Study On TERT Promoter Mutation In Uremic Tumor

BackgroundUrothelial carcinoma (UC) is the most common malignant tumor of urinary system. In recent years, incidence of urothelial carcinoma is increasing gradually in the world, but at present, the tumor pathogenesis is not yet clear. In addition to the imagological examination, the diagnosis and postoperative review of urothelial bladder cancer (UBC) also need cystoscopy, which is expensive and invasive, so looking for a noninvasive examination, such as biomarkers, already becomes a research hotspot. In eukaryotes, the ends of chromosomes are consist of 8 to 20kb tandem repeat DNA sequences (TTAGGG), called telomeres, which are protected by related proteins and form telomere protein complexes, and they are important structural units to seal chromosomes, avoid chromosomes fusion or degradation. In normal cells, telomeres replicate incompletely in mitosis, so as the cell division numbers increase, telomere sequences are gradually reduced. When telomere sequences are not enough to sustain normal structure and function of chromosomes, chromosome DNA will break down and finally cells enter into the process of senescence or apoptosis, which called Hayflick limit. Telomerase is an RNA-dependent DNA polymerase, which is composed of proteins and RNA, and its function is mainly completed by telomerase reverse transcriptase (TERT) and telomerase RNA template, also including some related binding factors, such as DKC, N0P10, GAR1 and NHP2. TERT is the activated component and limiting step of telomerase, and at the end of DNA replication, telomerase compounds telomere repeat sequences by RNA template reverse transcription. 85%-90% of the tumor cells have continuous high expression of telomerase, and can compound lost telomere repeat sequences through abnormal activation of telomerase and/or non-traditional telomere extension mechanism, which is considered to play an important role in the process of cell immortalization and tumor formation. Human TERT gene is on chromosome 5p15.33, and there are 2 copies of TERT gene in the normal somatic cells. The regulation of TERT gene is affected by many factors, including transcription, RNA cut, RNA mature, post-transcriptional modification, translation, assembling, epigenetic regulation and gene copy number change, especially reflected in transcription regulation. In recent years, some researches show that TERT promoter mutation is associated with a wide variety of tumors.ObjectiveOur research detects TERT promoter mutation in tumor tissues and urine from patients with urothelial carcinoma, to explore whether TERT promoter can be an ideal biomarker for urothelial carcinoma, which may contribute to diagnosis, postoperative follow-up and early detection of tumor recurrence. We will detect TERT promoter mutation in tumor tissues and urine, and compare expression differences of TERT promoter mutation in tumor tissues and urine, then analyze the correlation between TERT promoter mutation and clinical characteristics, also explore the correlation between TERT promoter mutation in postoperative follow-up urine and tumor recurrence.MethodsWe select patients with urothelial carcinoma in July 2015 to March 2016, who receives surgical treatment in Peking Union Medical College Hospital (PUMCH), involving 42 cases of tumor tissue specimen (paraffin embedding) and 99 cases of urine specimen before surgery. We also select 26 cases of urine specimen from patients without urothelial carcinoma, and 22 cases of urine specimen from postoperative patients with UBC before cystoscopy. We consult clinical medical records of these patients, to get the baseline data, clinical stage and pathological grade, and then detect TERT promoter mutation in tumor tissues and urine through Sanger sequencing method. Finally, we use SPSS 19.0 to analyze expression differences of TERT promoter mutation in other diseases of urinary system, to analyze the correlation between TERT promoter mutation and clinical characteristics, and to explore whether TERT promoter can be an ideal biomarker used in clinical diagnosis and prognosis of urothelial carcinoma.Result1. TERT promoter mutation in tumor tissues42 cases of tumor tissue specimen are composed of 7 cases of renal pelvis carcinoma (RPC),7 cases of ureteral carcinoma (UC) and 28 cases of UBC. In tumor tissue specimens,25 cases (59.5%) have TERT promoter mutation, among them,8 cases (32.0%) have-124bp G>A mutation,17 cases (68.0%) have-146bp G>A mutation. TERT promoter mutation rate in male patients (77.8%) is higher than female patients (26.7%) (P=0.001). In 7 cases of RPC tissue specimen,3 cases (42.9%) have TERT promoter mutation, which are all-146bp G>A mutation. In 7 cases of UC tissue specimen,3 cases (42.9%) have TERT promoter mutation, among them,1 case (33.3%) has-124bp G>A mutation,2 cases (66.7%) have-146bp G>A mutation. In 28 cases of UBC tissue specimen, 19 cases (67.9%) have TERT promoter mutation, among them,7 cases (36.8%) have-124bp G>A mutation,12 cases (63.2%) have-146bp G>A mutation, and TERT promoter mutation rate in tumor diameter≥3cm (90.9%) is higher than tumor diameter<3cm (52.9%) (P=0.049)2. TERT promoter mutation in urine2.1 TERT promoter mutation in urine with urothelial carcinoma99 cases of urine specimen are composed of 9 cases of RPC,16 cases of UC and 74 cases of UBC. In urine specimens,23 cases (23.2%) have TERT promoter mutation, among them,13 cases (56.5%) have-124bp G>A mutation, 10 cases (43.5%) have-146bp G>A mutation. TERT promoter mutation rate in male patients (30.8%) is higher than female patients (8.8%) (P=0.014), TERT promoter mutation rate in clinical stage≥ pT2 (40.7%) is higher than clinical stage pTa+pTl (16.7%) (P=0.012), TERT promoter mutation rate in tumor diameter≥ 3cm (38.7%) is higher than tumor diameter<3cm (16.2%) (P=0.014). In 9 cases of RPC urine specimen,2 cases (22.2%) have TERT promoter mutation, among them,1 case (50.0%) has-124bp G>A mutation,1 case (50.0%) has-146bp G>A mutation. In 16 cases of UC urine specimen, 2 cases (12.5%) have TERT promoter mutation, among them,1 case (50.0%) has-124bp G>A mutation,1 case (50.0%) has-146bp G>A mutation. In 74 cases of UBC urine specimen,19 cases (25.7%) have TERT promoter mutation, among them,11 cases (57.9%) have-124bp G>A mutation,8 cases (42.1%) have-146bp G>A mutation, and TERT promoter mutation rate in pathological grade G3 (35.0%) is higher than pathological grade G1+G2 (14.7%) (P=0.046). We choose Sanger sequencing method in tumor tissues as the gold standard, and in 42 cases of patients with urothelial carcinoma, sensibility of Sanger sequencing method to detect TERT promoter mutation in urine is 52.0%, specificity is 100%, and accuracy is 71.4%.2.2 TERT promoter mutation in postoperative urineWe detect TERT promoter mutation in 22 cases of urine specimen (6 cases of tumor recurrence,16 cases of no tumor recurrence) from postoperative patients with UBC before cystoscopy, only 3 cases (13.6%) have TERT promoter mutation, among them,2 cases (66.7%) have-124bp G>A mutation,1 case (33.3%) has-146bp G>A mutation. In 6 cases of urine specimen from patients with tumor recurrence,2 cases (33.3%) have TERT promoter mutation. There is no obvious correlation between TERT promoter mutation in postoperative follow-up urine and tumor recurrence.2.3 TERT promoter mutation in urine without urothelial carcinomaWe choose 26 cases of patients without urothelial carcinoma as control group, including 9 cases of renal tumor,6 cases of adrenal tumor,6 cases of urinary calculi,3 cases of prostatic hyperplasia and 2 cases of prostate cancer. There is no TERT promoter mutation detected in their urine, which accounts for relative organized specificity of TERT promoter mutation.ConclusionFrom the above, TERT promoter mutation is widespread, and can be detected in both tumor tissues and urine. In tumor tissues, TERT promoter mutation rate of urothelial carcinoma is 59.5%(25/42), which is a high probable event, and widely exists in various pathological grade and clinical stage, which means that TERT promoter mutation may occur in the early formation process of tumor, and be associated with the beginning of tumorigenesis. In urine, TERT promoter mutation rate of urothelial carcinoma is 23.2% (23/99), and there are close correlation between TERT promoter mutation and sex, clinical stage, pathological grade, tumor diameter. There is no TERT promoter mutation detected in urine from patients without urothelial carcinoma, so TERT promoter may be an ideal biomarker used in clinical diagnosis and prognosis of urothelial carcinoma with high pathological grade (35.0%) and/or high clinical stage (40.7%). TERT promoter mutation sites detected in tumor tissues and urine are exactly the same, specificity of Sanger sequencing method to detect TERT promoter mutation in urine is high but sensibility is low, so we need to find a higher sensitivity detection method to detect tiny proportion of TERT promoter mutation that exists in urine.