TERT promoter mutations and gene amplification: Promoting TERT expression in Merkel cell carcinoma

Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.     

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 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.     

TERT promoter mutations are frequent and show association with MED12 mutations in phyllodes tumors of the breast

Background:

Phyllodes tumors are rare fibroepithelial neoplasms of the breast, which carry the potential risk of local recurrence and metastasis. Phyllodes tumors share several histological features with fibroadenomas, and no widely accepted markers for distinguishing these lesions have been identified.

Methods:

We analyzed molecular abnormalities related to telomere elongation in tumors, including TERT promoter mutations, as well as loss of expression of ATRX and DAXX, in a total of 104 phyllodes tumors and fibroadenomas.

Results:

Sequencing analyses showed that TERT promoter mutations were frequent in phyllodes tumors (30/46, 65%), but rare in fibroadenomas (4/58, 7%). Among phyllodes tumors, the mutations were more frequent in borderline tumors (13/15, 87%), but were also common in benign (9/18, 50%) and malignant tumors (8/13, 62%). Remarkably, all but one TERT promoter-mutated tumor also contained MED12 mutations, indicating that these mutations are strongly associated (P=8.4 × 10⁻⁶). Expression of ATRX and DAXX, as evaluated by immunohistochemistry, was retained in all tumors.

Conclusions:

Our observations suggest a critical role of TERT promoter mutations, in cooperation with MED12 mutations, in the development of phyllodes tumors. Because TERT promoter mutations are rare among fibroadenomas, their detection may be of potential use in discriminating between phyllodes tumors and fibroadenomas.     

Construction of double suicide genes system controlled by MDR1 promoter with targeted expression in drug-resistant glioma cells

The multiple drug resistance protein (MDR1) is frequently overexpressed in human glioma. The aim of this study is to clone the MDR1 promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells. MDR1 promoter from C6/ADR genomic DNA, which was linked with T vector, was amplified by using Polymerase chain reaction (PCR). After cut by NdeI and HindIII, MDR1 promoter was cloned into pcDNA3-TK (thymidine kinase) plasmid. The cytosine deaminase (CD) gene from pcDNA3-CD-TK plasmid was directly cloned into the above vector to construct pcDNA3-MDR1-promoter-CD-TK vector. Then this vector was transfected into C6 and C6/ADR cells respectively by liposome. After selection by G418, the tumor cell lines were stably established. Then these cell lines were examined through PCR and RT-PCR to respectively detect the integration and expression of TK and CD genes. The results showed the length and sequence of MDR1 promoter amplified by PCR were confirmed by DNA sequencing. The pcDNA3-MDR1-promoter-CD-TK expression vectors were constructed successfully. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. RT-PCR reveled that CD and TK genes expressed in C6/ADR/CD-TK cells, whereas not in C6/CD-TK cells. In conclusions, construction of expressive vector containing double suicide genes controlled by MDR1 promoter with targeted expression in C6/ADR will provide a sound basis for targeted gene therapy for multidrug resistance (MDR) glioma.     

正常人及视网膜母细胞瘤患者Rb基因启动子结构、功能和突变的研究

目的检测正常人及视网膜母细胞瘤（ＲＢ）患者Ｒｂ基因５′端调控区的ＤＮＡ序列,以及不同的ＤＮＡ片段的转录调控活性,研究Ｒｂ基因启动子的结构、功能以及突变对功能的影响和与ＲＢ发病的关系。方法应用ＳＳＣＰ分析及ＤＮＡ序列测定检测正常人及ＲＢ患者Ｒｂ基因启动子的ＤＮＡ序列和点突变;分离Ｒｂ基因５′端不同位置及不同大小的ＤＮＡ片段,通过氯霉素乙酰基转移酶做报告基因,检测不同的ＤＮＡ片段的转录调控活性。结果Ｒｂ基因产物起始密码上游３２７ｂｐ至８７ｂｐ间２４０ｂｐ的ＤＮＡ片段具有基本的启动子功能,其上游和下游的ＤＮＡ序列内可能还存在正或负转录调控元件。１００例正常人Ｒｂ基因启动子ＤＮＡ序列未见有任何变异,但３０２例ＲＢ患者中５例有点突变并伴有ＣＡＴ活性明显降低。结论Ｒｂ基因启动子的ＤＮＡ序列非常稳定,其突变常常导致启动子活性下降,并与视网膜母细胞瘤的遗传易感性有关     

BRAF mutations are associated with increased iron regulatory protein‐2 expression in colorectal tumorigenesis

A role for iron in carcinogenesis is supported by evidence that iron metabolism proteins are modulated in cancer progression. To date, however, the expression of iron regulatory protein‐2 (IRP2), which is known to regulate several iron metabolism proteins, has not been assessed in colorectal cancer. Expression of IRP2 was assessed by quantitative RT‐PCR and immunohistochemistry in human colorectal cancer tissue. By interrogating The Cancer Genome Atlas (TCGA) database, expression of IRP2 and transferrin receptor‐1 (TfR1) was assessed relative to common mutations that are known to occur in cancer. The impact of suppressing IRP2 on cellular iron metabolism was also determined by using siRNA and by using the MEK inhibitor trametinib. IRP2 was overexpressed in colorectal cancer compared to normal colonic mucosa and its expression was positively correlated with TfR1 expression. In addition, IRP2 expression was associated with mutations in BRAF. The MEK inhibitor trametinib suppressed IRP2 and this was associated with a suppression in TfR1 and the labile iron pool (LIP). Moreover, epidermal growth factor stimulation resulted in decreased ferritin expression and an increase in the LIP which were independent of IRP2. Results presented here suggest that ablating IRP2 provides a therapeutic platform for intervening in colorectal tumorigenesis.     

胃癌组织中端粒酶活性与细胞周期蛋白D_1表达关系的研究

目的　探索胃癌组织中端粒酶活性与细胞周期蛋白D1表达之间可能的关系。方法　利用TelomerasePCR ELISAKit测量端粒酶活性 ;利用RT PCRELISA检测细胞周期蛋白D1mRNA表达水平。共检测了 84例胃癌及其相关性的组织标本。结果　周期蛋白D1异常高表达在胃良性疾病、胃癌前期病变和胃癌组织中均可检出 ,而端粒酶活性仅在胃癌前期病变和胃癌组织中检出。胃癌前期病变和胃癌组织端粒酶活性与周期蛋白D1表达程度明显正相关 (r =0 .713,P <0 .0 5 )。结论　至少在胃癌组织中 ,细胞周期蛋白D1异常高表达可能是端粒酶激活的前提之一。     

siRNA targeting LMP1-induced apoptosis in EBV-positive lymphoma cells is associated with inhibition of telomerase activity and expression

Epstein-Barr Virus (EBV) is closely associated with B cell malignancies. However, whether EBV appears to be absolutely required for cell proliferation and survival in lymphoma cells is still unknown. In this study, small interfering RNA (siRNA) targeting LMP1 was employed to investigate the effect of LMP1 on cell proliferation in EBV-positive lymphoblastoid B-cell line. A plasmid stable encoding 21-nt small RNA specifically and efficiently interfering LMP1 was constructed, resulting in a substantial loss of LMP1 mRNA and a significantly decreased LMP1 protein expression. Our data demonstrated that cell proliferation was completely inhibited and apoptosis was induced after knockdown of LMP1 gene in lymphoblastoid B-cell line. Also, we found that suppression of LMP1 caused downregulation of telomerase protein expression and decreased telomerase activity in lymphoma cells. In EBV-negative NPC cell line, transfection of plasmid expressing LMP1 greatly enhanced telomerase protein expression. Our results suggested that siRNA targeting LMP1 can induce apoptosis in EBV-positive lymphoma cells and is associated with inhibition of telomerase activity and expression. siRNA-directed LMP1 silencing may be of the therapeutic value for preventing and treating those EBV-associated tumors.     

胃癌组织中端粒酶活性与细胞周期蛋白D_1表达关系的研究

目的　探索胃癌组织中端粒酶活性与细胞周期蛋白D1表达之间可能的关系。方法　利用TelomerasePCR ELISAKit测量端粒酶活性 ;利用RT PCRELISA检测细胞周期蛋白D1mRNA表达水平。共检测了 84例胃癌及其相关性的组织标本。结果　周期蛋白D1异常高表达在胃良性疾病、胃癌前期病变和胃癌组织中均可检出 ,而端粒酶活性仅在胃癌前期病变和胃癌组织中检出。胃癌前期病变和胃癌组织端粒酶活性与周期蛋白D1表达程度明显正相关 (r =0 .713,P <0 .0 5 )。结论　至少在胃癌组织中 ,细胞周期蛋白D1异常高表达可能是端粒酶激活的前提之一。     

肝细胞癌端粒酶活性及hTERT mRNA表达与术后早期复发的关系

目的：研究肝细胞癌端粒酶活性及人端粒酶逆转录酶（hTERT）mRNA表达与肝细胞癌术后早期复发的关系。方法：采用ELISA—TRAP法检测60例肝癌组织及其癌旁组织端粒酶活性，RT—PCR法检测hTERT mRNA表达，5例正常肝脏组织作为对照。分析端粒酶活性及hTERT mRNA表达与临床病理之间的关系。结果：肝癌组织端粒酶活性及hTERT mRNA表达阳性率分别为86．7％（52／60）及90％（54／60），癌旁组织端粒酶活性及hTERT mRNA表达阳性率分别为40％（24／60）及43．3％（26／60）。正常肝脏组织均未检测到端粒酶活性及hTERT mRNA表达。癌旁组织端粒酶活性及hTERT mRNA表达与术后早期复发及包膜浸润、门静脉侵犯、肝内转移等恶性肿瘤的恶性生物学行为有关。结论：癌旁组织端粒酶活性及hTERT mRNA表达可能是肝细胞癌术后早期复发的预后指标。     

Circulating tumour cells are associated with increased risk of venous thromboembolism in metastatic breast cancer patients

Background:

Cancer is a risk factor for venous thromboembolism (VTE). Circulating tumour cells (CTCs) are an independent predictor of survival in metastatic breast cancer (MBC) patients. The aim of this study was to test the hypothesis that CTCs are associated with the risk of VTE in MBC patients.

Methods:

This retrospective study included 290 MBC patients treated in the MD Anderson Cancer Center from January 2004 to December 2007. Circulating tumour cells were detected and enumerated using the CellSearch system before starting new lines of therapy.

Results:

At a median follow-up of 12.5 months, 25 patients experienced VTE and 53 patients died without experiencing thrombosis. Cumulative incidence of thrombosis at 12 months was 8.5% (95% confidence interval (CI)=5.5%, 12.4%). Patients with CTCs ⩾1 and ⩾5 had a higher incidence of VTE compared with patients with 0 and <5 CTCs (12-month estimate, 11.7 and 11.6% vs 3 and 6.6%; P=0.006 and P=0.076, respectively). In the multivariate model, patients with CTCs⩾1 had a hazard ratio of VTE of 5.29 (95% CI=1.58, 17.7, P=0.007) compared with patients with no CTCs.

Conclusion:

These results suggest that CTCs in MBC patients are associated with increased risk of VTE. These patients should be followed up more closely for the risk of VTE.     

乳腺癌Runx3基因启动子甲基化状态及其与病理特征的关系

目的探讨乳腺癌组织中Runx3基因启动子甲基化状态及其与乳腺癌临床病理特征之间的关系。方法 2007年2月至2009年4月收集经病理确诊的56例乳腺癌患者癌组织及相应的癌旁组织。采用甲基化特异聚合酶链反应检测Runx3基因启动子区CpG岛的甲基化状态,并检测其对Runx3基因表达的影响和分析其与乳腺癌临床病理特征之间的关系。统计学分析采用χ2检验和Pearson相关分析法。结果乳腺癌组织中Runx3基因启动子区CpG岛甲基化率(55.4%)显著高于癌旁组织中的甲基化率(10.7%),二者之间差异有统计学意义(χ2=25.225,P=0.000)。Runx3基因的异常甲基化和乳腺癌患者的临床分期及淋巴结转移有关(P=0.018,P=0.010),但与患者的年龄、肿瘤直径大小及组织学类型无关(P0.050)。Runx3mRNA表达与Runx3启动子甲基化呈弱相关(r=0.343,P=0.010)。结论 Runx3基因启动子甲基化状态导致Runx3基因mRNA表达降低或缺失。Runx3基因启动子区甲基化可能是导致Runx3基因在乳腺癌中失活的分子机制之一。     

乙肝病毒衣壳运载体甲胎蛋白启动子驱动的白细胞介素-2在肝癌细胞中的靶向表达

目的 利用乙肝病毒衣壳（HBVE）和人甲胎蛋白（AFP）启动子的双重靶向性,观察白细胞介素-2（IL-2）基因是否可以在肝癌细胞中靶向表达.方法 体外培养HepG2、L02、HepG2.2.15细胞,PEG8000浓缩法提取HBVE作为基因运载体,聚合酶链反应（PCR）法制备人AFP启动子-IL-2重组基因,瞬时转染HepG2细胞与L02细胞,反转录-聚合酶链式反应（RT-PCR）观察IL-2 mRNA表达,Western blot观察IL-2蛋白表达情况.结果 以HBVE为基因的运载体,包裹人AFP启动子-IL-2重组基因后转染HepG2细胞和L02细胞,RT-PCR和Westem blot检测到HepG2细胞有IL-2的表达,而对照组L02细胞无IL-2的表达.结论 以HBVE为基因运载体,可以使AFP启动子驱动的IL-2基因在AFP阳性的肝癌细胞靶向表达,从而提高了外源基因导入肝癌细胞的安全性.     

Interleukin-1B gene promoter variants are associated with an increased risk of gastric cancer in a Chinese population

Studies suggest that IL-1beta (encoded by IL-1B gene) is a pro-inflammatory cytokine and potent inhibitor of gastric acid secretion, which is proposed as a key determinant in gastric carcinogenesis. Two potentially functional polymorphisms (C-31T and T-511C) in the IL-1B promoter were suggested to be correlated with alteration of Helicobacter pylori infection and IL-1beta expression and therefore may be associated with risk of gastric cancer. To test the hypothesis that these two polymorphisms are associated with gastric cancer risk, we performed a case-control study of 280 histologically confirmed gastric cancer patients and 258 age, sex frequency-matched cancer-free controls in a Chinese population. Multivariate logistic regression analyses revealed that the risks (adjusted odds ratio [OR] and 95% confidence interval [CI]) associated with the IL-1B variant genotypes were 1.64 (95% CI, 1.01-2.66) for -31TT and 1.52 (95% CI, 0.91-2.54) for -511CC, respectively, compared with their wild-type homozygotes. The risks were significantly more evident in individuals with H. pylori infection (adjusted OR, 2.14; 95% CI, 1.13-4.06 for -31TT; adjusted OR, 2.00; 95% CI, 1.02-3.89 for -511CC), which was consistent with the biological effects of IL-1beta. When we used the haplotype analyses and assumed the IL-1B -31T and -511C as risk alleles, no synergistic effect was found between these two loci. These findings indicate that these two IL-1B promoter variants may contribute to the risk of developing gastric cancer in the Chinese population, especially in individuals with H. pylori infection.     

抑癌基因促甲状腺激素受体和p16启动子甲基化与甲状腺乳头状癌关系的研究

 目的　研究促甲状腺激素受体（TSHR）和p16抑癌基因在甲状腺乳头状癌的表达及启动子甲基化的情况, 分析两种抑癌基因的甲基化状况与肿瘤发生的关系。方法　对50例甲状腺乳头状癌组织和32例对照组织（20例结节性甲状腺肿,12例甲状腺腺瘤）提取RNA后,反转录为cDNA,进行PCR,检测两种抑癌基因mRNA的表达情况,运用甲基化PCR（其中p16使用巢氏甲基化PCR）检测上述组织中两种抑癌基因启动子区甲基化的情况, 对两种抑癌基因甲基化和未甲基化的组织随机进行测序。结果　甲状腺乳头状癌组50例患者中,有34例（68 %）TSHR基因、27例（54 %）的p16 基因启动子发生了甲基化;对照组32例患者中,有7例（21.9 %）TSHR基因、5例（15.6 %）的p16基因启动子发生了甲基化;甲状腺乳头状癌组TSHR基因、p16基因启动子甲基化率均显著高于对照组,差异有统计学意义（χ2=16.61,P < 0.05;χ2＝12.08,P <0.05）。TSHR基因和p16基因mRNA在甲状腺乳头状癌中的表达量分别为0.41±0.11、0.51±0.17,相应的对照组织的mRNA的表达量分别为0.63±0.08、0.72±0.22,两种基因的mRNA在甲状腺乳头状癌中的表达明显低于对照组织,差异有统计学意义（t值分别为3.86和3.66,P <0.05）。最终,经DNA 测序证实,两种抑癌基因启动子发生甲基化的其CpG岛的碱基未发生改变,仍为CG;未发生甲基化的,碱基由CG变为TG。结论　两种抑癌基因启动子甲基化与甲状腺乳头状癌的发生和发展均相关。     

Germline mutations in the CHEK2 kinase gene are associated with an increased risk of bladder cancer

Germline mutations in CHEK2 have been associated with a range of cancer types but little is known about disease risks conveyed by CHEK2 mutations outside of the context of breast and prostate cancer. To investigate whether CHEK2 mutations confer an increased risk of bladder cancer, we genotyped 416 unselected cases of bladder cancer and 3,313 controls from Poland for 4 founder alleles in the CHEK2 gene, each of which has been associated with an increased risk of cancer at other sites. A CHEK2 mutation (all variants combined) was found in 10.6% of the cancer cases and in 5.9% of the controls (OR = 1.9; 95%CI 1.3-2.7; p = 0.0003). We conclude that CHEK2 mutations increase the risk of bladder cancer in the population.