Telomerase Reverse Transcriptase Gene Promoter Research Articles

DNA Hypermethylation Downregulates Telomerase Reverse Transcriptase (TERT) during H. pylori-Induced Chronic Inflammation.

Helicobacter pylori infection causes chronic gastritis and is the major risk factor of gastric cancer. H. pylori induces a chronic inflammation-producing reactive oxygen species (ROS) which is a source of chromosome instabilities and contributes to the development of malignancy. H. pylori also promotes DNA hypermethylation, known to dysregulate essential genes that maintain genetic stability. The maintenance of telomere length by telomerase is essential for chromosome integrity. Telomerase reverse transcriptase (TERT) is the catalytic component of telomerase activity and an important target during host-pathogen interaction. We aimed to investigate the consequences of H. pylori on the regulation of TERT gene expression and telomerase activity. In vitro, hTERT mRNA levels and telomerase activity were analysed in H. pylori-infected human gastric epithelial cells. In addition, C57BL/6 and INS-GAS mice were used to investigate the influence of H. pylori-induced inflammation on TERT levels. Our data demonstrated that, in vitro, H. pylori inhibits TERT gene expression and decreases the telomerase activity. The exposure of cells to lycopene, an antioxidant compound, restores TERT levels in infected cells, indicating that ROS are implicated in this downregulation. In vivo, fewer TERT-positive cells are observed in gastric tissues of infected mice compared to uninfected, more predominantly in the vicinity of large aggregates of lymphocytes, suggesting an inflammation-mediated regulation. Furthermore, H. pylori appears to downregulate TERT gene expression through DNA hypermethylation as shown by the restoration of TERT transcript levels in cells treated with 5′-azacytidine, an inhibitor of DNA methylation. This was confirmed in infected mice, by PCR-methylation assay of the TERT gene promoter. Our data unraveled a novel way for H. pylori to promote genome instabilities through the inhibition of TERT levels and telomerase activity. This mechanism could play an important role in the early steps of gastric carcinogenesis.

Identification of IDH and TERTp mutation status using 1 H-MRS in 112 hemispheric diffuse gliomas.

There is a growing interest in noninvasively defining molecular subsets of hemispheric diffuse gliomas based on the isocitrate dehydrogenase (IDH) and telomerase reverse transcriptase gene promoter (TERTp) mutation status, which correspond to distinct tumor entities, and differ in demographics, natural history, treatment response, recurrence, and survival patterns. To investigate whether metabolite levels detected with short echo time (TE) proton MR spectroscopy (1 H-MRS) at 3T can be used for noninvasive molecular classification of IDH and TERTp mutation-based subsets of gliomas. Retrospective. In all, 112 hemispheric diffuse gliomas (70 males/42 females, mean age: 42.1 ± 13.9 years). Short-TE 1 H-MRS (repetition time (TR) = 2000 msec, TE = 30 msec, number of signal averages = 192) and routine clinical brain tumor MR protocols were acquired at 3T. 1 H-MRS data were quantified using LCModel software. TERTp and IDH1 or IDH2 (IDH1/2) mutations in the tissue were determined by either minisequencing or Sanger sequencing. Metabolic differences between IDH mutant and IDH wildtype gliomas were assessed by a Mann-Whitney U-test. A Kruskal-Wallis test followed by a Tukey-Kramer test was used to analyze metabolic differences between IDH and TERTp mutational molecular subsets of gliomas. A Spearman rank correlation coefficient was used to assess the correlations of metabolite intensities with the Ki-67 index. Furthermore, machine learning was employed to classify the IDH and TERTp mutational status of gliomas, and the accuracy, sensitivity, and specificity values were estimated. Short-TE 1 H-MRS classified the presence of an IDH mutation with 88.39% accuracy, 76.92% sensitivity, and 94.52% specificity, and a TERTp mutation within primary IDH wildtype gliomas with 92.59% accuracy, 83.33% sensitivity, and 95.24% specificity. Short-TE 1 H-MRS could be used to identify molecular subsets of hemispheric diffuse gliomas corresponding to IDH and TERTp mutations. 3 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;51:1799-1809.

Clinical Routine TERT Promoter Mutational Screening of Follicular Thyroid Tumors of Uncertain Malignant Potential (FT-UMPs): A Useful Predictor of Metastatic Disease.

Mutations of the Telomerase reverse transcriptase (TERT) gene promoter are recurrently found in follicular thyroid carcinoma (FTC) and follicular tumors of uncertain malignant potential (FT-UMP), but nearly never in follicular thyroid adenoma (FTA). We, therefore, believe these mutations could signify malignant potential. At our department, postoperative TERT promoter mutational testing of FT-UMPs was implemented in 2014, with a positive mutation screening leading to vigilant follow-up and sometimes adjuvant treatment. To date, we screened 51 FT-UMPs and compared outcomes to 40 minimally invasive FTCs (miFTCs) with known TERT genotypes. Eight FT-UMPs (16%) displayed TERT promoter mutations, of which four cases underwent a completion lobectomy at the discretion of the patient, and a single patient also opted in for radioiodine (RAI) treatment. Three mutation-positive patients developed distant metastases, registered in one patient receiving a completion lobectomy and in two patients with no additional treatment. Three out of four patients who received additional surgery, including the RAI-treated patient, are still without metastatic disease. We conclude that FT-UMPs with TERT promoter mutations harbor malignant potential and exhibit at least similar recurrence rates to TERT-promoter-mutated miFTCs. Mutational screening should constitute a cornerstone analysis in the histopathological work-up of FT-UMPs.

Toward In Vitro Epigenetic Drug Design for Thyroid Cancer: The Promise of PF-03814735, an Aurora Kinase Inhibitor.

Thyroid cancer (TC) is a very common malignancy worldwide. Chief among the innovative molecular drug targets for TC are epigenetic modifications. Increased telomerase activity in cancer cells makes telomerase a novel target for epigenetic anticancer drug innovation. Recently, telomerase reverse transcriptase (TERT) gene promoter (TERTp) mutations (C228T and C250T) were reported at high frequency in TC cell lines and tumor biopsies. In this study, three representative TC cell lines, mutant TERTp (TPC1), mutant BRAF/TERTp (KTC2), and wild-type TERTp (WRO), were screened with a drug library composed of 51 epigenetic drugs: 14 Aurora kinase inhibitors; 23 histone deacetylase inhibitors; 5 sirtuin modifiers; 3 hypoxia-inducible factor inhibitors; 2 DNA methyltransferase inhibitors; 2 histone methyltransferase inhibitors, a histone demethylase inhibitor, and a bromodomain inhibitor. Effects of the drugs on cell growth at 48 and 72 h were compared. PF-03814735, a small-molecule inhibitor of Aurora kinase A (IC50 = 0.8 nM) and B (IC50 = 5 nM), was the most potent on KTC2 cells, whereas CUDC-101, a multitarget inhibitor, was effective on both WRO and KTC2 cells. Notably, PF-03814735 was found to be the most effective epigenetic drug on cell lines harboring the C228T mutation. In conclusion, these new findings offer specific guidance on dose and time course selection to design novel therapeutic interventions against TC using PF-03814735, and specifically target cells carrying the TERTpC228T mutation. In a larger context of drug discovery science, these findings inform new strategies to forecast optimal treatment regimens for TC, particularly with Aurora kinase inhibitors and in ways guided by epigenetic drug design.

Spectrum of TERT promoter mutations and mechanisms of activation in thyroid cancer.

BackgroundReactivation of telomerase reverse transcriptase (TERT) is an important event in cancer. Two hotspot mutations in the TERT promoter region, c.‐124C > T (C228T) and c.‐146C > T (C250T), occur in various cancer types including thyroid cancer. They generate de novo binding sites for E‐twenty‐six (ETS) transcription factors causing increased TERT transcription. The aim of this study was to search for novel TERT promoter mutations and additional mechanisms of TERT activation in thyroid cancer.MethodsWe studied 198 papillary thyroid carcinomas (PTCs), 34 follicular thyroid carcinomas (FTCs), 40 Hürthle cell carcinomas (HCCs), 14 poorly differentiated/anaplastic thyroid carcinomas (PDTC/ATC), and 15 medullary thyroid carcinomas (MTCs) for mutations in an −424 bp to +64 bp region of TERT. The luciferase reporter assay was used to functionally characterize the identified alterations. Copy number variations (CNVs) in the TERT region were analyzed using TaqMan copy number assay and validated with fluorescence in situ hybridization (FISH).ResultsWe detected the hotspot c.‐124C > T and c.‐146C > T mutations in 7% PTC, 18% FTC, 25% HCC, and 86% PDTC/ATC. One PTC carried a c.‐124C > A mutation. Furthermore, we identified two novel mutations resulting in the formation of de novo ETS‐binding motifs: c.‐332C > T in one MTC and c.‐104_‐83dup in one PTC. These genetic alterations, as well as other detected mutations, led to a significant increase in TERT promoter activity when assayed using luciferase reporter system. In addition, 5% of thyroid tumors were found to have ≥3 copies of TERT.ConclusionsThis study confirms the increased prevalence of TERT promoter mutations and CNV in advanced thyroid cancers and describes novel functional alterations in the TERT gene promoter, including a point mutation and small duplication. These mutations, as well as TERT copy number alterations, may represent an additional mechanism of TERT activation in thyroid cancer.

High frequency of H3K27M immunopositivity in adult thalamic glioblastoma.

Adult thalamic glioblastomas (GBM) are uncommon tumors with limited available molecular data. One of the reported molecular alterations in these tumors is the H3K27M mutation. It has been documented that H3K27M mutation is found in a high proportion of pediatric thalamic gliomas. In this study, we have analyzed the molecular alterations exclusive to adult thalamic GBM. This is a 6 years retrospective study of adult thalamic GBM patients who underwent surgical decompression of the tumor. Clinical data were obtained from the case records. Immunohistochemistry (IHC) was performed on the tumors using antibodies directed against the gene products of R132H mutant isocitrate dehydrogenase 1 (IDH1), alpha-thalassemia/mental retardation X-linked (ATRX), p53, H3K27M, H3K27me3, and V600E mutant BRAF. Molecular analyses were carried out to detect other IDH1 and IDH2 mutations, O6 -methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation, and epidermal growth factor gene (EGFR) and telomerase reverse transcriptase gene (TERT) promoter mutations. A total of 42 cases of adult thalamic GBM were studied. The mean age of presentation was 42 years with age range of 19-58 years. Male predominance was noted. All the tumors were IDH wild-type, BRAF (V600E)-immunonegative and unmethylated for MGMT promoter. H3K27M immunopositivity was noted in 60% of tumors. Of these 33.3% were from older adults above the age of 50 years. Of the H3K27M-immunopositive cases, ATRX loss of expression was seen in 32%, p53 immunopositivity in 24% and EGFR amplification in 12%. Higher frequency of TERT promoter mutations was noted in H3K27M-immunonegative cases (58.8%) compared to immunopositive cases (20%). Ours is one of the few studies elucidating the molecular alterations exclusive to adult thalamic GBM. We show a high frequency of H3K27M immunopositivity, suggestive of its mutational status in these tumors, including in older adults.

Functional comparison of the human epidermal growth factor receptor and telomerase reverse transcriptase promoters in canine tumor cells.

We previously showed that the promoter region of the human epidermal growth factor receptor (hEGFR) gene elicits high transduction efficiency, with transgene expression restricted to canine breast tumor cells. However, it was unclear whether this promoter induces tumor cell-specific transgene expression in canine urothelial carcinoma cells. Furthermore, compared with studies in human cancer cells, the utility of the telomerase reverse transcriptase (TERT) gene promoter for therapeutic transgene expression in canine cancer cells has not been evaluated thus far. Here, we compared the activity of these promoters in canine mammary tumor and urothelial carcinoma cells. Our results showed that compared with the TERT promoter, the hEGFR promoter was more useful as a tumor-specific promoter to induce efficient transgene expression in canine tumor cells.

TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell carcinoma

Telomerase reverse transcriptase gene (TERT) promoter (TERTp) mutations have been reported as potential predictors of poor prognosis in several cancers, but the prognostic value of TERTp mutations for cutaneous squamous cell carcinoma (cSCC) has not been determined. To evaluate the frequency of TERTp mutations and correlate it with clinicopathologic features and patient outcome. We performed genetic profiling of TERTp mutations in a retrospective series of cSCCs. The predictive value of TERTp mutations and clinicopathologic parameters were assessed by using logistic regression models. A total of 152 cSCCs from 122 patients were analyzed for TERTp mutations; the mutation rate was 31.6% (48 of 152), and it was higher in invasive cSCC (42 of 121 [34.7%]) than in in situ cSCC (6 of 31 [19.4%]). Age older than 75years (odds ratio [OR], 14.84; P=.013] and TERTp mutation (OR, 8.11; P=.002) were independent predictors of local recurrence. TERTp mutation (OR, 15.89; P=.022) was independently associated with higher risk of lymph node metastasis. The restricted number of metastatic cases. TERTp mutations may prove to be a molecular biomarker with prognostic significance in invasive cSCC, but larger studies are needed.

TERTpromoter mutations are associated with poor prognosis and cell immortalization in meningioma

Meningiomas are mostly benign tumors tending to progress to higher-grade lesions. Mutations in the telomerase reverse transcriptase (TERT) gene promoter are comparably rare in meningioma, but were recently suggested to predict risk of recurrence and progression. Here we have analyzed a cohort of World Health Organization grades I-III meningiomas regarding the impact of TERT promoter mutations on patient prognosis and in vitro cell propagation feasibility. From 110 meningioma patients, 128 tissue samples were analyzed for the TERT promoter mutations C228T and C250T by direct sequencing. Of the 128 samples, 121 were tested for cell propagation in vitro. Telomerase activity, TERT mRNA expression, and telomere lengths were investigated by telomeric repeat amplification protocol assay, reverse transcription PCR, and quantitative PCR, respectively. Impact of the E-twenty-six (ETS) transcription factor inhibitor YK-4-279 on cell viability and TERT promoter activity was analyzed. TERT promoter mutations were found in 5.5% of all samples analyzed and were associated with a significantly upregulated telomerase activity and TERT mRNA expression (P < 0.0001 both). Regarding telomere lengths, no significant difference between the TERT promoter wild-type and mutated subgroups was detected. Patients with TERT promoter mutated tumors exhibited significantly shorter overall survival (P = 0.0006; 53.8 vs 115.6 mo). The presence of TERT promoter mutations but not telomerase activity or TERT mRNA expression predicted indefinite cell growth in vitro. TERT promoter mutated meningioma cells were hypersensitive against the ETS transcription factor inhibitor YK-4-279, inducing a distinct downregulation of TERT promoter activity. TERT promoter mutations drive meningioma aggressiveness, resulting in reduced patient survival, but might also open novel therapeutic options for progressive disease.

Glioblastoma in neurofibromatosis 1 patients without IDH1, BRAF V600E, and TERT promoter mutations.

Pilocytic astrocytomas and low-grade gliomas are more common compared with glioblastomas in patients with neurofibromatosis 1 (NF1). A recent genome-wide analysis has shown frequent NF1 gene alterations in the mesenchymal subtype of a glioblastoma; however, little is known about clinicopathological features of glioblastomas in NF1 patients (NF1 glioblastomas). We analyzed four NF1 glioblastomas. Radiographical and intraoperative findings showed well-circumscribed tumors from surrounding brain. Pathological analysis presented a paucity of processes with an eosinophilic cytoplasm, bizarre nuclei, xanthomatous-like appearance, multinucleated giant cells, and histiocytoid appearance. During the follow-up period, one patient died at 49months and others remained alive for 60, 87, and 106months; thus, patients with NF1 glioblastoma presented a relatively favorable survival. None of the NF1 glioblastomas harbored isocitrate dehydrogenase 1 (IDH1) gene mutation, v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) gene mutation, and telomerase reverse transcriptase (TERT) gene promoter mutation. We identified that NF1 glioblastoma is a unique subset of glioblastoma.

The frequency and prognostic effect of TERT promoter mutation in diffuse gliomas

Mutations in the telomerase reverse transcriptase gene promoter (TERTp) are common in glioblastomas (GBMs) and oligodendrogliomas (ODGs), and therefore, have a key role in tumorigenesis and may be of prognostic value. However, the extent of their prognostic importance in various gliomas is controversial. We studied 168 patients separated into five groups: Group 1: 65 patients with ODG carrying an IDH1 or IDH2 mutation (IDH-mutant) and 1p/19q–codeletion, Group 2: 23 patients with anaplastic astrocytoma (AA), IDH-mutant, Group 3: 13 patients with GBM, IDH-mutant, Group 4: 15 patients with AA, IDH-wildtype (WT), and Group 5: 52 patients with GBM, IDH-WT. TERTp mutations were found in 96.9%, 4.4%, 76.9%, 20.0%, and 84.6% of patients in Groups 1, 2, 3, 4, and 5, respectively. The R132H mutation in IDH1 was found in 60.5% (23/38) of patients in the AA cohort (Groups 2 and 4) and 20.0% (13/65) of patients from our GBM cohort (Groups 3 and 5), whereas all patients with ODG (Group 1) had a mutation either in IDH1 (n = 62) or IDH2 (n = 3). Using Kaplan Meier survival analysis, we found that the TERTp mutation was correlated with poor overall survival (OS) in Groups 2 and 4 combined (P = 0.001) and in Group 4 (P = 0.113), and in multivariate analysis, the TERTp mutant group was associated with significantly poor survival in Group 5 (P = 0.045). However, IDH mutation, MGMT methylation, and younger patient age (<55 years old) were significantly correlated with favorable OS (all P < 0.05) in our cohort of astrocytic and ODGs. In patients with ODG (Group 1), mutant IDH and TERTp did not have prognostic value because these mutations were universally present. Based on the revised 2016 WHO classification of gliomas, we found that TERTp mutation was frequently present in patients with GBM or ODG and because it was strongly correlated with poor survival outcome in patients with IDH-WT GBM in multivariate analysis, it may be of prognostic value in this subgroup of patients with gliomas.

Diagnostic implications of TERT promoter mutation status in diffuse gliomas in a routine clinical setting.

IDH (isocitrate dehydrogenase) gene mutations are present in most diffuse low-grade gliomas and define the clinico-pathological core of the respective morphologically defined entities. Conversely, according to the 2016 WHO classification, the majority of glioblastomas belong to the IDH-wildtype category, which is defined by exclusion. TERT (telomerase reverse transcriptase gene) promoter mutations have been suggested as a molecular marker for primary glioblastomas. We analyzed molecular, histopathological, and clinical profiles of a series of 110 consecutive diffuse gliomas (WHO grades II-IV) diagnosed at our institution, in which TERT promoter mutation analysis had been performed as part of diagnostic work-up. A diagnostic algorithm based on IDH, TERT, ATRX, H3F3A, and 1p19q co-deletion status resulted in a consistent molecular classification with only 14 (13%) marker-negative tumors. TERT promoter mutations were present in 77% of IDH-wildtype tumors. The TERT/IDH-wildtype category was highly enriched for tumors with unconventional clinical or histological features. Molecular classes were associated with distinct rates of MGMT promoter methylation. We conclude that, in a routine diagnostic setting, TERT promoter mutations define a relatively homogeneous core group among IDH-wildtype diffuse gliomas that includes the majority of primary glioblastomas as well as their putative precursor lesions.

Erratum to: The role of histone modifications and telomere alterations in the pathogenesis of diffuse gliomas in adults and children.

Genetic profiling is an increasingly useful tool for sub-classification of gliomas in adults and children. Specific gene mutations, structural rearrangements, DNA methylation patterns, and gene expression profiles are now recognized to define molecular subgroups of gliomas that arise in distinct anatomic locations and patient age groups, and also provide a better prediction of clinical outcomes for glioma patients compared to histologic assessment alone. Understanding the role of these distinctive genetic alterations in gliomagenesis is also important for the development of potential targeted therapeutic interventions. Mutations including K27M and G34R/V that affect critical amino acids within the N-terminal tail of the histone H3 variants, H3.3 and H3.1 (encoded by H3F3A and HIST1H3B genes), are prime examples of mutations in diffuse gliomas with characteristic clinical associations that can help diagnostic classification and guide effective patient management. These histone H3 mutations frequently co-occur with inactivating mutations in ATRX in association with alternative lengthening of telomeres. Telomere length can also be maintained through upregulation of telomerase reverse transcriptase (TERT) expression driven by mutation within the TERT gene promoter region, an alteration most commonly found in oligodendrogliomas and primary glioblastomas arising in adults. Interestingly, the genetic alterations perturbing histone and telomere function in pediatric gliomas tend to be different from those present in adult tumors. We present a review of these mutations affecting the histone code and telomere length, highlighting their importance in prognosis and as targets for novel therapeutics in the treatment of diffuse gliomas.

The role of histone modifications and telomere alterations in the pathogenesis of diffuse gliomas in adults and children.

Genetic profiling is an increasingly useful tool for sub-classification of gliomas in adults and children. Specific gene mutations, structural rearrangements, DNA methylation patterns, and gene expression profiles are now recognized to define molecular subgroups of gliomas that arise in distinct anatomic locations and patient age groups, and also provide a better prediction of clinical outcomes for glioma patients compared to histologic assessment alone. Understanding the role of these distinctive genetic alterations in gliomagenesis is also important for the development of potential targeted therapeutic interventions. Mutations including K27M and G34R/V that affect critical amino acids within the N-terminal tail of the histone H3 variants, H3.3 and H3.1 (encoded by H3F3A and HIST1H3B genes), are prime examples of mutations in diffuse gliomas with characteristic clinical associations that can help diagnostic classification and guide effective patient management. These histone H3 mutations frequently co-occur with inactivating mutations in ATRX in association with alternative lengthening of telomeres. Telomere length can also be maintained through upregulation of telomerase reverse transcriptase (TERT) expression driven by mutation within the TERT gene promoter region, an alteration most commonly found in oligodendrogliomas and primary glioblastomas arising in adults. Interestingly, the genetic alterations perturbing histone and telomere function in pediatric gliomas tend to be different from those present in adult tumors. We present a review of these mutations affecting the histone code and telomere length, highlighting their importance in prognosis and as targets for novel therapeutics in the treatment of diffuse gliomas.

Avian Leukosis Virus Activation of an Antisense RNA Upstream of TERT in B-Cell Lymphomas.

Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering the expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep-sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) gene promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3,552 nucleotides (nt) upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation to TERT Transcriptome-sequencing (RNA-seq) analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is upregulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives the expression of a chimeric transcript containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving overexpression of the TERT antisense RNA suggest it may have a role in tumorigenesis. The data suggest that ALV integrations in the TERT promoter region drive the overexpression of a novel antisense RNA and contribute to the development of lymphomas.

Abstract 1356: Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity

Abstract Cellular immortality is a near-universal phenotype of cancer cells with the central role played by the multicomponent telomerase complex firmly established. The reactivation of telomerase is found in approximately 85% of all cancers. Furthermore, frequent activating non-coding mutations in the hTERT promoter region have been identified in multiple tumour types. A considerable body of evidence supports the concept of targeting the signal transduction pathways that contribute to telomerase overexpression in cancer cells. We have exploited our knowledge in this area to develop cell-based luciferase reporter gene assays to measure hTERT and hTR gene promoter activity. To this end we undertook high throughput screening in the A2780 human ovarian cancer cell line against both hTR and hTERT promoters. Secondary specificity and cytotoxicity assays reduced the initial hits to a single chemical series of dual inhibitors which strongly suppress both hTR and hTERT promoter activity without primary cytotoxicity, as predicted of telomerase inhibitors. Through a process of Hit to Lead we have further developed the chemical series. Lead compounds are active across a panel of cell lines from distinct histological origins, reduce endogenous hTERT mRNA levels and decrease telomerase catalytic activity at nM concentrations. Furthermore, these compounds repress both wild type and mutant C228T and C250T hTERT promoter activity. In specificity assays no off-target activity has been identified in an expanded promoter screen against a panel of 15 off-target promoter reporters. Citation Format: Alan Bilsland, Yu Liu, Sharon Burns, David Jenkinson, Jon Roffey, W. Nicol Keith. Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1356.

BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients.

The role of telomerase reverse transcriptase (TERT) gene promoter mutations in the aggressiveness of papillary thyroid cancer (PTC) remains to be further investigated. Here we examined the relationship of TERT promoter mutations and BRAF V600E with the clinicopathological features of PTC in 653 patients. Sanger sequencing of genomic DNA from primary PTC tumors was performed for mutation detection and genotype-clinicopathological correlation of the tumor was analyzed. BRAF V600E and TERT promoter mutations were found in 63.7% (416 of 653) and 4.1% (27 of 653) of patients, respectively; the latter became 9.8% when only tumors ≥ 1.5 cm were analyzed. TERT promoter mutations occurred more frequently in BRAF mutation-positive cases compared to wild-type cases, being 5.3% in the former versus 2.1% in the latter (P = 0.050). BRAF and TERT promoter mutations were each significantly associated with high-risk clinicopathological features of PTC, such as old patient age, large tumor size, extrathyroidal invasion, capsular invasion, and advanced disease stages. Coexistence of BRAF V600E and TERT promoter mutations was particularly associated with high-risk clinicopathological features, as exemplified by extrathyroidal invasion seen in 54.5% (12/22) of patients harboring both mutations versus 9.9% (23/232) of patients harboring neither mutation (P < 0.001). Thus, this study, the largest on TERT mutation so far, demonstrates a significant role of BRAF V600E and TERT promoter mutations in the aggressiveness of PTC, which is particularly robust and cooperative when the two mutations coexist. These results, together with previous studies, establish a significant role of these mutations in the aggressiveness of PTC.

Hotspot TERT promoter mutations are rare events in testicular germ cell tumors.

The abnormal activation of telomerase, codified by the telomerase reverse transcriptase (TERT) gene, is related to one of cancer hallmarks. Hotspot somatic mutations in the promoter region of TERT, specifically the c.-124:C>T and c.-146:C>T, were recently identified in a range of human cancers and have been associated with a more aggressive behavior. Testicular germ cell tumors frequently exhibit a good prognosis; however, the development of refractory disease is still a clinical challenge. In this study, we aim to evaluate for the first time the presence of the hotspot telomerase reverse transcriptase gene promoter mutations in testicular germ cell tumors. A series of 150 testicular germ cell tumor cases and four germ cell tumor cell lines were evaluated by PCR followed by direct Sanger sequencing and correlated with patient's clinical pathological features. Additionally, we genotyped the telomerase reverse transcriptase gene promoter single nucleotide polymorphism rs2853669 (T>C) located at -245 position. We observed the presence of the TERT promoter mutation in four patients, one exhibited the c.-124:C>T and three the c.-146:C>T. No association between TERT mutation status and clinicopathological features could be identified. The analysis of the rs2853669 showed that variant C was present in 22.8% of the cases. In conclusion, we showed for the first time that TERT promoter mutations occur in a small subset (~3%) of testicular germ cell tumors.

TERT promoter mutations in periocular carcinomas: implications of ultraviolet light in pathogenesis

Background/aimsUltraviolet light-signature mutations in the telomerase reverse transcriptase (TERT) gene promoter have been identified in cutaneous melanomas, basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs). Whether these mutations also...

Abstract 3535: A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer

Abstract Pancreatic cancer is one of the worst prognosis disease all over the world and has an overall 5-year survival rate of less than 5%. At the time of diagnosis, more than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis. Moreover, even after curative surgery, pancreatic cancers still show poor prognosis due to local recurrence and systemic metastasis. Gemcitabine is the standard chemotherapeutic agent to treat advanced pancreatic cancers, but median overall survival ranged from 5.0 to 7.2 months, suggesting that standard chemotherapy is less effective to most of pancreatic cancer patients. Therefore, the development of novel therapeutic strategy is needed for the treatment of pancreatic cancers. We developed a novel oncolytic adenovirus, OBP-702, in which a human telomerase reverse transcriptase gene promoter drives viral E1 gene for virus replication, and armed with the wild-type tumor suppressor p53 gene. Precise antitumor effects of OBP-702 have been evaluated in human pancreatic cancer cells. We used four pancreatic cancer cell lines with different invasion ability, including non-invasive type (MIA PaCa-2, Panc-1) and invasive type (BxPC-3, Capan-1). The antitumor effect of OBP-702 for each cell line was assessed using XTT assay. OBP-702 induced profound anti-tumor effect in all human pancreatic cancer cells, whereas OBP-301 lacking the p53 tumor suppressor gene showed differential effects among the cell types. OBP-301 induced moderate anti-tumor effect in MIA Paca-2, BxPC-3 and Capan-1 cells and strong anti-tumor effect in Panc-1 cells in a dose-dependent manner, suggesting the broad spectrum of OBP-702's efficacy. The molecular mechanism of anti-tumor effect was assessed by Western blot analysis. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. These results suggest that an oncolytic adenovirus, OBP-702, is a promising antitumor agent to induce profound cell death in invasive human pancreatic cancer cells. The clinical trial of intratumoral administration of OBP-702 in patients with invasive pancreatic cancer is warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Naoto Hori, Shuta Tamura, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2015-3535