Absence of TERT promoter mutations in colorectal precursor lesions and cancer

Abstract Hotspot mutations (c.-124bp G > A and c.-146bp G > A) in the promoter region of the TERT gene have been recently described in several types of solid tumors, including glioma, bladder, thyroid, liver and skin neoplasms. However, knowledge with respect to colorectal precursor lesions and cancer is scarce. In the present study we aimed to determine the frequency of hotspot TERT promoter mutations in 145 Brazilian patients, including 103 subjects with precursor lesions and 42 with colorectal carcinomas, and we associated the presence of such mutations with the patients clinical-pathological features. The mutation analysis was conclusive in 123 cases, and none of the precursor and colorectal carcinoma cases showed TERT promoter mutations. We conclude that TERT mutations are not a driving factor in colorectal carcinogenesis.

Colorectal cancer (CRC) is the third most frequent type of cancer worldwide (Ferlay et al., 2015). This scenario shows the importance to improve strategies for CRC prevention and early detection to decrease its incidence and mortality (Goss et al., 2013). CRC arises from a stepwise evolution of normal mucosa to precursor lesions and ultimately to a malignant tumor. The adenoma is the most commonly reported precursor lesion of CRC (Fearon and Vogelstein, 1990;Lieberman et al., 2000). However, alternative precursor lesions include the serrated polyp, which was recently described as a precursor lesion of CRC. Serrated polyps are known to be a heterogeneous group of colorectal lesions that include hyperplasic polyps (HPs), sessile serrated adenoma (SSA), traditional serrated adenoma (TSA) and mixed polyps. Clinically, HPs are the most common precursor serrated lesions of CRC . Serrated adenocarcinomas accounts for about 10% of all CRCs (Makinen, 2007). The classic genetic model for colorectal tumorigenesis is driven by the progressive accumulation of a series of critical mutations in cancer-related genes, such as APC and KRAS (Fearon and Vogelstein, 1990). Since the molecular alterations among serrated pathways are less understood, the BRAF gene has now emerged as a prevalent marker in this pathway . With the presence of these genetic alterations, molecular biomarkers have been widely proposed as a means of CRC screening and prevention (Imperiale and Ransohoff, 2010).
Recently, hotspot somatic mutations in the TERT promoter region (c.-124bp G > A and c.-146bp G > A) have been described in several tumors, particularly skin, brain, thyroid and bladder cancers (Horn et al., 2013;Huang et al., 2013;Killela et al., 2013;Vinagre et al., 2013;Heidenreich et al., 2014). The TERT gene encodes a telomerase reverse transcriptase, an essential protein for preserving telomere genomic integrity. These mutations result in the creation of new binding motif sites (GGAA) for ETS transcription factors, leading to an increase in TERT activity and subsequent telomere preservation (Horn et al., 2013;Huang et al., 2013). Additionally, these hotspot mutations have been associated with advanced tumor stages and poor prognosis for patients (Killela et al., 2013;Vinagre et al., 2013;Heidenreich et al., 2014). Currently, only one study evaluated TERT mutation frequency in CRC, and no muta-tions were found in colorectal adenocarcinomas (Killela et al., 2013).
Herein, we investigated the frequency of TERT mutations in a series of Brazilian patients with colorectal precursor and cancer lesions. We analyzed 145 Brazilian patients from the Barretos Cancer Hospital. The clinico-pathological and molecular features of the patients were previously reported (Table 1)  . All included patients were over 50 years old, with a mean age of 66 years (ranging from 51 -89), with similar frequency for both genders. Patients with known family history, hereditary CRC, or bowel inflammatory disease were excluded. All cases were reviewed by an expert pathologist and categorized according to the WHO classification. Tumor DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue, as previously reported . TERT promoter mutations were identified by PCR followed by direct sequencing as described elsewhere (Vinagre et al., 2013;Batista et al., 2016).
Of the 145 samples analyzed, 22 were inconclusive due to poor quality/quantity of DNA. The evaluation of hotspot TERT promoter mutations showed that all precursor and cancer lesions (123 samples), which included 45 adenoma polyps, 15 serrated polyps, 22 hyperplastic polyps and 41 adenocarcinomas, were wild-type (Figure 1). Our results are in agreement with a previous report that showed the absence of TERT promoter mutation in colorectal adenocarcinomas (Killela et al., 2013). We also showed for the first time, that these mutations are absent in precursor lesions as well. Furthermore, it is the first study to analyze the TERT mutation status in Brazilian colorectal disease patients.
Telomere length is a major tumor hallmark (Heidenreich et al., 2014). Besides hotspot TERT promoter mutations, other pathways are involved with an increase in telomere length (Heidenreich et al., 2014). One such mechanism is the alternative lengthening of telomeres (ATL) (Cesare and Reddel, 2010;Killela et al., 2013). However, a previous study reported the absence this pathway in CRC (Heaphy et al., 2011). Therefore, the mechanisms of telomere length variation in colorectal tumors are still unknown. Cruvinel-Carloni et al. 83  Concluding, we analyzed for the first time the presence of TERT promoter mutations in precursor and carcinoma colorectal lesions in Brazilian patients. The results showed the lack of TERT promoter mutations, suggesting that these alterations are not involved in CRC carcinogenesis.