TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375−146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Blc-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

MPC-2 Clinical course and prognosis of lower-grade glioma, IDH wildtype and pTERT mutant

Background and Purpose: In the cIMPACT-Now update 3, it was proposed that grade 2 astrocytic gliomas without IDH-mutations and grade 3 astrocytic gliomas with TERT promoter mutations should be designated as diffuse IDH wildtype astrocytic glioma with molecular features of WHO grade IV glioblastoma. Therefore, we investigated whether this group of tumors actually corresponds to grade IV prognostically in cases that we encountered ourselves. Cases and Methods: Among the 65 patients having primary astrocytic glioma who were operated in our hospital from January 2016 to March 2021, the prognostic values of seven patients with lower-grade glioma, IDH wildtype, and pTERT mutant were investigated. Results: Among the seven patients, the median age was 59 years (50–66 years). Four of them had anaplastic astrocytoma, two had diffuse astrocytoma, and no tumor lesion could be identified upon histological examination for one patient. The male-to-female ratio was 1:6. MGMT methylation was observed in two patients (29%). The median survival was 20 months, with a significantly worse prognosis when compared with lower-grade glioma without the TERT promoter mutation (13 patients: median survival 40 months), but a better prognosis when compared with glioblastoma (45 patients: median survival 13 months) (Log-rank p = 0.0051). Conclusion: Although EGFR amplification, combined whole chromosome 7 gain, and whole chromosome 10 loss were not examined, the prognostic value of lower-grade glioma, IDH wildtype, and pTERT mutant was not as poor as that of glioblastoma. Further investigation is required to confirm whether these groups of tumors should be treated in the same way as grade IV glioblastoma.

MPC-1 DNA methylome analysis suggested the presence of “true” IDH-wildtype lower-grade gliomas

Background: There will be significant changes in the diagnosis of IDH-wildtype adult-type gliomas in the upcoming 5th edition of the WHO Classification of Central Nervous System Tumours. IDH-wildtype lower grade gliomas (IDHwt LGGs) that harbor molecular features of glioblastoma (EGFR amplification, the combination of whole chromosome 7 gain and whole chromosome 10 loss (7+/10-), or TERT promoter mutations) will be diagnosed as glioblastomas (GBMs), while IDH-wildtype astrocytomas will not be included as a separate tumor type. However, IDHwt LGGs are a very heterogeneous group of tumors, and further investigation is warranted particularly in those without molecular features of glioblastoma. To elucidate the biology of IDHwt LGGs, we analyzed DNA methylation profile and survival time. Materials and Methods: Of the 724 adult-type diffuse glioma samples from a multi-institutional study, 64 IDHwt LGG, including 54 without any of molecular features of GBM and 10 with PDGFRA amplification or TERT promoter mutation, were examined using Infinium MethylationEPIC BeadChip. The raw data files (IDAT files) were analyzed by the web-based DNA methylation classifier provided by DKFZ (MolecularNeuropathology.org) or by R (Version 4.0.4) using the minfi (1.34.0) and Rtsne (0.15) packages. [Result] Twenty-three out of 54 IDHwt LGGs matched known methylation classes using the DKFZ methylation classifier. In t-Distributed Stochastic Neighbor Embedding clustering analysis, 20 cases formed a cluster within the methylation class family glioblastoma, IDH-wildtype, mainly subclass RTK I (“GBM” cluster). Another 29 IDHwt LGGs formed an independent cluster (“LGG” cluster) separate from any of the existing reference groups near but not overlapping with several subtypes of pediatric-type lower grade gliomas. The “LGG” cluster cases had significantly longer overall survival than the “GBM” cluster cases. Discussion: Methylation profiling showed that IDHwt LGGs without molecular features of GBM were heterogeneous group of tumors. Our data suggested the presence of “true” IDHwt LGGs with intermediate prognosis.

TERT Promoter Mutations Increase Sense and Antisense Transcription from the TERT Promoter

Background: Chief among mechanisms of telomerase reverse transcriptase (TERT) reactivation is the appearance of mutations in the TERT promoter. The two main TERT promoter mutations are C>T transitions located −146C>T and −124C>T upstream from the translational start site. They generate a novel Ets/TCF binding site. Both mutations are mutually exclusive and −124C>T is strikingly overrepresented in most cancers. We investigated whether this mutational bias and mutual exclusion could be due to transcriptional constraints. Methods: We compared sense and antisense transcription of a panel of TERT promoter-luciferase vectors harboring the −124C>T and -146C>T mutations alone or together. lncRNA TAPAS levels were measured by RT-PCR. Results: Both mutations generally increased TERT transcription by 2–4-fold regardless of upstream and downstream regulatory elements. The double mutant increased transcription in an additive fashion, arguing against a direct transcriptional constraint. The −146C>T mutation, alone or in combination with −124C>T, also unleashed antisense transcription. In line with this finding, lncRNA TAPAS was higher in cells with mutated TERT promoter (T98G and U87) than in cells with wild-type promoter, suggesting that lncRNA TAPAS may balance the effect of TERT promoter mutations. Conclusions: −146C>T and −124C>T TERT promoter mutations increase TERT sense and antisense transcription, and the double mutant features higher transcription levels. Increased antisense transcription may contain TERT expression within sustainable levels.

Gliosarcoma with Osteosarcomatous Component: A Case Report and Short Review Illustration

Background: Gliosarcoma (GS) represents a rare variant of glioblastoma in the central nervous system, characterized by biphasic histopathological features of gliomatous and sarcomatous components. Here, we present an unusual case of GS, which also demonstrated osteosarcomatous component. Case presentations: The patient underwent gross total resection (GTR) of right temporal lobe lesion. Subsequently received external beam radiation therapy with 60 Gy and chemotherapy, postoperatively. The sarcomatous portion of the typical fibrosarcoma pattern mingled with areas of osteoid structure in this 65-year-old feminine case. The molecular pathological analysis demonstrated IDH1/2 wild-type and MGMT promoter island methylated phenotype. Target Enrichment Sequencing (TES) was performed on the gliomatous and sarcomatous components of the tumor tissues. TERT promoter, RB1, NF1, TP53 mutations and copy number variations (CNVs) on chromosome 7, 10q, 11q, 12, 13, 17 and 22 were observed in gliomatous and fibro-sarcomatous mixed tumor tissue; While we found TERT promoter, RB1, TP53 mutations and CNVs on chromosome 2q, 3q, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19 and 22 in osteosarcomatous tumor tissue. Noteworthy, EGFR amplification was not observed in both gliomatous and sarcomatous tumor tissues. Conclusions: In conclusion, integrated with histopathology, molecular pathology, and genomic alteration analysis, we report a case of GS with an extremely rare histopathologic phenotype of osteosarcomatous differentiation, also suffered lung multi-metastases. Additionally, by reviewing the literature, our study of this unusual differentiation of GS into osteosarcoma provides novel insight into the natural history of GS.