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.

Prediction of hypocalcemia in postoperative total thyroidectomy using single measurement of intra-operative parathyroid hormone level.

OBJECTIVE: Many varieties of methods using intra-operative PTH have been applied to predict hypocalcemia after total thyroidectomy. The present study prospectively evaluated the reliability of intra-operative PTH through a single assay to predict hypocalcemia after total thyroidectomy. MATERIAL AND METHOD: Intra-operative PTH were performed before and 20 minutes after total thyroidectomy for 30 new patients. The calcium level was measured at 24 and 72 hours after surgery. Patients who had serum calcium < 8.5 mg/dl at 24 or 72 hrs. (hypocalcemia) were compared with normocalcemic patients. The level of intra-operative PTH was determined to predict post operative calcium level. RESULTS: Post operative hypocalcemia developed in 20 of 30 patients. Those hypocalcemic patients showed statistically significant lower intraoperative PTH at 20 minute post total thyroidectomy compared with normocalcemic patients (mean 9.2 vs 24.7, p = 0. 006). Sensitivity, specificity, and accuracy for prediction of post total thyroidectomy hypocalcemia were 85%, 80%, and 83.3% respectively. The positive predictive value = 89.5% and negative predictive value = 72.7%. Patients with intra-operative PTH at 20 minute post-total thyroidectomy < 15 pg/ml required close monitoring. CONCLUSION: Intra-operative PTH at 20 minutes after total thyroidectomy can predict impending post operative hypocalcemia. Based on the result, this can guide which patients will be considered safe, and who can be discharged early. Furthermore, it is used to closely monitor the calcium level and provide early calcium supplementation.

Micrometastasis and recurrent neck node in supraomohyoid neck dissection field.

OBJECTIVES: Supraomohyoid neck dissection (SOHND) is one of the treatment for clinically negative neck disease in carcinoma of the oral cavity when primary is treated with surgery. Neck tumor recurrence in these patients who were treated by SOHND with and without post-operative radiotherapy was evaluated. MATERIAL AND METHOD: Forty-four patients with squamous cell carcinoma of the oral cavity aged between 33-78 years was studied Every case had clinically negative neck and was treated with SOHND. RESULTS: There were 53 supraomohyoid neck dissections. The overall recurrence rate was 11.3% (6/53). The duration of recurrence ranged from six to nine months and was seen in five of pathologically negative neck and one in pathologically positive neck. Five of six of the recurrence cases were in the field of SOHND without post-operative radiotherapy. Histopathological review with immunohistochemistry study of all recurrent cases that was previously reported as negative by H&E stain showed no micrometastasis. The 5-yr survival of neck node metastasis was 64% versus 82% of these with no lymph node metastasis which showed no statistically significant difference. CONCLUSION: The SOHND was useful for treating clinical negative neck of oral cancer with high percentage of occult lymph node. The pathological report influenced the adjuvant treatment. Micrometastasis and other indicators for this pathologically negative neck is still await further study to improve survival of this particular group of patients.