Template-ready PCR method for detection of human telomerase reverse transcriptase mRNA in sputum.

Human telomerase reverse transcriptase (hTERT) mRNA in tissue is a biomarker of lung cancer, but hTERT mRNA in sputum had not been successfully detected with conventional reverse transcription PCR methods. Here, we developed a novel PCR protocol: Template-Ready PCR (TRPCR), to detect sputum hTERT mRNA, in which probes serve as templates of amplification. While free probes and dsDNA were removed in template preparation through aspiration and restriction digestion, probes that formed into heterocomplex with target RNA remained intact for PCR amplification. By fishing out the heterocomplex and amplifying the probes, TRPCR achieved sensitivity higher than reverse transcription-quantitative PCR (RT-qPCR). ROC curve of sputum hTERT mRNA by TRPCR assay showed the discrimination in high sensitivity and specificity between patients with lung cancer and lung cancer-free donors at the PCR Ct cutoff of 33. We further validated this approach through TRPCR assay of sputum from 858 lung cancer patients and 480 non-malignant pulmonary disease patients. 722 (84.2%) cases from 858 with lung cancer patients were detected as positive, whereas 461 (96.0%) cases from 480 non-malignant pulmonary disease patients were detected as negative, suggesting that TRPCR assay of sputum hTERT mRNA can serve as a non-invasive molecular diagnosis of lung cancer.

Diagnostic Value of hTERT mRNA and in Combination With AFP, AFP-L3%, Des-γ-carboxyprothrombin for Screening of Hepatocellular Carcinoma in Liver Cirrhosis Patients HBV or HCV-Related.

Diagnosis of hepatocellular carcinoma (HCC) in early-stage, to give an effective treatment option and improve quality of life for cancer patients, is an important medical mission globally. Combination of AFP with some biomarkers may be more supportive in both diagnosis and screening of HCC, but the range value of these markers can be applied as daily markers were unclearly. In some studies, human telomerase reverse transcriptase (hTERT mRNA) was reported as an advantage marker to diagnose cancer. The present study identified serum of 340 patients that were infected chronic hepatitis B virus or hepatitis C virus and divided in 2 groups including Hepatocellular carcinoma (HCC) and liver cirrhosis (LC) to measure their values of hTERT mRNA, AFP, AFP-L3%, and DCP, as well as combination of them. As a result, the concentration of hTERT mRNA, AFP, AFP-L3%, and DCP in HCC groups were significantly higher than that in LC group (P < .01). For detecting HCC, hTERT mRNA had sensitivity of 88% and specificity of 96% (at the cutoff value of 31.5 copies/mL), AFP sensitivity of 73% and specificity of 92% (at the cutoff value of 5.1 ng/mL), AFP-L3% sensitivity of 69% and specificity of 90% (at the cutoff value of 1.05%), DCP sensitivity of 82% and specificity of 92% (at the cutoff value of 29.01 mAU/mL). The largest area under the curve (AUC) of combination hTERT mRNA with DCP was 0.932 (sensitivity of 98.2% and specificity of 88.2%). New combination of DCP with hTERT mRNA gave a useful choice for screening of HCC in chronic HBV or HCV patients associated liver cirrhosis.

Diagnostic and prognostic value of the detection of hTERT mRNA in renal tumors.

INTRODUCTION: Elevated mRNA expression of human telomerase reverse transcriptase (hTERT mRNA) is common in many types of tumors, participating in tumor growth and progression. Such expression has not been sufficiently examined in renal cancer. The goal of the present study was to quantify it and analyze its possible clinical value in the management of this pathology. PATIENTS AND METHODS: The study included 111 patients who underwent surgery for renal cell carcinoma (RCC) between 2015 and 2017. Tumor samples were taken from all patients and, in 94 of them, healthy renal tissue adjacent to the tumor was also sampled. The 2 types of tissue were histologically confirmed, after which mRNA was extracted. Using real-time quantitative PCR, the expression of hTERT and glyceraldehyde-3-phosphate dehydrogenase (as endogenous control) were indirectly quantified using the crossing point (CP), which is inversely correlated with the number of sample replicates yielding positive results. These values were correlated with patient socio-demographic variables and clinical-pathological factors of the RCC. RESULTS: The majority of patients were males, with an average age of 60.5 years (SD: 14.02). Most tumors (69.4%) were clear cell carcinomas. The most frequent stages were pT2 or lower (73%), while 5% were pN1 and 12% pM1. The majority of tumors (58%) were Fuhrman grades 1 or 2 (low grade). All samples of tumor and nontumor tissue expressed glyceraldehyde-3-phosphate dehydrogenase mRNA, with the CP in the tumor sample significantly lower than in the nontumor tissue (P < 0.001). The expression of hTERT mRNA was detected in 68% of tumor tissues and significantly correlated with histopathology: 100% in sarcomatoid RCC and 77.9% in clear cell carcinomas (P < 0.0001). The CP was lower in pN1 (P = 0.018), pM1 (P = 0.046), and TNM IV stages (P = 0,041). A greater number of hTERT mRNA replicas were detected in M1 patients (P = 0.0005) and TNM IV stage (P = 0.017). There was no correlation of hTERT mRNA expression with Fuhrman grade. CONCLUSIONS: The quantitation of hTERT mRNA expression in RCC might be useful as a complementary diagnostic tool as well as for assessing aggressiveness of the tumor.

Human telomerase reverse transcriptase messenger RNA (TERT mRNA) as a tumour marker for early detection of hepatocellular carcinoma.

BACKGROUND AND STUDY AIMS: Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world. Although tumour markers such as α-foetoprotein (AFP) are widely used and important for HCC detection in clinical scenes, they still do not provide a satisfactory solution to detect HCC at the early stage. The aim of our study was to illustrate the significance of serum human telomerase reverse transcriptase messenger RNA (hTERT mRNA) as a novel biomarker for early detection of HCC. PATIENTS AND METHODS: Thirty-five patients with HCC, 15 patients with liver cirrhosis, and 10 healthy subjects were sex and age matched. History taking, full physical examination, and laboratory investigations including liver function tests, hepatitis markers, AFP, and quantification of serum human telomerase reverse transcriptase mRNA (hTERT mRNA) using real-time reverse transcription polymerase chain reaction (RT-PCR) were conducted. Ultrasonography (US), triphasic computed tomography (CT), and liver biopsy were carried out. RESULTS: The hTERT was above the cutoff point (>144 copies/ml) in 27 HCC patients with a sensitivity of 77.14% and a specificity of 100% and with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 65.2%. AFP was above the cutoff point (>50 ng/ml) in 23 HCC patients with a sensitivity of 65.71% and a specificity of 96% and with a PPV of 96.3% and an NPV of 53.8%. Our study also showed a statistically significant relationship between the size of the tumour in HCC patients and both AFP and hTERT, and hTERT appears to be more correlated with the size of the tumour than AFP. There is no direct correlation between hTERT or AFP and the number of focal lesions with p value>0.05. CONCLUSION: Serum hTERT mRNA is more sensitive and specific than AFP in the early detection of HCC and its level correlates with the size of the tumour.

EGFR and hTERT Expression as a Diagnostic Approach for Non-small Cell Lung Cancer in High Risk Groups.

OBJECTIVE: The early detection of NSCLC is of importance because it provides chances for better outcomes. The aim of the study was to explore the clinical utility of EGFR and hTERT mRNA expression as markers for diagnosis of NSCLC. METHODS: EGFR and hTERT mRNA were quantified by quantative reverse transcription real time polymerase chain reaction in plasma of 45 non-small cell lung cancer (NSCLC) and 40 chronic obstructive pulmonary disease (COPD) patients, selected by certain spirometric characteristics that made them at high risk of developing lung cancer in future. RESULTS: The gene expression level of each gene was calculated and given as a relative quantity-RQ. EGFR gene expression was found in all lung cancer patients. The mean level of expression was RQ = 29.39. hTERT mRNA could be detected in 88% of patients. The mean expression ratio in them was RQ = 17.31. Only 50% of the high risk patients turned to be positive for EGFR. The level of their expression was RQ = 2.09. The plasma levels of hTERT could be detected in 17 (42.5%) patients of the high risk COPD group. Their mean level of expression was RQ = 1.02. A statistically significant difference in EGFR and hTERT mRNA expression could be observed between the two groups of patients-p = 0.0001. CONCLUSION: EGFR and hTERT mRNA are potential markers for lung cancer diagnosis, whose clinical importance should be replicated in a larger cohort of patients.

Serum hTERT Level as Sensitive Biomarker With Prognostic Implications in Breast, Lung, Gastric and Liver Cancers

@#Human telomerase reverse transcriptase (hTERT) plays an important role in telomere restitution and gene regulation. Evidences suggest that hTERT is linked with the risk and progression of several types of malignancies. Detection of hTERT mRNA levels, as one of tumor markers, may reflect the tumor burden and the clinical status of the patient. Present paper emphasizes the potency of hTERT mRNA detection in serum as a sensitive tumor biomarker in different types of cancer. Detection of serum hTERT mRNA levels has been found highly sensitive and specific for varied cancers. A number of reports reflect its superiority to other conventional tumor markers including alfa-fetoprotein, EGFR, lens culinaris agglutinin-reactive AFP and Des-gamma carboxy prothrombin. Serum hTERT has been found linked with the risk and progression of different cancer types. hTERT levels in combination with other tumor markers may be used to improve cancer detection, tumor size and level of cancer progression.

Clinical significance of blood micrometastasis in pN0 esophageal squamous cell carcinoma / 肿瘤研究与临床

Objective To explore the clinical significance and the impact on prognosis of blood micrometastasis in the patients with pN0 esophageal squamous cell carcinoma. Methods Total RNA was extracted with TRIzol and mRNA was transcribed reversely into cDNA. RT-PCR was used to detect MMP-7 mRNA and hTERT mRNA in blood. △△Ct sample values were calculated with post-operative follow-up of 3 month, 6 month, 12 month. Results Statistical results suggested that blood micrometastasis was related to differentiation grade and pTNM staging (P=0.000, P=0.000 respectively), but not to age, sex, length of turnout (P0.05). Follow-up results suggested that the degree of invasion and tumor metastasis (recurrence) was no correlation; follow-up to 6 month and 12 month, tumor metastasis (recurrence) was associated with blood micrometastasis, and follow-up to 12 month, compared with the tumor metastasis (recurrence) probability of blood micrometastasis-positive patients and negative patients, the former was as 6.44 times as the latter. (OR=6.440, 95 % CI 1.547-26.822). Conclusion Blood micrometastasis testing is of great significance to early diagnosis and prognosis judgment in pN0 esophageal squamous cell carcinoma patients.

Correlation of Telomerase Activity, hTERT mRNA Expression and HPV E6 Detection in Cervical Cancer Tissue / 대한산부인과학회잡지

OBJECTIVE: Telomerase is a ribonucleoprotein enzyme which stabilizes chromosomal structure, thereby inducing cellular immortality. We investigated telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression in relation to high-risk human papillomavirus (HPV) DNA presence in cervical carcinomas. METHODS: From December 1995 to December 1999, at the department of obstetrics and Gynecology of Kyung-Hee University Hospital, 32 cervical carcinomas and 5 corresponding nontumor cervical tissues were obtained and the samples were immediately frozen and stored at -70 degree C. Telomerase activity was measured by using telomerase PCR ELISA, a modified version of the TRAP. Analysis of the expression of hTERT mRNA was performed by quantitative RT-PCR and the analysis of the HPV E6 gene was performed by DNA-PCR. RESULT: All of the carcinomas examined exhibited strongly positive for telomerase activity (OD>0.24), whereas telomerase activity was week or not found in the 5 corresponding nontumor cervical tissues. Quantitative RT-PCR analysis demonstrated significantly increased hTERT mRNA expression levels (>0.024) in most carcinomas comparing to control groups. There was no obvious relationship between telomerase activity levels and the clinical parameters examined including age, clinical stage, pathology, differentiation, tumour size, LN involvement and invasion depth except lymphovascular space invasion (p=0.03). In the correlation between the levels of hTERT mRNA expression and telomerase activity, correlation index which was 0.916, shows high correlation (p=0.01). According to the analysis of HPV E6 gene, 29 of 32 (90.6%) carcinomas showed HPV E6 positivity. CONCLUSION: There is a strong association between telomerase activity and hTERT mRNA expression, and up-regulation of hTERT probably plays a role in the progression of cervical carcinomas. Telomerase is at least partially activated by viral oncogenes of high-risk types. There is no obvious relationship between telomerase activity levels and the clinical parameters except LSVI (p=0.03). These findings provides that telomerase may play an important role in the early stage of carcinogenesis.