Detection of Serum CSLEX Levels in Breast Cancer Patients

Breast cancer (BRC) is an important malignant disease. Its incidence is highest among the female cancers in Thailand, with a trend to increase annually. The discovery of new tumor markers is urgently needed, to enable BRC to be detected in its early stages, to improve prognosis, to monitor treatment, and to detect recurrence, and thereby control this serious neoplasm. It has been reported that CSLEX (sialyl LewisX), a cancer-associated carbohydrate antigen on cancer-cell membrane, can be detected in the serum of cancer patients at higher levels than healthy controls; it can be used for monitoring the treatment of patients with cancers of the lung, stomach, colon, and breast. The aims of this study were to investigate differences in the serum CSLEX levels of BRC patients and healthy controls, and the correlation of serum CSLEX levels with stages and histological types. The level of serum CSLEX was measured in 200 breast-cancer patients and 200 healthy controls by enzyme immunoassay technique. The average level of serum CSLEX in the breast-cancer group was 12.14 + 15.70 U/ml (mean + SD) which was significantly higher than the healthy control group (3.56 + 2.41 U/ml) (p\<0.001). In the healthy control group, the cutoff value was 8.38 U/ml (mean + 2SD). The sensitivity and specificity of serum CSLEX for the detection of BRC were 69.0% and 98.0%, respectively. The serum CSLEX levels increased among the advanced-stage BRC patients. However, no correlation was found between serum CSLEX level and histological type. The findings of this study suggest that the detection of serum CSLEX level may be a useful tool for the diagnosis and prognosis of breast cancers among Thai women. (Thai Cancer J 2010;30:145-152)

Detection of GSTM1 and GSTT1 Polymorphisms in Nasopharyngeal Carcinoma Patients by Multiplex PCR Assay

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)

Association between GSTM1 Gene Polymorphism and Oral Cavity Cancer in Suratthani Province

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.

Detection of p53 codon 72 gene polymorphism in patients with oral cancer

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.

Detection of P53 Codon 72 Polymorphism in Lung Cancer Patients by Real Time Polymerase Chain Reaction - SimpleProbe Probe Aassay

The P53 tumor suppressor gene (P53) is a key gene involved in cancer control by producing P53 protein to inhibit proliferation and enhance apoptosis of cancer cells. It has been reported that the polymorphism at the codon 72 on the exon 4 of P53 resulted in 3 different genotypes of the P53: Arginine/Arginine (Arg/Arg), Arginine/Proline (Arg/Pro) and Proline/Proline (Pro/Pro) genotypes, and found that individual with Pro/Pro of P53 codon 72 polymorphism had a higher risk for lung, bladder and nasopharyngeal cancers than those with Arg/Arg. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay used to detect P53 codon 72 polymorphism is not suitable for a mass screening since it is complicated, time-consuming and at risk for carcinogen exposure. Recently, real-time polymerase chain reaction-SimpleProbe probe (R-PCR-SP) assay has been suggested to be suitable for a mass screening of various genetic polymorphisms with a faster and safer way. The aim of this study is to detect P53 codon 72 polymorphism in Thai patients with lung cancer by the R-PCR-SP assay using SimpleProbe probe and melting curve analysis and the PCR-RFLP assay. The findings from these two methods were then comparable. Seventy DNA samples from peripheral blood leukocyte of lung cancer cases were recruited in this study. The results of P53 codon 72 polymorphism detected by the R-PCR-SP and the PCR-RFLP assays were completely accordant (κ=1.0, 95%CI = 1.0-1.0). The findings demonstrated 12 individuals with Arg/Arg, 41 cases with Arg/Pro and 17 cases with Pro/Pro. In addition, we found that the R-PCR-SP assay was a faster and safer method for detection of P53 codon 72 polymorphism than the PCR-RFLP assay. Results of the present study suggest that the R-PCR-SP assay with SimpleProbe probe and melting curve analysis may be a useful screening tool for detection of P53 codon 72 polymorphism in the high risk group of lung cancer in Thais.

Detection of P53 Codon 72 Polymorphism in Lung Cancer High-Risk Subjects in Lampang Province

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.

Quantitative Detection of Plasma Cell-free Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma Patients by Real-time PCR

Nasopharyngeal carcinoma (NPC) is obviously a serious cancer in southern China, Taiwan, and Southeast Asia including Thailand. The Epstein-Barr virus (EBV) is a human oncogenic virus that is strongly associated with NPC development. Recently, plasma cell-free EBV DNA levels detected by the real-time polymerase chain reaction (R-PCR) have been reported to be a useful marker for rapid diagnosis and monitoring of NPC patients. The objectives of this study are to measure the amount of plasma cell-free EBV DNA in NPC patients and healthy controls by using the R-PCR and to determine the relationship between plasma cell-free EBV DNA levels and NPC status in Thai population. The concentrations of plasma cell-free EBV DNA of 60 NPC patients and 60 healthy controls were determined by using the R-PCR assay with the LightCycler® EBV quantification kit. Plasma cell-free EBV DNA levels in NPC patients [4679.88 ± 1381.99 copies/ml (mean ± standard error, SE)] were significantly higher than healthy controls (3.93 ± 2.32 copies/ml) (p \< 0.001). The median concentration of EBV DNA in NPC and healthy control groups was 1135.00 and 0.00 copies/ml, respectively. The cutoff value of plasma cell-free EBV DNA in Thais is 50 copies/ml (mean+2standard deviation, SD). Fifty-nine NPC patients (98%) and 3 healthy controls (5%) had plasma cell-free EBV DNA levels over the cutoff value (50 copies/ml). However, there were no significant differences between the levels of plasma cell-free EBV DNA and histological types, and TNM staging of NPC. The results of this study suggest that plasma cell-free EBV DNA may be a useful molecular marker for diagnosis of EBV-associated NPC in Thailand. Further investigation on the relationship of plasma cell-free EBV DNA levels and the clinical outcome needs to be explored.

Association between p53 codon 72 and GSTM1 polymorphisms and risk of hepatocellular carcinoma

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.

Detection of GSTT1 Polymorphisms in Cancer Patients by Real-Time PCR

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.

Detection of Iga Antibody Against Epstein-Barr Virus Nuclear Antigen 1 in Nasopharyngeal Carcinoma Patients by ELISA

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.