hTERT gene methylation in circulating DNA, tumor, and surrounding tissue in breast cancer: a prospective study.

BACKGROUND: The human telomerase reverse transcriptase (hTERT) enzyme, encoded by the hTERT gene, synthesizes protective telomeric sequences on chromosomes and plays a fundamental role in cancer formation. Methylation of the hTERT gene has an upregulatory effect, increasing hTERT enzyme synthesis and allowing continuous tumor cell division. OBJECTIVE: In a group of patients with breast cancer, we aimed to analyze the methylation status of hTERT in the tumor, surrounding tissue, and circulating free deoxyribonucleic acid (cfDNA) of blood collected on the day of mastectomy and then approximately one year later. DESIGN AND SETTING: A prospective study was conducted at a university hospital in Rio de Janeiro, Brazil. METHODS: Samples were collected from 15 women with breast cancer on the day of mastectomy and approximately one year postoperatively. cfDNA was analyzed by sodium bisulfite conversion, followed by polymerase chain reaction, electrophoresis, and silver nitrate staining. RESULTS: Methylation of hTERT was detected in the tumors and surrounding tissues of all 15 patients. Five patients displayed hTERT methylation in the cfDNA from the blood of the first collection. Of the ten patients who returned for the second collection, three showed methylation. Two patients with methylation in the first collection did not display methylation in the second collection. One patient with no methylation in the first collection displayed methylation in the second collection, and one patient had a diminished level of methylation in the second collection. CONCLUSION: Only one-third of patients displayed methylation in their cfDNA, which may be related to the success of chemotherapy.

Telomerase as a Therapeutic Target in Cardiovascular Disease.

Shortened telomeres have been linked to numerous chronic diseases, most importantly coronary artery disease, but the underlying mechanisms remain ill defined. Loss-of-function mutations and deletions in telomerase both accelerate telomere shortening but do not necessarily lead to a clinical phenotype associated with atherosclerosis, questioning the causal role of telomere length in cardiac pathology. The differential extranuclear functions of the 2 main components of telomerase, telomerase reverse transcriptase and telomerase RNA component, offer important clues about the complex relationship between telomere length and cardiovascular pathology. In this review, we critically discuss relevant preclinical models, genetic disorders, and clinical studies to elucidate the impact of telomerase in cardiovascular disease and its potential role as a therapeutic target. We suggest that the antioxidative function of mitochondrial telomerase reverse transcriptase might be atheroprotective, making it a potential target for clinical trials. Graphic Abstract: A graphic abstract is available for this article.

The influence of TERC, TERT and ACYP2 genes polymorphisms on plasma telomerase concentration, telomeres length and T2DM.

Telomeres are duplex tandem repeats of DNA sequence 5'-TTAGGG-3' at chromosomal ends synthesized by telomerase enzyme (TE). Telomeres length (TL) shortening is associated with age and age-related disorders. Recently, we demonstrated marked leukocytes TL (LTL) shortening in T2DM. To set the relationship between the TE, LTL and T2DM, we analyzed samples from 212 Kuwaiti subjects, 112 patients withT2DM and 100 non-diabetic subjects. The plasma TE and fasting insulin were measured by ELISA, the LTL was estimated by qPCR and three SNPs of genes related to TL; TERC rs12696304 (C/G), TERT rs2736100 (C/A) and ACYP2 rs6713088 (C/G) were genotyped by rtPCR. Results revealed comparable TE levels and alleles/genotypes between the cases and controls with no influence of either on the LTL. Interestingly, although the plasma concentration of the TE was generally low, it was significantly influenced by the TERT and ACYP2 but not TERC polymorphisms. The CC genotype carriers of rs2736100 (C/A) had significantly higher plasma TE levels compared to CA and AA carriers, p 0.009 and p 0.047, respectively, and the A-allele was associated with low TE, p 0.018. Similarly, significantly higher TE levels were detected in CC carriers of ACYP2 rs6713088 (C/G) compared with GC carriers, p 0.002, and the G-allele was associated with low TE, p 0.009. Finally, the TERT and ACYP2 polymorphisms had an influence on blood glucose levels. In conclusion, the telomeres shortening in T2DM was not due to TE deficiency or gene polymorphisms, while the TE levels were significantly associated with the TERT and ACYP2 but not TERC polymorphisms.

Functional skeletal muscle constructs from transdifferentiated human fibroblasts.

Transdifferentiation of human non-muscle cells directly into myogenic cells by forced expression of MyoD represents one route to obtain highly desirable human myogenic cells. However, functional properties of the tissue constructs derived from these transdifferentiated cells have been rarely studied. Here, we report that three-dimensional (3D) tissue constructs engineered with iMyoD-hTERT-NHDFs, normal human dermal fibroblasts transduced with genes encoding human telomerase reverse transcriptase and doxycycline-inducible MyoD, generate detectable contractile forces in response to electrical stimuli upon MyoD expression. Withdrawal of doxycycline in the middle of 3D culture results in 3.05 and 2.28 times increases in twitch and tetanic forces, respectively, suggesting that temporally-controlled MyoD expression benefits functional myogenic differentiation of transdifferentiated myoblast-like cells. Treatment with CHIR99021, a Wnt activator, and DAPT, a Notch inhibitor, leads to further enhanced contractile forces. The ability of these abundant and potentially patient-specific and disease-specific cells to develop into functional skeletal muscle constructs makes them highly valuable for many applications, such as disease modeling.

The predictive role of circulating telomerase and vitamin D for long-term survival in patients undergoing coronary artery bypass grafting surgery (CABG).

BACKGROUNDS: Cardiovascular disease (CVD) is the leading cause of mortality all over the globe. Inflammation is believed to play a pivotal role in the pathophysiology of CVD. While there are studies on the interrelationship of telomerase and vitamin D and their involvement in CVD, their independent contributions to long-term outcomes in patients with CVD are not well-defined. This study aimed to investigate the association of both telomerase and vitamin D concentrations with 10-year survival among candidates of coronary artery bypass grafting (CABG) surgery. METHODS: Participants were 404 patients from Tehran Heart Center-Coronary Outcome Measurement (THC-COM) cohort who were recruited from CABG surgery candidates in 2006. In addition to demographic and clinical data including risk factors for coronary artery disease, laboratory parameters such as markers of inflammation as well as baseline serum 25-hydroxy vitamin D [25(OH)D] and telomerase concentrations were measured. Cardiac function indexes alongside outcome measures such as mortality and survival days were recorded for every patient up to 10 years after CABG. Cox-proportional hazard model was used to study the association between all-cause mortality and research parameters. RESULTS: The mean serum telomerase enzyme level was 24.92 ±21.4 nmol/L and the mean serum 25(OH)D was 27.27±10.3 ng/mL. 10-year mortality was reported in 64 (15.8%) patients. 25(OH)D was categorized into three groups (<20, 20-30, and >30) and the cut-point for telomerase was set at 25.0 nmol/L. In Cox regression analysis, higher levels of telomerase (>25 nmol/L) were significantly associated with longer survival (p = 0.041), whereas 25(OH)D concentrations were not associated with survival time. Further analysis showed that telomerase concentrations significantly predicted survival only in the presence of insufficient levels of 25(OH)D (20-30 ng/mL) (p = 0.037). CONCLUSIONS: Telomerase can be regarded as a potential predictor of long-term outcomes in patients who underwent CABG. However, the association of telomerase with the mortality may be modified by vitamin D concentrations.

Accelerated Biologic Aging, Chronic Stress, and Risk for Sepsis and Organ Failure Following Trauma.

Chronic stress and accelerated aging have been shown to impact the inflammatory response and related outcomes like sepsis and organ failure, but data are lacking in the trauma literature. The purpose of this study was to investigate potential relationships between pretrauma stress and posttrauma outcomes. The hypothesis was that pretrauma chronic stress accelerates aging, which increases susceptibility to posttrauma sepsis and organ failure. In this prospective, correlational study, chronic stress and accelerated biologic aging were compared to the occurrence of systemic inflammatory response syndrome, sepsis, and organ failure in trauma patients aged 18-44 years. Results supported the hypothesis with significant overall associations between susceptibility to sepsis and accelerated biologic aging (n = 142). There were also significant negative associations between mean cytokine levels and chronic stress. The strongest association was found between mean interleukin-1ß (IL-1ß) and human telomerase reverse transcriptase (hTERT), r(101) = -0.28), p = .004. Significant negative associations were found between mean cytokine levels, IL-12p70, r(108) = -0.20, p = .034; and tumor necrosis factor-α (TNF-α), r(108) = -0.20, p = .033, and positive life events via the behavioral measure of chronic stress. Results may help identify individuals at increased risk for poor outcomes of trauma and inform interventions that may reduce the risk for sepsis and organ failure.

Human Telomerase Reverse Transcriptase Gene Promoter Mutation in Serum of Patients with Hepatocellular Carcinoma.

BACKGROUND: Mutations in the human telomerase reverse transcriptase (hTERT) gene promoter have been reported in hepatocellular carcinoma (HCC); however, analyses of these mutations in liquid biopsies have been technically difficult because of the high GC content of the regions of interest within this promoter. We evaluated the feasibility and prognostic value of hTERT promoter mutations identified in circulating cell-free DNA (cfDNA) from the serum of patients with HCC. OBJECTIVE: A cohort of HCC patients (n = 36) who were curatively treated by surgical resection between June 2003 and September 2014 were enrolled in this study. METHODS: The presence of hTERT promoter mutations in cfDNA from the patients' serum was analyzed via modified droplet digital polymerase chain reaction, and associations were sought between specific promoter mutations and patients' disease-free survival (DFS). RESULTS: The G>A hTERT mutation at -124 bp was detected in the serum of 25 patients (69%). Although no marked differences were observed between the characteristics of the serum mutation-positive and serum mutation-negative patient groups, the DFS of patients with the mutation was significantly shorter than that of the serum mutation-negative patients (p = 0.02). Among 18 clinicopathologic and background liver factors examined, the presence of the -124 bp G>A mutation was an independent and significant predictor of patients' DFS (hazard ratio = 3.01, 95% confidence interval 1.11-10.5, p = 0.03) in multivariate analyses. CONCLUSIONS: The -124 bp G>A hTERT promoter mutation was observed in the serum of 69% of HCC patients who underwent surgical resection and was an independent predictor of disease progression in HCC.

Visual and sensitive detection of telomerase activity via hydrogen peroxide test strip.

The point of care testing (POCT) of telomerase activity is critical for early diagnosis of cancer. Herein, a colorimetric method was developed for visual detection of telomerase activity via hydrogen peroxide test strip. It is based on the telomerase-controlled in-situ formation of hydrogen peroxide. Firstly, biotinylated telomerase substrate (TS) primer was attached on the surface of magnetic beads (MBs) via the streptavidin-biotin reaction to form MB-TS complex. Then, TS primers were elongated by telomerase to form long telomere elongated products (TEP) which contains TTAGGG repeat units. The in-situ formed MB-TEP complex specifically hybridized with glucose oxidase modified cDNA (GOD-cDNA). After magnetic separation and washing, the MB-TEP/GOD-cDNA complex incubated with glucose solution to in-situ produce hydrogen peroxide which was detected by hydrogen peroxide test strip. One long TEP hybridized with multiple GOD-cDNAs, which enriched GOD to highly efficiently catalyze glucose for generating hydrogen peroxide. Thus, the visual assay achieved sensitive detection of telomerase activity, and the limit of detection (LOD) reached as low as 10 HeLa cells/µL by naked eyes and 4.5 HeLa cells/µL by absorbance measurements. Therefore, it offers a sensitive and low-cost method for visual detection of telomerase activity, which also, widens the application of commercial hydrogen peroxide test strip in the development of non-H2O2 biosensors.

The association between dietary quality indices and serum telomerase activity in patient candidates for CABG.

BACKGROUND: Diet and dietary habits are major determinants of human telomere length. Telomerase activity is affected mostly by oxidative stress and inflammation. However, the association of telomerase activity with dietary quality indices has not been evaluated before. In the current work, we aimed to test the association of telomerase activity with dietary antioxidant quality score (DAQ), dietary inflammatory index and dietary patterns in patients who were candidate for coronary artery bypass grafting surgery (CABG). METHODS AND MATERIALS: In the current cross-sectional study, 454 candidates for the CABG were enrolled from Tehran Heart Center-Coronary Outcome Measurement (THC-COM) cohort. Laboratory measurements included Hb-A1C, serum lipid profile, creatinine, blood urea nitrogen, hematocrit, lipoprotein (LP)-a, telomerase activity, serum vitamin D and C-reactive protein. Dietary status was measured by semiquantitative food frequency questionnaire, and dietary indices were calculated. Dietary patterns were extracted by factor analysis method. RESULTS: High telomerase activity was associated with lower prevalence of myocardial infarction (MI) (P = 0.04), high dietary vitamin E and high total dietary antioxidant quality scores. Telomerase activity in top quartile of neo-traditional dietary pattern was higher than other quartiles (P = 0.021). No significant association between telomerase activity and other dietary patterns was obtained. Higher telomerase activity was also associated with higher serum creatinine and lower LP-(a) concentrations (P < 0.05). CONCLUSION: To our findings, higher telomerase activity was associated with higher DAQ and lower MI prevalence. It seems that adherence to healthy diet increases serum telomerase activity and reduced telomerase concentration is associated with increased cardio-metabolic risk factors. Moreover, adherence to neo-traditional pattern with higher intake of low-fat dairy products was associated with higher telomerase activity. LEVEL OF EVIDENCE: Level V: A well-designed observational cross-sectional study.

LncRNA TERC alleviates the progression of osteoporosis by absorbing miRNA-217 to upregulate RUNX2.

OBJECTIVE: To elucidate the role of telomerase RNA elements (TERC) in alleviating osteoporosis (OP) by absorbing microRNA-217 (miRNAs-217) to regulate runt-related transcription factor 2 (RUNX2) level. MATERIALS AND METHODS: The serum levels of TERC and miRNA-217 in OP patients and healthy controls were determined. During the osteogenic process, the relative levels of alkaline phosphatase (ALP), RUNX2, and Osterix were determined in hMSCs. The regulatory effects of TERC, miRNA-217, and RUNX2 on ALP and RUNX2 levels in hMSCs were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. In addition, the changes in ALP activity and calcification ability in hMSCs influenced by them were assessed through ALP activity determination and alizarin red staining, respectively. The interaction of TERC/miRNA-217/RUNX2 regulatory loop and its role in influencing hMSCs osteogenesis were assessed by Dual-Luciferase Reporter Gene Assay and a series of rescue experiments, respectively. RESULTS: The downregulated TERC and upregulated miRNA-217 were identified in the serum of the OP patients. Consistently, the downregulated TERC and upregulated miRNA-217 were discovered in in vitro osteogenic process of hMSCs. The silence of TERC, or RUNX2 downregulated ALP and RUNX2 levels, decreased ALP activity and attenuated the calcification ability in hMSCs. The overexpression of miRNA-217 gave similar results. The binding relationship in TERC/miRNA-217/RUNX2 regulatory loop was verified. At last, rescue experiments suggested that TERC accelerated hMSCs osteogenesis by absorbing miRNA-217 to upregulate RUNX2. CONCLUSIONS: The serum level of TERC is lowly expressed in OP patients. TERC influences hMSCs osteogenesis by absorbing miRNA-217 to upregulate RUNX2, thus alleviating the progression of OP.

Circulating Tumor Cells Are Associated with Recurrent Disease in Patients with Early-Stage Non-Small Cell Lung Cancer Treated with Stereotactic Body Radiotherapy.

PURPOSE: Although stereotactic body radiotherapy (SBRT) is effective in early-stage non-small cell lung cancer (NSCLC), approximately 10%-15% of patients will fail regionally and 20%-25% distantly. We evaluate a novel circulating tumor cell (CTC) assay as a prognostic marker for increased risk of recurrence following SBRT. EXPERIMENTAL DESIGN: Ninety-two subjects (median age, 71 years) with T1a (64%), T1b (23%), or T2a (13%) stage I NSCLC treated with SBRT were prospectively enrolled. CTCs were enumerated by utilizing a GFP-expressing adenoviral probe that detects elevated telomerase activity in cancer cells. Samples were obtained before, during, and serially up to 24 months after treatment. SBRT was delivered to a median dose of 50 Gy (range, 40-60 Gy), mostly commonly in four to five fractions (92%). RESULTS: Thirty-eight of 92 subjects (41%) had a positive CTC test prior to SBRT. A cutoff of ≥5 CTCs/mL before treatment defined favorable (n = 78) and unfavorable (n = 14) prognostic groups. Increased risk of nodal (P = 0.04) and distant (P = 0.03) failure was observed in the unfavorable group. Within 3 months following SBRT, CTCs continued to be detected in 10 of 35 (29%) subjects. Persistent detection of CTCs was associated with increased risk of distant failure (P = 0.04) and trended toward increased regional (P = 0.08) and local failure (P = 0.16). CONCLUSIONS: Higher pretreatment CTCs and persistence of CTCs posttreatment is significantly associated with increased risk of recurrence outside the targeted treatment site. This suggests that CTC analysis may potentially identify patients at higher risk for regional or distant recurrences and who may benefit from either systemic therapy and/or timely locoregional salvage treatment.

Beta-carotene, telomerase activity and Alzheimer's disease in old age subjects.

PURPOSE: Advancing age represents the strongest risk factor for Alzheimer's disease (AD), and the identification of biomarkers able to define what characterizes physiological aging from AD may represent a potential starting point for novel preventive strategies. Among these biomarkers, telomeres seem to be a promising target. Interestingly, high intake of carotenoid-rich food may play a role in protecting telomeres by oxidative stress reduction. Accordingly, low plasma ß-carotene concentrations have been found in AD subjects when compared with cognitively healthy subjects. In this study, we aim at investigating the hypothesis that low ß-carotene might be associated with markers of accelerated cellular aging, including leucocyte telomere length (LTL) and peripheral mononuclear cell (PBMC) telomerase activity in a cohort of old age subjects. METHODS: The study was conducted in 68 old age subjects, 37 AD, and 31 age-matched healthy controls. In all subjects, ß-carotene plasma level, LTL and peripheral telomerase activity were measured. RESULTS: In all populations, ß-carotene significantly and positively (r = 0.320, p = 0.008) correlated with telomerase activity, independent of gender. A model having telomerase activity levels as the dependent variable, and age, gender, smoking habit, and ß-carotene as independent variables, confirmed that ß-carotene was independently associated with telomerase activity (ß = 0.319, p = 0.012). Subjects affected by AD had significantly lower plasmatic levels of ß-carotene (448 ± 66 mg/ml vs 497 ± 59 mg/ml, p = 0.001) and LTL (0.53 ± 0.25 vs 0.69 ± 0.29; p = 0.009) as compared with healthy controls. Β-carotene plasma level was associated with AD diagnosis (OR 0.988; IC95% 0.978-0.997; p = 0.013) independently of age, gender, smoking habit, ApoE genotype, and LTL. CONCLUSION: Our data show that ß-carotene may modulate telomerase activity in old age. Moreover, lower plasma ß-carotene levels, correlating with peripheral telomerase activity, are associated with AD diagnosis independent of multiple covariates.

Telomerase-specific attenuated viruses, a definitive strategy or just one more in circulating tumor cells detection anthology?

The quantification and isolation of Circulating Tumor Cells (CTC) is being the battleground during last years. There are many groups that are investing economic resources in trying to solve this jigsaw. Technological platforms based on different proofs of concept have been developed achieving in some cases excellent results despite not having been able to detect the total compute of the patient's CTC population. The handicap of this matter has been the lack of universal markers. Several years have gone so that in detection of CTC is take into account a basic characteristic that possesses the most of tumor cells, the loss of inactivation of the enzyme telomerase. Gene therapy has been combined with telomerase activity concept for develop a molecular tool that makes it possible to identify CTC: Telomerase-specific replication-selective viruses. This review includes for the first time all the scientific studies that have been published to date with this advanced technology. Furthermore, it describes the role in the diagnosis and prognosis that Telomerase-specific attenuated viruses have been playing in cancer patients study during this last decade.

Improvement in indices of cellular protection after psychological treatment for social anxiety disorder.

Telomere attrition is a hallmark of cellular aging and shorter telomeres have been reported in mood and anxiety disorders. Telomere shortening is counteracted by the enzyme telomerase and cellular protection is also provided by the antioxidant enzyme glutathione peroxidase (GPx). Here, telomerase, GPx, and telomeres were investigated in 46 social anxiety disorder (SAD) patients in a within-subject design with repeated measures before and after cognitive behavioral therapy. Treatment outcome was assessed by the Liebowitz Social Anxiety Scale (self-report), administered three times before treatment to control for time and regression artifacts, and posttreatment. Venipunctures were performed twice before treatment, separated by 9 weeks, and once posttreatment. Telomerase activity and telomere length were measured in peripheral blood mononuclear cells and GPx activity in plasma. All patients contributed with complete data. Results showed that social anxiety symptom severity was significantly reduced from pretreatment to posttreatment (Cohen's d = 1.46). There were no significant alterations in telomeres or cellular protection markers before treatment onset. Telomere length and telomerase activity did not change significantly after treatment, but an increase in telomerase over treatment was associated with reduced social anxiety. Also, lower pretreatment telomerase activity predicted subsequent symptom improvement. GPx activity increased significantly during treatment, and increases were significantly associated with symptom improvement. The relationships between symptom improvement and putative protective enzymes remained significant also after controlling for body mass index, sex, duration of SAD, smoking, concurrent psychotropic medication, and the proportion of lymphocytes to monocytes. Thus, indices of cellular protection may be involved in the therapeutic mechanisms of psychological treatment for anxiety.

Predictive and prognostic significance of telomerase levels/telomere length in tissues and peripheral blood in head and neck squamous cell carcinoma.

A growing body of evidence indicates that the expression of TERT, the catalytic subunit of telomerase, is a biological marker of progression in several cancers. We investigated the predictive and prognostic role of TERT levels and telomere length in tissues and peripheral blood in patients with head and neck squamous cell carcinoma (HNSCC). High TERT levels in cancer tissues were independently associated with worse response to therapy (odds ratio [OR]:6.26), regional failure (hazard ratio [HR]:5.75), progression (HR:2.12), and death (HR:3.53). Longer telomeres in the mucosa surrounding the tumor (SM) were independently associated with a lower risk of mucosal failure (HR:0.39). While telomere length in peripheral blood mononuclear cells (PBMC) significantly decreased with age, no correlation was found between age and telomere length in SM. No associations were found between TERT levels in plasma and telomere length in PBMC and the prognostic variables. High levels of TERT transcripts in cancer cells represent a reliable prognostic marker for identifying HNSCC patients with risk of progression. The altered relationship of telomere length to age in SM compared with PBMC suggests that in a subset of cases the phenotypically normal SM constitutes an acquired telomere-shortened epithelial field prone to genetic instability.

Potential associations of testosterone/estradiol ratio, leukocyte hTERT expression and PBMC telomerase activity with aging and the presence of coronary artery disease in men.

The present study aimed to examine associations of the testosterone/estradiol ratio, human leukocyte telomerase reverse transcriptase (hTERT) expression and telomerase activity of peripheral blood mononuclear cells (PBMCs) with aging and the presence of coronary artery disease (CAD). Telomeres in leukocytes are shorter in individuals with CAD than in healthy individuals of the same age. Levels of sex hormones are related to aging, and the ratio of testosterone to estradiol has been linked to CAD in men. Here we compared younger men (22 ±â€¯2 yr, n = 26), middle-aged men (31 ±â€¯5 yr, n = 35), older men without CAD (60 ±â€¯10 yr, n = 30) and older men with CAD (63 ±â€¯8 yr, n = 30) in terms of testosterone/estradiol ratio, leukocyte telomerase reverse transcriptase (hTERT) expression, activity of telomerase in peripheral blood mononuclear cells (PBMCs), and length of PBMC telomeres. Levels of hTERT mRNA of leukocyte and PBMC telomerase activity were significantly lower in older men than in younger or middle-aged men (p < 0.05). These two parameters, as well as testosterone/estradiol ratio, were significantly lower in older men with CAD than in all the other groups (p < 0.05). The sex hormone ratio correlated significantly with age, hTERT mRNA levels, PBMC telomerase activity and telomere length (p < 0.05). These results support the hypothesis that sex hormone balance is a biomarker of telomerase function, and that both of these parameters change as men age or develop CAD.

Epidemiological, clinical and genetic characterization of aplastic anemia patients in Pakistan.

Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.

Telomerase gene expression bioassays indicate metabolic activation of genotoxic lower chlorinated polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are ubiquitously occurring pollutants with different chemical and toxicological properties. In this study we evaluated blood plasma samples of two PCB-exposed cohorts for their ability to alter telomerase (hTERT) gene expression. Blood plasma from PCB-exposed individuals inhibited hTERT expression depending solely on the concentration of lower chlorinated PCBs, with the lowest observed adverse effect level (LOAEL) at a plasma concentration between 0.5 and 2 µg/L of LC PCBs. Individual OH-metabolites derived from the WHO indicator congeners PCB 28 and PCB 101 mimicked these effects on hTERT expression in vitro with high toxicity, including DNA damage. However, by the combination of different OH-metabolites, the bio effective PCB concentration was reduced and the respective effects on hTERT expression could be increased. At a concentration which showed no toxic activity in MTT assay, hTERT inhibition reflected the interference of OH-PCBs with the mitochondrial respiratory chain, which could lead to the production of reactive oxygen species (ROS). As individual OH-metabolites already showed a much stronger inhibition of hTERT gene expression at a lower concentration than their parental compounds, the hTERT gene expression bioassay described in this study seems to indicate metabolic activation of LC PCBs rather than the mere effect of LC PCBs on their own. In summary, this study provides dose-response linkages between effects of lower chlorinated PCBs and their concentrations in human plasma.

Circulating Tumor Cell Assessment in Presumed Early Stage Non-Small Cell Lung Cancer Patients Treated with Stereotactic Body Radiation Therapy: A Prospective Pilot Study.

PURPOSE: In patients treated with stereotactic body radiation therapy (SBRT) for presumed early stage non-small cell lung cancer (NSCLC), detection and monitoring of circulating tumor cells (CTCs) may be useful for assessing treatment response safely and noninvasively. No published reports of CTC trends in this patient population exist to date. METHODS AND MATERIALS: Patients with clinically diagnosed stage I NSCLC treated with SBRT were eligible for this institutional review board-approved prospective clinical trial. Peripheral blood samples were assayed for CTCs via a green fluorescent protein-expressing adenoviral probe. CTC positivity was defined as 1.3 green fluorescent protein-positive cells/mL of collected blood. Samples were obtained before (pre-radiation therapy [RT]), during, and after SBRT (post-RT; months 1, 3, 6, 12, 18, and 24). SBRT was delivered in ≤5 fractions (median dose of 50 Gy in 12.5 Gy fractions) to a biological equivalent dose of ≥100 Gy in all cases. RESULTS: Forty-eight consecutive patients (T1a [73%], T1b [21%], and T2a [6%]) were enrolled. Median follow-up was 14.2 months. Twenty patients (42%) had a positive CTC level pre-RT, with a median CTC count of 4.2 CTCs per mL (interquartile range [IQR], 2.2-18.7). Of these 20 patients, 17 had evaluable post-RT CTC evaluations showing reduced CTC counts at 1 month (median, 0.2; IQR, 0.1-0.8) and 3 months (median, 0.6; IQR, 0-1.1). Three of these 17 patients experienced disease progression at a median of 19.9 months; all 3 experienced ≥1 positive post-RT CTC test predating clinical progression by a median of 16 months (range, 2-17 months). In contrast, among patients presenting with CTC-detectable disease and for whom all post-RT CTC tests were negative, none experienced recurrence or progression. CONCLUSIONS: CTC monitoring after SBRT for presumed early stage NSCLC may give lead-time notice of disease recurrence or progression. Conversely, negative CTC counts after treatment may provide reassurance of disease control. CTC analysis is thus potentially useful in enhancing clinical diagnosis and follow-up in this population.