Probes for TERT

Calcifying nested stromal-epithelial tumor (CNSET) is a rare hepatic tumor that occurs in children and young adults. With <40 cases in the literature, the mechanism for tumorigenesis and the biological behavior of CNSET remain uncertain. Here, we studied the clinicopathologic and molecular genetic features of eight CNSETs. Six patients (75%) were female, and the median age at presentation was 22.5 years (range 14-34 years). The median tumor size was 14 cm (range 2.7-18 cm). All tumors had fibrous stroma that contained organoid nests of epithelioid to spindled tumor cells with moderate amounts of palely eosinophilic cytoplasm and ovoid, vesicular nuclei. Five tumors showed calcifications, and one showed lymphovascular invasion. Necrosis was absent in all. Immunohistochemistry demonstrated nuclear β-catenin expression in five of five tested tumors and focal to diffuse nuclear WT-1 positivity in five of seven. Hepatocellular markers (HepPar-1, arginase-1, and albumin in situ hybridization) and neuroendocrine markers (synaptophysin, chromogranin, and INSM1) were uniformly negative. Next-generation sequencing demonstrated CTNNB1 alterations in all seven sequenced tumors. Sanger sequencing demonstrated TERT promoter mutations in all six sequenced tumors. Clinical follow-up was available for seven patients (median duration 4.4 years; range 1.2-6.2 years): four (57%) developed metastatic disease; all four developed lung metastases; and two also had abdominal metastases. All four patients with metastatic disease also had persistent or recurrent liver tumors. Three patients with metastases were alive with disease at the most recent follow-up and one died of disease. The other three patients with available follow-up did not develop metastasis or recurrence. One tumor treated with neoadjuvant chemotherapy showed no response, and another showed 90% tumor fibrosis; the latter patient remained disease-free at 6.2 years of follow-up. Our series demonstrates the presence of TERT promoter mutations and CTNNB1 alterations in all sequenced tumors and suggests that CNSET might perhaps be more aggressive than previously reported.

Telomeres consist of repetitive DNA sequences and telomere-associated proteins. Telomeres located at the ends of eukaryotic chromosomes undergo shortening due to DNA replication, genotoxic factors and reactive oxygen species. The short telomeres are elongated by the enzyme telomerase expressed in the germ line, embryonic and stem cells. Telomerase is in the structure of ribonucleoprotein composed of telomerase reverse transcriptase (TERT), telomerase RNA component (Terc) and other components. Among telomere-associated proteins, telomeric repeat binding factor 1 (TRF1) and 2 (TRF2) exclusively bind to the double-stranded telomeric DNA to regulate its length. However, protection of telomeres 1 (POT1) interacts with the single-stranded telomeric DNA to protect from DNA damage response. Herein, we characterised the spatial and temporal expression of the TERT, TRF1, TRF2 and POT1 proteins in the postnatal mouse testes at the ages of 6, 8, 16, 20, 29, 32 and 88 days by using immunohistochemistry. Significant differences in the spatiotemporal expression patterns and levels of these proteins were determined in the postnatal testes (p < .05). These findings indicate that TERT and telomere repeat binding proteins seem to be required for maintaining the length and structural integrity of telomeres in the spermatogenic cells from newborn to adult terms.

While P16 immunohistochemistry (IHC) is a well-established surrogate marker of Human Papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC), Retinoblastoma 1 (RB1) loss may lead to p16 overexpression in the absence of HPV. We determined the proportion of p16-positive/HPV-negative OSCC with RB1 loss and other alterations in RB1/p16 pathway, and tested RB1 IHC as a prognostic biomarker for OSCC, along with the 8th edition of AJCC staging manual. P16 and RB1 IHC and HPV DNA in situ hybridization (ISH) were performed on 257 OSCC. High risk HPV RNA ISH, RB1 fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) were done on p16-positive/HPV DNA ISH-negative OSCC. Disease free survival (DFS) was used as an endpoint. In the entire cohort and in p16-positive (n = 184) and p16-negative (n = 73) subgroups, AJCC 8th edition staging correlated with DFS (p < 0.01). RB1 IHC showed RB1 loss in p16-positive OSCC only (79/184, 43%). RB1 loss by IHC is associated with a better DFS, without providing additional prognostic information for patients with p16-positive OSCC. HPV RNA ISH was positive in 12 of 14 HPV DNA ISH-negative cases. RB1 IHC showed loss in 10 of 15 HPV DNA ISH-negative cases and in 1 of 2 HPV RNA ISH-negative cases. Overall, only one case of p16-positive/HPV RNA ISH-negative OSCC showed RB1 loss by IHC (1/184, 0.5%). Of the 10 p16-positive and HPV DNA ISH-negative cases with RB1 loss by IHC, 2 had RB1 hemizygous deletion and 3 showed Chromosome 13 monosomy by FISH. No RB1 mutations were detected by NGS. Other molecular alterations in p16-positive/HPV DNA ISH-negative cases included TP53 and TERT mutations and DDX3X loss. HPV-independent RB1 inactivation rarely results in false positive p16 IHC. RB1 inactivation by high risk HPV E7 oncoprotein may co-exist with RB1 deletion. RB1 loss is a favorable prognosticator and occurs exclusively in p16-positive OSCC. The 8th edition of the AJCC staging manual satisfactorily predicts DFS of OSCC patients.

Dysregulation of alternative splicing of hTERT gene to generate full-length Htert (hTERT-FL) that reactivate telomerase has been recognized as a major pathological alteration in pancreatic cancer (PrCa). Mechanism about the factors that regulate hTERT-FL splicing is lacking. Through bioinformatics approach, we focus on a candidate splicing factor RBM10, which leads to a switch in hTERT transcripts to generate a function-less isoform hTERT-s in PrCa, suppressed both telomerase activity and subsequent telomere shortening. RBM10 expression is negatively associated with PrCa progression. Gain or loss of RBM10 also significantly changed PrCa cell proliferation in vitro and in xenografts. RNA-IP and RNA pull-down assays reveal that RBM10 promotes the exclusion of exons7 and 8 which results in the production of TERT-s transcripts. This study may increase knowledge about potentially targetable cancer associated splicing factors and provide novel insights into therapeutic approach in PrCa. AJCR