Comparative study of TERT promoter mutation status within spatially, temporally and morphologically distinct components of urothelial carcinoma.

Abstract

Sir: Telomerase reverse transcriptase (TERT) is a ribonucleoprotein involved in maintaining the length of telomeres. In the absence of TERT expression, differentiated cells can only divide a finite number of times before undergoing cellular senescence – often referred to as the Hayflick limit. Mutations within the promoter region of TERT that create consensus binding sequences for ETS family transcription factors are a common mechanism by which neoplastic cells increase TERT expression and overcome this limit.1 TERT promoter mutations are common in many cancer types, including 60–80% of urothelial carcinomas (UC).2, 3 Given the high frequency of these mutations in UC and the absence of these mutations in non-neoplastic/benign mimics of UC,4 TERT promoter mutations may serve as potential biomarkers for monitoring patients with a history of malignancy. Multiple studies have reported detecting TERT mutations in specimens used commonly for monitoring UC patients, such as urine.2, 3, 5 However, in order to be a reliable marker of residual/recurrent disease, TERT mutation status must be a stable and uniform attribute shared among all neoplastic cells and preserved over time. To our knowledge, no previous study has compared TERT promoter genotypes within spatial, temporal and morphologically distinct components of UC. In order to evaluate the stability and uniformity of TERT mutations within a given UC, we developed an allele-specific polymerase chain reaction (PCR) assay targeting the most common TERT promoter mutations: c.-146C>T (Chr.5:1295250C>T), c.-124C>T (Chr.5:1295228C>T), c.-138_139CC>TT (Chr.5:1295242_1295243CC>TT) and c.-124_125CC>TT (Chr.5:1295228_1295229CC>TT). Using this assay, we evaluated 102 DNA samples extracted from formalin-fixed paraffin-embedded tissues from 50 patients with invasive, high-grade UC. The age range of the patients in this cohort was 50–88 years, with the majority of the patients demonstrating pathological stage pT2b–pT4 at the time of cystectomy.6 In order to determine if TERT mutation status varies among spatially distinct regions of UC, microdissection was performed to isolate distinct regions within the same block for 19 cases and within separate blocks for 20 cases, including three metastatic foci (Figure 1, Supporting information, Table S1). For 26 UC patients, microdissection was performed in order to evaluate conventional UC and components with divergent differentiation separately, including sarcomatoid (five), nested and tubular (eight), micropapillary (seven), squamous (nine), glandular (two), single cell/diffuse/plasmacytoid (two) and neuroendocrine (one). The variant morphologies were assigned as instructed by the WHO classification of tumours of the urinary system and male genital organs, 4th edition.7 To evaluate the temporal stability of TERT mutations, specimens from multiple time-points were evaluated for 11 patients (mean: 2.9 years apart; range: 0.2–8.8 years). Fourteen single-sample cases (conventional UC and divergent differentiation) were also included to establish the frequency of TERT promotor mutations in comparison with previous studies. Overall, TERT mutations were found in in 76.0% (38 of 50) of UC cases, similar to previous studies;2, 3 -124C>T was the most common (34 patients), followed by -146C>T (seven patients) and a single instance of -138_-139CC>TT. TERT status was conserved temporally in all cases evaluated. For morphologically and spatially disparate components, we found TERT mutation status to be conserved perfectly in all but one case. This case harboured a -138_-139CC>TT mutation within conventional UC and a -124C>T mutation within a separate block showing squamous differentiation. These results were confirmed after re-extraction and repeated TERT testing. Further evaluation of these two specimens using the Ion AmpliSeq Cancer Hotspot Panel showed that both components shared a PIK3CA E542K mutation. However, a PTEN R130Q mutation was present with the squamous component, but not in the conventional UC. These results suggest that while these two components are related clonally to one another, each component represents a morphologically and molecularly distinct subclonal population. In conclusion, TERT promoter mutations are conserved in the majority of morphologically, spatially and temporally distinct components of a given urothelial carcinoma. These findings corroborate the notion that components of UC with divergent differentiation remain related clonally to the conventional UC. In rare cases, spatially and morphologically distinct components of UC also show differing TERT genotypes. In this study, we showed that these genotypical differences reflect subclonal populations (intratumoral heterogeneity) within some cases of UC. TERT promoter mutations represent secondary alterations in the pathogenesis of UC and other neoplasms.1, 8 That TERT status is conserved spatially and temporally in most cases reflects the fact that these secondary mutations generally occur early in the pathogenesis of UC.3 Overall, TERT promoter mutation status is a stable biomarker in most cases and may therefore be useful in disease monitoring. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.