Dear Editors, Telomerase is a ribonucleoprotein that consists of an RNA subunit and a reverse transcriptase catalytic subunit. The latter is encoded by telomerase reverse transcriptase (TERT) gene. Telomerase adds telomeric repeats to chromosome ends, thereby maintaining telomere length. It is frequently upregulated in human cancers, contributing to human tumorigenesis. 1 Mutations in the TERT gene promoter have been reported in melanoma, follicular cellderived thyroid cancer, bladder cancer, glioblastoma and other cancers. 2–7 Two of them, 1,295,228 C> T and 1,295,250 C> T (termed C228T and C250T hereafter, respectively), are particularly common. These two mutations have been demonstrated to confer the TERT promoter increased transcriptional activity. 2 Importantly, these mutations are found to be absent in benign tumors and normal human subjects, further implicating their potentially critical roles in human tumorigenesis. However, until now, TERT promoter mutations have not been investigated in a large cohort of gallbladder and gastric cancers. Although a previous study has shown no mutations in TERT gene promoter in gallbladder cancer, the limited number (N5 10) yielded an inconclusive results. 6 In this study, a total of 154 gallbladder cancer tissues and 268 gastric cancer tissues were randomly obtained from the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, with approval by the institutional review board of the Hospital. Clinicopathological data were obtained from the patients’ files or by interview with the patients or their relatives, and were presented in Supporting Information Table 1. Genomic DNA was isolated using standard procedures of protease K digestion, phenol-chloroform extraction and ethanol precipitation. A pyrosequencing assay was performed to examine these two TERT promoter mutations. The following primers were used to amplify a fragment of 162 bp, which contained the sites of C228T and C250T mutations: 50-GCG CTG CCT GAA ACT CGC-30 (sense, biotinylated at the 50-end) and 50-CGT CCT GCC CCT TCA CCT-30 (antisense). PCR was performed with an initial denaturation at 95 C for 3 min, followed by 40 cycles of 95 C denaturation for 30 sec, 59 C annealing for 30 sec and 68 C elongation for 1 min. Quality of PCR products was determined by gel electrophoresis. The primer for pyrosequencing (50-CCC GCC CCC TCC CGA-30) was designed immediately upstream of C250T so that these two mutations are analyzed in the same assay. Pyrosequencing assay was performed on a PyroMark Q24 system using PyroMark Gold Q24 reagent (Qiagen). Sanger sequencing was then performed to confirm the results of pyrosequencing using the same primer pair as for pyrosequencing assay. As shown in Figure 1 and Table 1, similar to the findings in a previous study, the frequency of TERT promoter mutations in esophageal squamous cell carcinoma (ESCC) was very low, 8 we also found low frequency of TERT promoter mutations in primary gallbladder and gastric cancer tissues, and gastric cancer cell lines. C228T and C250T mutations were found in 6 and 8 of 154 (3.9% and 5.2%) gallbladder cancer tissues, respectively, and these two mutations were mutually exclusive in this cancer. Notably, only two C250T mutations were found in 268 gastric cancer tissues, and no C228T or C250T mutations were found in eight gastric cancer cell lines. Given extremely low frequency of TERT promoter mutations in gastric cancer, we only investigated the association of these two mutations with clinicopathological characteristics of gallbladder cancer patients, including gender, age, tumor size …