breaking bad news. In conclusion, even though breaking bad news was very hard for doctors, patients wanted their doctors to do it; then they would be able to cope with the bad news they received. They expected good communication from their doctors during this process. (Thai Cancer J 2009;29:143-151.)
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    Cervical Cancer Survival at the National Cancer Institute, Thailand
    (Thai Cancer Journal, 2011-02-03) Sukumarn Sanersak Swangvaree; Nithinai Tangprasert; Araya Adulbhan; Sujira Foongfaung; Siriporn Talacheep; Orawan Muangsamran; Daoroong Thepsuwan; Chariya Sanguansai; Kanokwan Tabtimsri; Pentipya Chaowalit; Porntip Boonpen; Paranee Ratanaphasura; Watchraporn Boonrung; Lumyong Krairittichai; Ousa Naksuwan
    This retrospective descriptive study assessed cervical-cancer patient survival at the National Cancer Institute (NCI) of Thailand. All patients with cervical cancer, admitted to the NCI during the period 1987-2005, were included in the study. A total of 318 patients who met the study criteria were selected. The mean age of the patients was 52.24 years. Most of the cervical cancers were FIGO stage IIB. Our findings revealed that the 5-year overall survival rate for patients who received treatment at the NCI was 64.9%. The 5-year survival rates were 88.6% for stage I, 63.7% for stage II, 41.2% for stage III, and 33.3% for stage IV. In conclusion, we found that the survival rates of cervical-cancer patients who received treatment at the NCI were comparable to, or better than, those in other Asian countries. (Thai Cancer J 2010;30:87-93)
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    Characterization and Antibiotic Susceptibility Profile of Nosocomial Pathogens Isolated from Cancer Patients
    (Thai Cancer Journal, 2011-01-24) Warayupa Thompat; Yuttana Sudjaroen
    Cancer patients are particularly susceptible to nosocomial infections because of their compromised immune systems and treatment regimens. This study analyzed the distribution and antimicrobial resistance of nosocomial pathogens (n=365) isolated from cancer patients admitted to the National Cancer Institute, Thailand during the period September 2007- October 2008. Nosocomial pathogens from cancer patients were isolated and their susceptibility analyzed accounting for microbial spectrum and antimicrobial resistance. The most prevalent sources of infection were non-ICU (56.6%) and ICU (31.8%). Most of the sites of the isolated nosocomial pathogens were the respiratory tract (27.8%) and skin (26%). The main Gram-negative bacteria causing nosocomial infections were Escherichia coli (19.9%), Klebsiella pneumoniae (15.0%), Pseudomonas aeruginosa (10.6%), and Enterobacter cloacae (7.4%); the main Gram-positive bacterium was Staphylococcus aureus (10%). The most isolated yeasts were Candida spp. (14.8%), mainly Candida albicans (50.6%). The extendedspectrum Beta-lactamase (ESBL)-producing bacteria were E. coli (29.9%), K. pneumoniae (15.1%) and Proteus mirabilis (8.3%). The prevalence of methicillin resistance was 18.4% for Staphylococcus aureus. Non-fermentative Gram-negative bacteria with multidrug-resistant properties were P. aeruginosa (48.6%), Acinetobacter baumannii (18.9%), and Stenotrophomonas maltophilia (5.4%). Most of the Gram-negative bacteria were resistant to the cephalosporins. Penicillin resistance was high among all bacteria. This study demonstrated the risk of nosocomial infection and the pathogens with drug-resistant profiles prevalent among cancer patients. These data may be useful in preventive clinical practice, and inform the choice of the most appropriate antibiotics for the treatment and prophylaxis of cancer patients. (Thai Cancer J 2009;29:176-183.)
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    Chemopreventive Potential of Rhinacanthus nasutus Kurz. On 7, 12-Dimethylbenz[a] anthracene (DMBA)-induced Mammary Carcinogenesis in Female Sprague-Dawley Rats
    (Thai Cancer Journal, 2010-04-07) Pongpun Siripong; Piengchai Kupradinun; Suratsawadee Piyaviriyakul; Sirirat Tunsakul; Rittichai Chanpai; Jantana Yahaufai
    The chemopreventive potential of Rhinacanthus nasutus Kurz. (family Acanthaceae), traditionally used in Thai folk medicine for cancer treatment, on 7, 12-Dimethylbenz[a] anthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague-Dawley rats was investigated. Seventy-five rats were randomly divided into seven groups consisting of one control group (15 animals) and six treated groups (10 animals for each group). The control group received 0.3% carboxymethylcellulose in normal saline and each treated group received the aqueous extracts of roots and stems (500 mg/kg body weight/day), chloroform extract and rhinacanthin C from roots (25 and 50 mg/kg body weight/day) intragastrically everyday two weeks prior to the administration of a single oral dose of DMBA (30 mg/kg body weight) in corn oil and continuously for 9 weeks. Rats were weighed and monitored twice weekly for tumor development after DMBA administration. At the end of experiment, all rats were sacrificed and the tumor volume, number of tumor per rat, tumor incidence, histopathological and blood biochemical parameters were evaluated. The results revealed that all extracts and rhinacanthin C isolated from R. nasutus Kurz. at the given doses had no affects in the growth rate between the treated and control groups. Oral administration of aqueous extracts of roots and stems (500 mg/kg/day) and chloroform extract (25 mg/kg/day) in tumor-bearing rats significantly suppressed the growth of mammary gland carcinogenesis (p \< 0.01). Moreover, rhinacanthin C (25 and 50 mg/kg/day) suppressed the tumor growth in dose-dependently (p \< 0.001), whereas the high dose of chloroform extract (50 mg/kg/day) had no effect when compared with the control group. The aqueous extract of roots (500 mg/kg/day), chloroform extract and rhinacanthin C (25 mg/kg/day) markedly reduced the number of tumors per rat at 4.2±0.5, 4.37±0.4 and 4.89±0.2, respectively whereas the aqueous extract of stems and high doses of chloroform extract and rhinacanthin C (50 mg/kg/day) had slightly effect when compared with the control group (7.25±0.8). Likewise, rats receiving the aqueous extract of roots (500 mg/kg/day), chloroform extract and rhinacanthin C (25 mg/kg/day) had lower tumor incidence (72.41%, 60.34% and 75.86%) than the control group (100%). For the biochemical analysis, no significant difference of the biochemical parameters was occurred in rats receiving both of aqueous extracts of roots and stems (500 mg/kg/day). In contrast, the levels of blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in rats receiving the high dose of chloroform extract (50 mg/kg/day) were markedly increased. Tumor-bearing rats treated with rhinacanthin C (25 mg/kg/day) also had increasing levels of AST and ALT, but with no significant difference when compared with the control group. Based on these available data, our findings first demonstrate that the aqueous extracts of roots and stems, chloroform extract and rhinacanthin C isolated from roots R. nasutus Kurz. markedly suppress tumor growth, number of tumor per rat and tumor incidence of the mammary gland carcinogenesis in the experimental rats induced by 7, 12-dimethylbenz[a] anthracene (DMBA). Interestingly, aqueous extracts of both roots and stems, traditionally usage forms for the treatment of various cancers, provided significantly effective results. Therefore, Rhinacanthus nasutus Kurz. may be a useful Thai medicinal plant for preventing the mammary carcinogenesis, however, prolonged use and overdoses of chloroform extract and rhinacanthin C should be avoided to prevent hepatoxic and nephrotoxic effects of the drugs.
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    Comparison of Aflatoxin-Albumin Adduct Levels in Sera of School Children from Bangkok and Rural Area
    (Thai Cancer Journal, 2010-04-07) Siripat Chutimataewin; Srisurang Jitchinakul
    Aflatoxin-albumin (AF-alb) adduct can be detected in urine, breast milk, serum and tissue of human. Correlation of aflatoxin B1 exposure with hepatocellular carcinoma (HCC) has been reported. In Thailand, the highest AF exposure was observed in Bangkok residents followed by Ubonrachthani, Chiangmai, Songkla and Nakornratchasima of which the AF levels were between 5-2510 mg AF-equivalent/ml. However, no report on AF-alb adducts in sera of school children has been revealed. Therefore, this study aimed to compare the level of AF-alb adducts in sera of school children from Bangkok and rural area. Sera of school children were collected from six schools : two in Bangkok, two in rural area and the other two in bordered area. The study group comprised 133 boys and 117 girls. Their age ranged from 9 to 16 years, and 125 children resided in Bangkok and the other 125 ones in rural and bordered area. The exposure of AF-alb adducts among the 250 school children was determined. No exposure of the AF-alb adducts was found in one child, but it was found in the other 249 children with levels between 1.2 and 59.0 pg/mg, averaged 18.8 pg/mg. When compared the AF-alb adduct levels between the two study groups, no statistical difference was demonstrated (P=0.068).
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    Comparison of Amount of Phenolic Compounds and Antioxidant Activity Between Green Tea Beverage and Tea Freshly Prepared from Green Tea Leaf
    (Thai Cancer Journal, 2011-02-11) Pornsri Patimanukasea; Yuttana Punya-Ngarm
    Free radical damage may lead to cancer. Green tea contains antioxidant phenolic compounds that interact and stabilize free radicals. Green tea is widely consumed around the world. Conventionally, tea has been prepared by brewing dried tea leaves with hot water. Due to their worldwide acceptance, ready-to-drink green-tea beverages with different concentrations of tea have been manufactured for consumer convenience. This study aimed to compare amount of phenolic compounds and antioxidant activity between green tea beverages and tea freshly prepared from dried green-tea leaf. Green-tea beverages without artificial flavoring (n=16) and dried green-tea leaf (n=5), available in Bangkok, were collected. Phenolic compounds and antioxidant activity were determined by Folin-Ciocalteu and Ferric Reducing Antioxidant Power methods. The differences in the amounts of phenolic compounds and antioxidant activity, between green tea beverages and tea freshly prepared from dried green-tea leaf, were analyzed by Mann-Whitney U Test using a 95% confidence limit. The correlation between the amount of phenolic compounds and antioxidant activity of green-tea beverages, and tea freshly prepared from dried green tea leaf, were determined by Spearman Rank Correlation Coefficient. It was found that the amount of phenolic compounds and antioxidant activity of tea freshly prepared from dried green tea leaf were significantly higher than the green tea beverages (p\<0.05). A positive correlation between the amount of phenolic compounds and antioxidant activity were found in both green tea beverages (r=0.845) and tea freshly prepared from dried green-tea leaf (r=0.975). (Thai Cancer J 2010;30:127-134)
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    Correlation between Serum AFP Level and HBsAg, Age and Sex in Subjects of the National Cancer Institute, Thailand
    (Thai Cancer Journal, 2010-04-07) Aree Prasitthipayong; Anant Karalak; Vichuda Triratapichat; Phongphan Romhirun
    Hepatocellular carcinoma (HCC) is one of the most common cancer in Thailand. Serum alpha-fetoprotien (AFP) is widely used for HCC screening and has been suggested as a tumor marker for monitoring and diagnosis of hepatocellular carcinoma (HCC) in high-risk population. In this study, we aimed to demonstrate the correlation between serum AFP level and HBsAg, age and sex in HCC and normal subjects of the National Cancer Institute, Thailand. The laboratory data from computerized records of the laboratory information system (LIS) were collected. Two groups of sample comprised 545 non-HCC subjects as control group (215 with viral hepatitis B infection and 330 without viral hepatitis B infection) and 61 patients with HCC. Elevation of serum AFP level (\> 20 ng/ml) was detected in 0.9% in controls and 62.3% in HCC patients. In addition, high serum AFP levels were found in 2.3% of controls who were infected with hepatitis B virus. Mean age was higher in controls who had high serum AFP levels, and male had higher serum AFP level than female. In HCC patients, no correlation of serum AFP level with HBsAg with age and sex was found. From our findings, 2.3% of the HBsAg carriers had elevation of AFP level, therefore, these carriers should be further investigated for early detection of HCC.
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    Detection of DNA methylation of p16INK4a in hepatocellular carcinoma by methylation specific-polymerase chain reaction
    (Thai Cancer Journal, 2010-04-07) Pensri Saelee; Songsak Petmitr; Sopit Wongkham; Sunanta Chariyalertsak; Patcharin Homcha-cm
    The purpose of this study is to investigate aberrant methylation of p16INK4a in hepatocellular carcinoma (HCC). We determined the methylation status of CpG island of p16INK4a in 29 HCC and corresponding normal liver tissues by methylation specific-PCR method. Aberrant methylation status was detected in 41.4% of tumors and 6.9% of corresponding normal liver tissues. No significant correlations between methylation status and clinico-pathological data were found. In addition, survival analysis by multivariate Cox regression analysis showed that aberrant methylation status of p16INK4a was not an independent prognostic factor for poor survival among HCC patients. Our findings demonstrated that methylation of p16INK4a were detectable in HCC tissues of Thai patients and suggested that hypermethylation of p16INK4a may contribute to the hepatocellular carcinogenesis.
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    Detection of GSTM1 and GSTT1 Polymorphisms in Nasopharyngeal Carcinoma Patients by Multiplex PCR Assay
    (Thai Cancer Journal, 2011-02-03) Danai Tiwawech; Somjin Chindavijak; Yaninee Jarratwisarutporn; Takafumi Ishida
    Nasopharyngeal carcinoma (NPC) is a common cancer and a serious public-health problem in Thailand. Glutathione S-transferase Mu1 and Theta1 gene (GSTM1 and GSTT1) are involved in the prevention of cancer, by encoding GSTM1 and GSTT1 enzymes to detoxify various electrophiles derived from environmental carcinogens. GSTM1 and GSTT1 polymorphisms are reportedly associated with several malignancies and can be used as a genetic risk marker for cancer. Nevertheless, GSTM1 and GSTT1 polymorphism detection using the conventional polymerase chain reaction (C-PCR) assay is complicated and time-consuming, and thus unsuitable for mass screening. A faster multiplex PCR (M-PCR) assay has been developed to help overcome these problems. The present study aimed to establish the M-PCR assay for GSTM1 and GSTT1 polymorphism detection in NPC patients, and confirm the results of the new assay with the C-PCR assay. Eighty DNA samples, extracted from the peripheral blood leukocytes of Thai NPC patients, were examined for GSTM1 and GSTT1 polymorphism [GSTM1 normal genotype (GSTM1+), GSTM1 null genotype (GSTM1-), GSTT1 normal genotype (GSTT1+), and GSTT1 null genotype (GSTT1-)] by M-PCR and C-PCR assays. The GSTM1 and GSTT1 polymorphism-detection results in all NPC cases by M-PCR assay agreed with the C-PCR assay ( = 1.0). In addition, the M-PCR assay was a simple, faster, and less costly method for GSTM1 and GSTT1 polymorphism detection than the C-PCR assay. The present study suggests that GSTM1 and GSTT1 polymorphism detection by M-PCR assay is a reliable and suitable tool for screening high-risk NPC groups. (Thai Cancer J 2010;30:94-103)
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    Detection of GSTT1 Polymorphisms in Cancer Patients by Real-Time PCR
    (Thai Cancer Journal, 2010-04-07) Danai Tiwawech; Somjin Chindavijak; Adisak Sornprom; Wutthi Sumetchotimaytha; Aree Prasitthipayong; Yaninee Jarratwisarutporn; Takufumi Ishida
    Cancer is a huge public health problem in Thailand. Glutathione S-transferase T1 gene (GSTT1) plays a crucial role in prevention of cancer by encoding GSTT1 enzyme to detoxify the electrophiles form of carcinogens. GSTT1 polymorphism has been reported to be associated with several malignancies and able to be used as a potential genetic risk marker for cancer. However, GSTT1 polymorphism detection using the conventional polymerase chain reaction (C-PCR) assay is not suitable for a mass screening since it is time consuming and not for safe since it uses a carcinogen in the post PCR. To date, real-time PCR (R-PCR) assay has been proposed as a quicker and safer method to solve these problems. This study aims to establish the R-PCR assay by using SYBR green I fluorescence and melting curve analysis for GSTT1 polymorphism detection in cancer patients by confirming the results of this assay with the results of the C-PCR assay. Two-hundred DNA samples, extracted from peripheral blood leukocyte of Thai patients with cancers of nasopharynx, lung, breast, colon and liver (40 cases in each group) were examined for GSTT1 polymorphism, GSTT1 normal genotype (GSTT1+) and GSTT1 null genotype (GSTT1-) by using the R-PCR assay with SYBR green I and melting curve analysis and the C-PCR assay. The results of GSTT1 polymorphism detection by the R-PCR assay were in concordance with the C-PCR assay (Kappa value, K=1.0). One hundred-forty-one individuals with GSTT1+ in the R-PCR assay showed 2 peaks of melting point at 91.0°C and 88.5°C that correlated with the appearance of 2 DNA bands of GSTT1 [480 base pair (bp)] and β-globulin (268 bp) in the C-PCR assay, respectively. By contrast, fifty-nine individuals with GSTT1- in the R-PCR assay showed a peak of melting point at 88.5°C that associated with the appearance of 1 DNA band of β-globulin (268 bp) in the C-PCR assay. In addition, it has been found that the R-PCR assay was a faster method for GSTT1 polymorphism detection than the C-PCR assay. The present study suggests that the R-PCR with SYBR Green I and melting curve analysis may be a useful screening tool for more convenient, rapid, reliable and safer detection of GSTT1 polymorphism in patients with cancer as compared to the C-PCR assay.
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    Detection of Iga Antibody Against Epstein-Barr Virus Nuclear Antigen 1 in Nasopharyngeal Carcinoma Patients by ELISA
    (Thai Cancer Journal, 2010-04-07) Danai Tiwawech; Somjin Chindavijak; Pensri Saelee; Soawakon Sukarayodhin; Takafumi Ishida; Park S.P. Ng
    Nasopharyngeal carcinoma (NPC) is a multi-factorial disease caused by genetic, viral (Epstein Barr virus, EBV) and environmental factors. The elevation of IgA antibody titers against EBV viral capsid antigen (VCA) measured by indirect immunofluorescence assay (IFA) had been use as ‘gold standard’ for NPC diagnosis for over thirty years. However, IFA is unsuitable for mass screening among population since it is time-consuming, inconvenient to perform and difficult to standardize. To date, these difficulties of IFA have been solved by using recombinant protein-based enzyme-linked immunosorbent assay (ELISA). The EBV nuclear antigen 1 (EBNA1) is the only latent EBV antigen consistently expressed in NPC tissues. Recently, it has been found that IgA antibody against EBNA1 (IgA/EBNA1) measured by ELISA may be a useful marker for NPC and the early detection of this cancer. The purpose of this study is to evaluate the usefulness of IgA/EBNA1 from a commercial kit in Thai NPC cases. The concentration of serum IgA/EBNA1 was measured in 54 NPC patients and 122 age match healthy controls by using Sinoclone EBV IgA ELISA kit. The normal cut off value (mean+2SD) of serum IgA/EBNA1 showed a relative optical density (rOD) at 1.26 units. Serum IgA/EBNA1 level was positive in 52 (96.30%) out of 54 NPC patients and in 5 (4.10%) out of 122 healthy controls. NPC cases showed significantly higher serum IgA/EBNA1 level than healthy controls (P \< 0.001). In NPC patients, the serum IGA/EBNA1 level was increased with aggressiveness and advance stages of the disease. Detection of IgA/EBNA1 by Sinoclone EBV IgA ELISA kit in serum had a sensitivity, a specificity, positive predictive values and negative predictive values of 96.30, 95.90, 91.23 and 98.32%, respectively, for the diagnosis of NPC. The results of our study suggest that serum IgA/EBNA1 may be a suitable marker for diagnosis and prognosis of NPC in Thailand and that this test may be a useful addition to the panel of tests used for this purpose. Further studies are currently underway to evaluate the effectiveness of this marker as an early detection tool for NPC in Thailand.
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    Detection of p53 codon 72 gene polymorphism in patients with oral cancer
    (Thai Cancer Journal, 2010-04-06) Danai Tiwawech; Somjin Chindavijak; Anant Karalak; Takafumi Ishida
    Oral cancer is a serious malignant disease that caused vastly losses in Thailand annum. The potential risk factor for predicting and screening of high-risk populations that developed early stage oral cancer followed by immediately intensive counseling and efficiency treatment is an important strategy to control this harmful cancer. To address on the genetic risk factor for oral cancer was investigated. The association between the p53 codon 72 gene polymorphism and oral cancer susceptibility in Thai people. The frequency of p53 codon 72 gene polymorphism (Arginine/Arginine, Arginine/Proline and Proline/Proline genotypes) in 80 oral cancer patients, 80 chronic oral disease patients and 80 age-matched healthy controls was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Statistically significant difference in the overall genotype frequencies between cases and controls comprising chronic oral disease patients and healthy controls was observed (p \< 0.05). Proline/Proline genotype carriers had 2.8-fold increased risk for oral cancer as compared with Arginine/Arginine genotype carriers (Odds ratio = 2.8, 95% confidence interval = 1.0-4.7). Among oral cancer patients, statistical significant difference in p53 genotype frequencies between clinical stages was also observed. The results in this study suggest that the p53 codon 72 gene polymorphism may associate with oral cancer susceptibility in Thai population, particularly the Proline/Proline genotype carrier. The suggestion is that the detection of p53 polymorphism may be a useful tool for screening of the high-risk group as well as prognosis of oral cancer in Thai people.
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    Detection of P53 Codon 72 Polymorphism in Lung Cancer High-Risk Subjects in Lampang Province
    (Thai Cancer Journal, 2010-04-07) Surathat Pongnikorn; Danai Tiwawech; Supumas Laingoen; Duriya Fongmoon; Wichit Suwan
    The P53 tumor suppressor gene (P53) encodes for P53 protein to inhibit cell proliferation and induceapoptosis of cancer cell. P53 is one of the most commonly mutated genes (somatic mutation) in all types of human cancer. Actually, the P53 exhibits germ-line mutation or polymorphism at the codon 72 on the 4th exon, which produces 2 different proteins (variants) with an arginine amino acid (Arg, CGC: wild type) or proline amino acid (Pro, CCC: mutant type). In general, there are 3 genotypes of the P53 codon 72 polymorphism: Arg/Arg (A/A), Arg/Pro (A/P) and Pro/Pro (P/P) genotypes. It has been reported that P53 variant with an Arg induced cell death more effectively than those with the Pro. Individuals with P/P genotype have been reported to be at higher risk for lung cancer compared to those with A/A genotype. It is suggested that P/P genotype of the P53 codon 72 polymorphism is the risk genotype. Therefore, it may be used as a genetic risk marker for lung cancer in Thai population. The purpose of the present study is to detect the frequency of individuals with P/P genotype of the P53 codon 72 polymorphism in 850 healthy subjects in Lampang province by detecting A/A, A/P and P/P genotypes of the P53 codon 72 polymorphism using the Real-Time Polymerase Chain Reaction-Simple Probe Probe assay. The frequencies of individuals with A/A, A/P and P/P genotypes of the P53 codon 72 polymorphism were 23.1% (196/850), 52.7% (448/580) and 24.2% (206/850), respectively. The allele frequencies of Arg and Pro were 0.49 and 0.51, respectively. Among all subjects, there were 559 males and 291 females. The frequencies of A/A, A/P and P/P genotypes of the P53 codon 72 polymorphism in males were 22.5% (126/559), 55.5% (310/559) and 22.0% (123/559), respectively, whereas in females were 24.1% (70/291), 47.4% (138/291) and 28.5% (83/291), respectively. When all subjects were divided into 2 groups of 586 smokers and 264 non smokers with the occupational high-risk, the frequencies of A/A, A/P and P/P genotypes of the P53 codon 72 polymorphism in smokers were 23.2% (136/586), 53.6% (314/586) and 23.2% (136/586), whereas in non smokers, but with the occupational high-risk, there were 22.7% (60/264), 50.8% (134/264) and 26.5% (70/264), respectively. Among all subjects, there were no significant differences of the frequency of P53 codon 72 polymorphisms between males and females and between smokers and non-smokers who work in the high-risk factory. However, females seem to have a higher frequency of P/P genotype than males. In addition, no significant difference of the frequency of P53 codon 72 polymorphisms was observed between males and females in smokers group. On the other hand, there were significant differences of the frequency of P53 codon 72 polymorphisms between males and females in the non-smoker group who work in the high-risk factory and females had higher frequency of P/P genotype than males. The results of this study showed that the frequency of individuals with P/P genotype of the P53 codon 72 polymorphism in healthy subjects in Lampang province was higher than in Bangkok from the previous study and females had tendency to have higher frequency of P/P genotype than males. Further study on the intensive follow-up, counseling and surveillance in these P/P genotype carriers for prevention and early detection of lung cancer is needed.