The C228T mutation of TERT promoter frequently occurs in bladder cancer stem cells and contributes to tumorigenesis of bladder cancer

Bladder cancer is one of the most common malignant tumors worldwide. Bladder cancer stem cells (BCSCs) have been isolated recently but have not been defined yet. Here we sorted BCSCs from bladder tumor tissues or normal bladder stem cells (NBBCs) from adjacent normal bladder tissues. We found that the C228T mutation (chr5, 1, 295, 228 C > T) of TERT promoter frequently occurs in BCSCs, but not exist in NBBCs. Importantly, introducing the C228T mutation in NBBCs causes TERT overexpression and transformation of bladder cancer. Restoration of the C228T mutation to T228C in BCSCs can recover the TERT expression to a basal level and abolish tumor formation. Additionally, the C228T mutation of TERT promoter triggers TERT expression leading to increased telomerase activity. TERT expression levels are consistent with clinical severity and prognosis of bladder cancer.     

TERT promoter mutations are associated with distant metastases in upper tract urothelial carcinomas and serve as urinary biomarkers detected by a sensitive castPCR

TERT promoter C228T and C250T mutations occur in various malignancies including bladder cancer (BC) and may serve as urinary tumor markers. However, the mutation association with clinical variables in upper tract urothelial carcinomas (UTUCs) is unclear. There is also a lack of sensitive tools to detect the minor mutant TERT promoter in bulk urinary DNA. Here we analyzed 220 UTUC patients [98 with renal pelvic carcinoma (RPC) and 122 with ureter carcinoma (UC)] and developed a Competitive Allele-Specific TaqMan PCR (castPCR) for urinary assay. We identified C228T or C250T mutations in 42 of 98 (43%) RPC and 23 of 122 (19%) UC tumors. Distant metastases were significantly correlated with UTUC patients harboring TERT promoter mutations (P = 0.001). C228T were detected in 6/10 and 9/10 of urine samples from patients with mutation-carrying tumors using Sanger sequencing and castPCR, respectively. When urine samples from 70 BC patients were analyzed together, the sensitivity of urinary C228T assay was 89% and 50% for castPCR and Sanger sequencing, respectively (P < 0.001). Collectively, TERT promoter mutations occur in UTUCs with a high frequency in RPCs and predict distant metastasis. castPCR assays of the mutation are a useful tool for urine-based diagnostics of urological malignancies.     

TERT Promoter Mutations and TERT mRNA but Not FGFR3 Mutations Are Urinary Biomarkers in Han Chinese Patients With Urothelial Bladder Cancer

The TERT promoter and FGFR3 gene mutations are two of the most common genetic events in urothelial bladder cancer (UBC), and these mutation assays in patient urine have been shown to be promising biomarkers for UBC diagnosis and surveillance. These results were obtained mainly from studies of patients with UBC in Western countries, and little is known about such information in Han Chinese patients with UBC. In the present study, we addressed this issue by analyzing tumors from 182 Han Chinese patients with UBC and urine samples from 102 patients for mutations in the TERT promoter and FGFR3 and TERT mRNA expression in tumors and/or urine. TERT promoter and FGFR3 mutations were identified in 87 of 182 (47.8%) and 7 of 102 (6.7%) UBC cases, respectively. In 46 urine samples from patients with TERT promoter mutation‐carrying tumors, the mutant promoter was detected in 24 (52%) prior to operation and disappeared in most examined urine samples (80%) taken 1 week after operation. TERT mRNA was detected in urine derived from 46 of 49 patients (94%) that was analyzed before operation independently of the presence of TERT promoter mutations. Collectively, FGFR3 mutations occur at a very low rate in Han Chinese UBC and cannot serve as diagnostic markers for Chinese patients. Han Chinese patients with UBC have relatively low TERT promoter mutation frequency compared with patients in Western countries, and simultaneous detection of both mutant TERT promoter and TERT mRNA improves sensitivity and specificity of urine‐based diagnosis.     

TERT promoter mutations in renal cell carcinomas and upper tract urothelial carcinomas

TERT promoter mutations are identified in many malignancies including bladder cancer (BC) and upper tract urothelial carcinoma (UTUC). In contrast, no mutations were found in renal cell carcinoma (RCC) as reported in a recent study. Because the mutant TERT promoter in urine DNA was recently tested as a marker for BC, it is important to ascertain whether these mutations are truly absent in RCCs. Here we determined TERT promoter mutations in 109 patients with RCC and 14 patients with UTUC. The mutations were found in 9/96 (9.3%) clear cell RCC (ccRCC) tumors and 1/8 (13%) chromophobe RCC tumors. Among ccRCC patients, the mutation was correlated with the advanced stages and metastasis, and higher TERT expression. Among UTUCs, the mutation was detected in tumors from 3/5 (60%) patients with renal pelvic cancer and 1/9 (11%) patients with ureter cancer. The mutation was also detected in 1 of 4 urine samples from patients with mutation+ UTUC. Collectively, TERT promoter mutations do occur in RCCs and are associated with aggressive disease. The mutation is more frequent in renal pelvic cancer. Thus, the mutant TERT promoter found in urine may come from not only BC, but also RCC or UTUC.     

DNA variants of DLC-1, a candidate tumor suppressor gene in human hepatocellular carcinoma

The DLC-1 gene encoding a regulator of the Rho family of small GTPases is altered in breast, prostate, colon, and liver cancer and has several characteristics of a tumor suppressor gene. DLC-1 overexpression causes inhibition of in vitro growth of liver tumor cells and complete suppression of in vivo tumorigenicity of breast tumor cells. Inactivation and aberrant expression of DLC-1 in human hepatocellular carcinoma (HCC) is frequently associated with hemizygous and homozygous genomic deletion and promoter methylation. Since inactivation of tumor suppressor genes in cancer cells is also commonly associated with point mutation, we evaluated the incidence of mutation of the DLC-1 gene by PCR-SSCP in 17 primary HCC and 18 HCC cell lines. One missense mutation was detected at codon 991 of exon 12 (C-->T transition, Val-->Ile) in an HCC cell line. In addition, two types of polymorphisms were identified: a G-->T at codon 745 of exon 9, a T-->C at 17 bp downstream of exon 2. While the pathogenic relevance of the intronic polymorphism is not known, the low rate of mutation of the DLC-1 gene in HCC implies that genomic deletion and promoter methylation primarily account for the altered expression and tumor suppressive inactivation of the DLC-1 gene.     

FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis.

PURPOSE: Tumor-infiltrating lymphocytes represent the host immune response to cancer. CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress the immune reaction. The aim of the present study was to investigate the clinicopathologic significance and roles of Tregs and CD8+ T cells during hepatocarcinogenesis. EXPERIMENTAL DESIGN: We examined the infiltration of FOXP3+ Tregs and CD8+ T cells in the tumor stroma and nontumorous liver parenchyma using 323 hepatic nodules including precursor lesions, early hepatocellular carcinoma (HCC), and advanced HCC, along with 39 intrahepatic cholangiocarcinomas and 59 metastatic liver adenocarcinomas. We did immunohistochemical comparative studies. RESULTS: The prevalence of Tregs was significantly higher in HCC than in the nontumorous liver (P<0.001). The patient group with a high prevalence of Tregs infiltrating HCC showed a significantly lower survival rate (P=0.007). Multivariate analysis revealed that the prevalence of Tregs infiltrating HCC was an independent prognostic factor. The prevalence of Tregs increased in a stepwise manner (P<0.001) and that of CD8+ T cells decreased during the progression of hepatocarcinogenesis (P<0.001). Regardless of the presence of hepatitis virus infection or histopathologic evidence of hepatitis, the prevalence of Tregs was significantly increased in nontumorous liver bearing primary hepatic tumors. CONCLUSIONS: Tregs play a role in controlling the immune response to HCC during the progression of hepatocarcinogenesis. It has been suggested that primary hepatic cancers develop in liver that is immunosuppressed by a marked infiltration of Tregs. A high prevalence of Tregs infiltrating HCC is thought to be an unfavorable prognostic indicator.     

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.     

CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

Cancer up-regulated drug resistant (CUDR) is a novel non-coding RNA gene. Herein, we demonstrate excessive CUDR cooperates with excessive CyclinD1 or PTEN depletion to accelerate liver cancer stem cells growth and liver stem cell malignant transformation in vitro and in vivo. Mechanistically, we reveal the decrease of PTEN in cells may lead to increase binding capacity of CUDR to CyclinD1. Therefore, CUDR-CyclinD1 complex loads onto the long noncoding RNA H19 promoter region that may lead to reduce the DNA methylation on H19 promoter region and then to enhance the H19 expression. Strikingly, the overexpression of H19 increases the binding of TERT to TERC and reduces the interplay between TERT with TERRA, thus enhancing the cell telomerase activity and extending the telomere length. On the other hand, insulator CTCF recruits the CUDR-CyclinD1 complx to form the composite CUDR-CyclinD1-insulator CTCF complex which occupancied on the C-myc gene promoter region, increasing the outcome of oncogene C-myc. Ultimately, excessive TERT and C-myc lead to liver cancer stem cell and hepatocyte-like stem cell malignant proliferation. To understand the novel functions of long noncoding RNA CUDR will help in the development of new liver cancer therapeutic and diagnostic approaches.     

Recurrent TERT promoter mutations in non-small cell lung cancers

IntroductionThe recurrent TERT promoter mutations have been recently described in diverse human cancers. We previously showed that over 60% of non-small cell lung cancer from East Asian harbored well-known oncogenic mutations in EGFR and KRAS. Here, we sought to determine the incidence, clinicopathologic characteristics, and association with known oncogenic mutations.Patients and methodsA total of 467 patients treated surgically for primary lung cancer were examined for mutations in TERT promoter using polymerase chain reaction (PCR) followed by Sanger sequencing. Immunohistochemical (IHC) staining was performed to detect the expression of TERT. Clinical characteristics including gender, age, smoking status, tumor size, differentiation, lymph node metastasis, TNM stage, overall survival and relapse-free survival were analyzed.ResultsOf 467 patients with non-small cell lung cancer, the TERT promoter mutation was detected in 12 patients. Of the 12 patients, 3 with C228T, 2 with C250T, 2 with C216T, 1 with C228A, 1 with C229G, 1 with G267C, 1 with C295T and 1 with G233C. Compared to the TERT mutation negative group, patients with TERT promoter mutation were significantly associated with older age (≥60 years, p = 0.039). No significant difference was found in overall survival (OS) or relapse-free survival (RFS) between TERT with mutation and TERT without mutation.ConclusionsTERT promoter mutations are recurrent mutated in 2.57% of NSCLCs and are highly enriched in older patients. It may play an important role in the pathogenesis of NSCLC and may serve as a potential target for therapy.     

Generation of combined hepatocellular-cholangiocarcinoma through transdifferentiation and dedifferentiation in p53-knockout mice

The two principal histological types of primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma, can coexist within a tumor, comprising combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Although the possible involvement of liver stem/progenitor cells has been proposed for the pathogenesis of cHCC-CCA, the cells might originate from transformed hepatocytes that undergo ductular transdifferentiation or dedifferentiation. We previously demonstrated that concomitant introduction of mutant HRAS^V12 (HRAS) and Myc into mouse hepatocytes induced dedifferentiated tumors that expressed fetal/neonatal liver genes and proteins. Here, we examine whether the phenotype of HRAS- or HRAS/Myc-induced tumors might be affected by the disruption of the Trp53 gene, which has been shown to induce biliary differentiation in mouse liver tumors. Hepatocyte-derived liver tumors were induced in heterozygous and homozygous p53-knockout (KO) mice by hydrodynamic tail vein injection of HRAS- or Myc-containing transposon cassette plasmids, which were modified by deleting loxP sites, with a transposase-expressing plasmid. The HRAS-induced and HRAS/Myc-induced tumors in the wild-type mice demonstrated histological features of HCC, whereas the phenotype of the tumors generated in the p53-KO mice was consistent with cHCC-CCA. The expression of fetal/neonatal liver proteins, including delta-like 1, was detected in the HRAS/Myc-induced but not in the HRAS-induced cHCC-CCA tissues. The dedifferentiation in the HRAS/Myc-induced tumors was more marked in the homozygous p53-KO mice than in the heterozygous p53-KO mice and was associated with activation of Myc and YAP and suppression of ERK phosphorylation. Our results suggest that the loss of p53 promotes ductular differentiation of hepatocyte-derived tumor cells through either transdifferentiation or Myc-mediated dedifferentiation.     

Autoimmune diseases as comorbidities for liver,gallbladder, and biliary duct cancers in Sweden

Background

Autoimmune diseases are associated with many cancers but there is a lack of population-based studies with different autoimmune diseases that have a long follow-up. This is also true of hepatobiliary cancers, which include hepatocellular cancer (HCC) and rarer entities of gallbladder cancer (GBC), intra- and extrahepatic cholangiocarcinoma (iCCA and eCCA), and ampullary cancer.

Methods

Diagnostic data on 43 autoimmune diseases were collected from the Swedish Inpatient Register from 1987 to 2018, and cancer data were derived from the national cancer registry from 1997 onward. Relative risks were expressed as standardized incidence ratios (SIRs).

Results

In a population of 13.6 million, 1.1 million autoimmune diseases were diagnosed and subsequent hepatobiliary cancer was diagnosed in 3191 patients (17.2% of cancers). SIRs for HCC were 2.73 (men) and 2.86 (women), 3.74/1.96 for iCCA, 2.65/1.37 for GBC, 2.38/1.64 for eCCA, and 1.80/1.85 for ampullary cancer. Significant associations between autoimmune disease and HCC were observed for 13 autoimmune diseases, with the highest risks being for autoimmune hepatitis (48.92/73.53, men/women) and primary biliary cirrhosis (38.03/54.48). GBC was increased after six autoimmune diseases, with high SIRs for ulcerative colitis (12.22/3.24) and men with Crohn disease (9.16). These autoimmune diseases were also associated with a high risk of iCCA, which had seven other associations, and eCCA, which had five other associations. Ampullary cancer occurrence was increased after four autoimmune diseases.

Conclusion

An autoimmune disease is a common precursor condition for hepatobiliary cancers. This calls for careful control of autoimmune disease symptoms in each patient and encouragement to practice a healthy lifestyle.     

Ang-1和Ang-2在肝细胞癌组织中的表达及其与血管生成的关系

目的 探讨Ang-1和Ang-2在肝细胞癌(HCC)组织中的表达及其与肿瘤血管生成的关系.方法 使用免疫组化法检测36例HCC组织和5例正常肝组织中,Ang-1和Ang-2的表达,检测HCC组织的微血管密度(MVD).结果 5例正常肝组织中,Ang-1阳性者3例,Ang-2均为阴性.HCC组织中Ang-2阳性率为83.33％,明显高于癌旁组织的33.33％ (P ＜0.05).Ang-2表达与MVD有关,Ang-2阳性者MVD为28.48±10.36,阴性者为14.22±11.02,差异有统计学意义(P＜0.05).Ang-2表达与血管侵犯、侵袭转移程度有关(P＜0.05).结论 HCC组织中Ang-2高表达,且与肿瘤血管生成有关,检测Ang-2有助于判断肿瘤侵袭转移能力,但不能反映恶性程度.     

Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma

Unlike other types of cancer, hepatocellular carcinoma (HCC) is usually preceded by chronic inflammation caused by viral infection. The mutation of mitochondrial DNA (mtDNA) in hepatocarcinogenesis associated with viral infection was investigated. Compared with control liver tissue, the frequency of mtDNA mutations was markedly increased in both noncancerous and cancerous liver specimens from individuals with HCC. The accumulation of mtDNA mutations in HCC tissue reflected the degree of malignancy. The frequency of mtDNA mutations in HCC tissue was also greater than that described previously for other types of tumors. These observations suggest that the repeated destruction and regeneration of liver tissue associated with chronic viral hepatitis lead to the accumulation of mtDNA mutations. The genetic instability that results in the high rate of mtDNA mutation in cancerous liver tissue is also consistent with the multicentric hepatocarcinogenesis detected clinically.