The Association of pre-miR27a Gene Polymorphism and Clinicopathological Data in Thai Breast Cancer Patients.

BACKGROUND: MiR27a plays an important role in carcinogenesis, cell proliferation, apoptosis, invasion, migration and angiogenesis. Several studies have identified an important role of pre-miR27a (rs895819) A>G polymorphism in several types of cancer. This research aims to investigate the association of pre-miR27a (rs895819) A>G and breast cancer susceptibility, clinicopathological data and survival. Blood DNA samples of 143 Thai breast cancer patients and 100 healthy Thai women were studied for pre-miR27a (rs895819) A>G polymorphism using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). RESULTS: The results revealed that the frequency of pre-miR27a (rs895819) A>G genotypes was not statistically significant different between breast cancer patient and normal control subjects. The rs895819 A>G genotype was significantly associated with clinicopathological parameter of grade III differentiation (P = 0.006), progesterone receptor (P = 0.011) and triple negative (P = 0.031) in breast cancer patients, but not with breast cancer susceptibility. CONCLUSION: The pre-miR27a (rs895819) A>G genotype was significantly associated with poorly differentiated, progesterone receptor and triple-negative in breast cancer patients. Therefore, pre-miR27a (rs895819) A>G may be used as a biomarker for poor prognosis.

Identification of Genomic Alterations in Thai Patients With Colorectal Cancer Using Next-Generation Sequencing-Based Multigene Cancer Panel.

Introduction Colorectal cancer (CRC) is one of the leading causes of death and illness in the general population. Although the incidence of CRC is steadily decreasing worldwide, it is being diagnosed more in individuals under 50 years of age. Multiple disease-causing variants have been reported to be involved in the development of CRC. This study aimed to investigate the molecular and clinical characteristics of Thai patients with CRC. Methods NGS-based multigene cancer panel testing was performed on 21 unrelated patients. Target enrichment was performed using a custom-designed Ion AmpliSeq on-demand panel. Thirty-six genes associated with CRC and other cancer were analyzed for variant detection. Results Sixteen variants (five nonsense, eight missense, two deletions, and one duplication) in nine genes were identified in 12 patients. Eight (66.7%) patients harboring disease-causing deleterious variants in genes APC, ATM, BRCA2, MSH2, and MUTYH. One of the eight patients also carried additional heterozygous variants in genes ATM, BMPR1A, and MUTYH. In addition, four patients carried variants of uncertain significance in genes APC, MLH1, MSH2, STK11, and TP53. Among all detected genes, APC was the most frequent causative gene observed in CRC patients, which is consistent with previous reports. Conclusion This study demonstrated the comprehensive molecular and clinical characterization of CRC patients. These findings showed the benefits of using multigene cancer panel sequencing for pathogenic gene detection and showed the prevalence of genetic aberrations in Thai patients with CRC.

APC Promoter Hypermethylation as a Prognostic Marker in Breast Cancer Patients.

BACKGROUND: Adenomatous polyposis coli (APC) promoter hypermethylation implicated in breast cancer development through Wnt signaling pathway, hypermethylation may result in inactivation of APC expression. This study aimed to investigated whether hypermethylation of APC promoter, the aggressive behavior of breast cancer cells, and correlated them with clinicopathological parameters and survival. METHODS: Sixty-one fresh tissues of breast tumor were evaluated for APC promoter hypermethylation with methylation-specific PCR techniques (MS-PCR) and  APC mRNA expression level analysis by quantitative real-time reverse transcription-PCR. RESULTS: Our results show aberrant APC hypermethylation status was founded in 27 of 61 cases (44%), and significantly associated with chemotherapy treatment (OR= 6.9, 95%CI=1.5-31.01, P = 0.01), distant metastasis (OR = 5.52, 95%CI = 1.27-24.08, P = 0.04) as well as APC methylated status also associated with shorter overall survival than those without (8.4 and 11.0 years respectively, P = 0.02). CONCLUSION: The findings indicated hypermethylation of APC promoter may be used as a useful prognostic biomarker in breast cancer patients.

Frequency and Association Of GSTM1 and GSTT1 Gene Polymorphisms with Survival in Breast Cancer Patients.

OBJECTIVE: Glutathione S-transferase M1 and T1 (GSTM1 and GSTT1) are the key detoxification enzymes of xenobiotics, including chemotherapeutic drugs. The deletion polymorphisms of GSTM1 and GSTT1 genes are associated with reduced enzyme activity that influenced clinical outcomes of chemotherapeutic agents in breast cancer. However, there is limited information among Thai patients. This research aims to explore the frequency and role of GSTM1 and GSTT1 polymorphisms on survival among Thai patients with breast cancer. METHODS: The retrospective cohort study was performed. Demographic data and clinicopathology characteristics were collected from hospital base registry data and medical records. A multiplex qualitative real-time PCR method was used to detect the presence or absence of the GSTM1 and GSTT1 gene in the genomic DNA samples of the participants. RESULTS: The frequencies of the GSTM1 and GSTT1 null genotypes in 198 breast cancer patients were 65.70% and 33.30%, respectively. The overall survival at 1, 3 and 5 years were 95.00%, 83.00%, 71.00% respectively. The log rank test and Cox proportional hazards revealed a significant different in the 5-years overall survival according to lymph node metastasis and tumor stage (P = 0.014 and P < 0.001). No associations between overall survival and GSTM1 or GSTT1 genotype were found in single or combined genotypes analyses (P = 0.76 and P= 0.15). CONCLUSION: The results of our study provided the epidemiological information for prognostic of survival in breast cancer patients treated with chemotherapy.
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Glutathione S-Transferase P1 Polymorphism on Exon 6 and Risk of Hepatocellular Carcinoma in Thai Male Patients.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the fourth leading cause of cancer-related deaths worldwide. HCC cases are two to four times more common in males than in females, and the highest incidence is found in Asia and Sub-Saharan Africa. This gender disparity is the result of different behavioral risk factors, such as smoking and drinking alcohol. Glutathione S-Transferase P1 (GSTP1) is an enzyme that is involved in the detoxification of carcinogenic electrophiles. GSTP1 codon 105 in exon 5 and codon 114 in exon 6 polymorphisms result in decreased enzyme detoxification activity, which is the cause of many cancers. OBJECTIVES: This study aims to investigate the associations between GSTP1 polymorphism, HCC patients, and the risk factors for HCC. It is hoped that this research will provide useful knowledge on the effects of genetic GSTP1 polymorphism in Thai HCC patients. METHODS: DNA from 44 Thai HCC patients and 52 healthy controls was analyzed for GSTP1 exon 5 and exon 6 polymorphisms by PCR-RFLP. The associations between GSTP1 polymorphism, the control group, and clinicopathological parameters were determined. RESULTS: The results show that GSTP1 exon 6 polymorphism genotypes (C/T) were correlated with an increased risk of HCC susceptibility (OR = 4.40). Moreover, exon 6 polymorphism genotypes (C/T) were associated with the gender of patients (p = 0.015), but no relationships were found between GSTP1 exon 5 polymorphism and the clinicopathological data of patients. CONCLUSIONS: The results suggest that the GSTP1 exon 6 polymorphism genotype was associated with an increase in the risk of HCC in male patients and that it tended to be related to cancer differentiation. No association was found between GSTP1 exon 5 polymorphism and the risk of HCC.

Reduced Expression of GPX3 in Breast Cancer Patients in Correlation with Clinical Significance.

Glutathione peroxidase 3 (GPX3) is the main antioxidant enzyme in plasma. Its biological roles are to protect cells from oxidative stress-induced damage. Several studies have been reported the association between GPX3 expression and its correlation with cancer carcinogenesis including breast cancer. The aim of this research was to investigate the GPX3 messenger ribonucleic acid (mRNA) expression in 82 breast tumors and paired normal breast tissues by SYBR green quantitative real-time reverse transcription-polymerase chain reaction and the association with clinicopathological data. Our results show that GPX3 reduced expression was found significantly associated with number of metastatic lymph nodes (odds ratio [OR] = 3.41, 95% confidence interval [CI] = 1.35-8.64, p = 0.01), no distant metastasis (OR = 5.52, 95% CI = 3.74-11.89, p = 0.04), and nonhormone usage breast cancer patients (OR = 0.19, 95% CI = 0.04-0.93, p = 0.04). This finding suggested that GPX3 plays a role in breast carcinogenesis, and might serve as a prognostic biomarker in breast cancer patients.

GSTM1 and GSTT1 copy number variants and the risk to Thai females of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignancy found throughout the world that most often occurs in males. The cancer is associated with many risk factors such as viral infection, cirrhosis, alcohol, smoking, and fungal toxins. GSTM1 and GSTT1 are detoxification enzymes activated by the cleansing of carcinogenic compounds. Low DNA copy numbers of Glutathione S-transferases M1 and T1 result in a loss of enzyme activity, which causes carcinogenesis factors. DNA copy number variants (CNVs) were determined to compare the differences between the frequencies of GSTM1 and GSTT1 in a control group and patients. Then, the association of these genes with the pathological/survival status of HCC patients was investigated. METHODS: Forty-nine Thai HCC patients' DNA and the genomic DNA of 66 healthy controls were investigated for GSTM1 and GSTT1 CNVs by real-time polymerase chain reaction (PCR). Then, the correlations between GSTM1 and GSTT1 patients' CNVs, the control group, and clinico-pathological parameters were determined. RESULTS: The results show that were no differences between the CNVs of GSTM1 and GSTT1 in the controls and patients (P≥0.05). Only GSTT1 genotypes 0/0 correlated to an increase in the risk of hepatocellular carcinogenesis (OR value was 1.88). GSTM1 CNVs were associated with the gender of patients (P=0.002). However, no correlations were found between GSTT1 CNVs and any of the clinico-pathological parameters. CONCLUSIONS: The results suggest that only GSTT1 CNVs are associated with increased risk factors of HCC in Thais. GSTM1 copy numbers had a dominant correlation with female HCC patients.

Role of GSTM1 Copy Number Variant in the Prognosis of Thai Colorectal Cancer Patients Treated with 5-FU-based Chemotherapy

Background: Glutathione S-transferase M1 (GSTM1) is involved in the detoxification of carcinogenic agents. DNA copy number variants of GSTM1 may be associated with cancer progression and may result in reduced survival time of various cancers. Determination of DNA copy number variants was here used to assess the association between GSTM1 copy number variant and pathological status and survival time of colorectal-cancer patients treated with 5-fluorouracil-based chemotherapy. Methods: One hundred thirteen Thai colorectal-cancer patients were investigated for GSTM1 copy number variant by real-time PCR. Relationships between gene copy number variants and clinico-pathological parameters were determined. Result: Associations were evident between GSTM1 copy number and stage of tumor (P = 0.026) and metastasis at diagnosis (P = 0.049), with odds ratio values of 0.2 and 0.3 respectively. Conclusions: GSTM1 copy number variant was here not related with reduced overall survival for the colorectal-cancer patients receiving 5-FU-based chemotherapy.

Clinicopathological significance of BRCA1 promoter hypermethylation in Thai breast cancer patients.

Breast cancer susceptibility gene 1 (BRCA1), mapped on chromosome 17q21, is implicated in the mechanisms of cellular DNA repair. Inactivation of this gene is involved in the development of many human cancers, including breast cancer. This study aimed to investigate the prognostic value of BRCA1 promoter hypermethylation and expression in breast cancer cases. Sixty-one breast cancers were examined for BRCA1 hypermethylation by methylation-specific polymerase chain reaction (PCR), and 45 paired normal breast tissues were analyzed for altered BRCA1 mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction (qRT- PCR). Aberrant methylation status in BRCA1 was detected in 15 of 61 cases (24.6%), while reduced expression was found in 7 of 45 (15.6%). BRCA1 hypermethylation was statistically associated with tumor grade III (p=0.04), a high frequency of stage IIB (p=0.02), and triple-negative phenotype (OR= 3.64, 95%CI =1.1-12.3, p=0.03). Our findings indicated that BRCA1 promoter hypermethylation is a useful prognostic marker for breast cancer.

Hypermethylation of suppressor of cytokine signaling 1 in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC), the most common primary hepatic tumor, is highly prevalent in the Asia-Pacific region, including Thailand. Many genetic and epigenetic alterations in HCC have been elucidated. The aim of this study was to determine whether aberrant methylation of the suppressor of cytokine signaling 1 gene (SOCS1) occurs in HCCs. Methylation specific-PCR assays were performed to identify the methylation status of SOCS1 in 29 tumors and their corresponding normal liver tissues. An abnormal methylation status was detected in 17 (59%), with a higher prevalence of aberrant SOCS1 methylation significantly correlating with HCC treated without chemotherapy (OR=0.04, 95%CI=0.01-0.31; P=0.001). This study suggests that epigenetic aberrant SOCS1 methylation may be a predictive marker for HCC patients.

Histological type of intrahepatic cholangiocarcinoma differentiated by genetic alteration from AP-PCR fingerprint.

Cholangiocarcinoma (CCA), the malignant neoplasm of the biliary epithelium, is usually fatal due to difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of CCA are not well understood and only a few cytogenetic studies have been published. In this study, genomic instability in 30 Thai cases of intrahepatic cholangiocarcinoma (ICC) was assessed using an arbitrarily primed- polymerase chain reaction (AP-PCR) method. Genetic alterations were analyzed as banding pattern changes between tumors and corresponding normal DNA. The abnormal band present at the highest frequency (23/30 cases, 77%) appeared with the AO16 primer. Statistical analysis also showed that DNA alteration from this primer was significantly associated with the moderately to poorly differentiated histological type (P=0.038). Kaplan-Meier survival curves showed borderline significance for this DNA aberration (P=0.06 by the log-rank test). This DNA fragment may thus be of use to predict degree of malignancy of the disease.

Ubiquitin-specific protease 14 expression associated with intrahepatic cholangiocarcinoma cell differentiation.

The purpose of this study was to identify the gene alterations amplified from AO16 primer and examine whether the expression patterns of USP14 in clinical specimens from patients with intrahepatic cholangiocarcinoma (ICC) is associated with cancer cells. DNA from tumor and corresponding normal tissues of 52 patients was amplified with 33 arbitrary primers. The DNA fragment that altered most frequently in ICC was cloned, sequenced, and identified by comparison with known nucleotide sequences in the genome database. The DNA copy numbers of the allelic alterations in cholangiocarcinoma were determined by quantitative real-time PCR and interpreted as allelic loss or DNA amplification by comparison with the reference gene. Associations between allelic imbalance and clinicopathological parameters of ICC patients were evaluated by X²-tests. The Kaplan-Meier method was used to analyze survival rates. Immunohistochemically, USP14 showed weak cytoplasmic staining in normal bile duct epithelial cells. It was strongly detected in 21 cancer patients (43.8%). There were correlations between USP14 expression level and the clinicopathological features of ICC, histological grade (P < 0.05). However, there were no significant differences in age, gender, tumor size, metastasis, lymph node metastasis, and staging. USP14 expression was related to cholangiocarcinoma cell differentiation. Due to their emerging role in control of multiple signaling pathways and oncoproteins, USP14 inhibitors may be useful for anticancer agents.

RASSF1A promoter hypermethylation as a prognostic marker for hepatocellular carcinoma.

This study was performed to determine whether epigenetic aberrant methylation of RASSF1A might be associated with hepatocarcinogenesis. Methylation specific-PCR was performed to identify RASSF1A promoter hypermethylation in 29 tumors and corresponding normal liver tissues. In addition, RASSF1A mRNA levels were analyzed by quantitative real-time reverse transcription-PCR. Aberrant methylation of RASSF1A was detected in 25 of 29 cases (86%), with loss of RASSF1A expression evident in 8 of 22 cases (36%). No correlation between loss of RASSF1A mRNA and promoter hypermethylation of the RASSF1A gene was observed. There was a significant correlation between the methylation status of RASSF1A and hepatocellular carcinoma (HCC) patients who did not undergo chemotherapy (P = 0.03). Multivariate analysis, adjusted for tumor size, treatment, RASSF1A hypermethylation, and RASSF1A under-expression, showed RASSF1A hypermethylation to be assocaited with a better prognosis for HCC patients (HR= 0.089, 95%CI = 0.013-0.578; P = 0.012). Our findings showed that RASSF1A promoter hypermethylation occurs frequently, and may serve as a good prognostic factor.

Novel PNLIPRP3 and DOCK8 gene expression and prognostic implications of DNA loss on chromosome 10q25.3 in hepatocellular carcinoma.

Our previous study of gene alterations in 29 hepatocellular carcinoma (HCC) using AP-PCR amplified with 59 different 10-mer arbitrary primers and gene cloning, indicated DNA alterations by DNA fingerprints from 34 primers. Among these, the altered DNA fragment from primer U-8 predominated (62%). The aim of this report is to identify the gene alterations on chromosomal banding and gene expression in these patients, including the association of these alterations with patient demographic data. Seven different sequences, mapped to chromosomes 5q33.3, 7q31.33, 7q34, 9p24.3, 10q25.3, 13q31.3, and 16p11.2, were identified by gene cloning and nucleotide sequencing. Novel PNLIPRP3 gene over-expression and DOCK8 gene under-expression were observed in 41% and 44% of these patients, respectively, which point to an association of these genes and the development of HCC. Likewise, allelic loss on chromosome 10q25.3 was associated with shorter survival among HCC patients (P=0.03); this indicated that allelic loss on chromosome 10q25.3 may serve as a prognostic marker in patients with HCC.