Clinical implications of single cell sequencing for bladder cancer

Bladder cancer(BC)is the 10th most common cancer worldwide,with about 0.5 million reported new cases and about 0.2 million deaths per year.In this scoping review,we summarize the current evidence regarding the clinical implications of single-cell sequencing for bladder cancer based on PRISMA guidelines.We searched PubMed,CENTRAL,Embase,and supplemented with manual searches through the Scopus,and Web of Science for published studies until February 2023.We included original studies that used at least one single-cell technology to study bladder cancer.Forty-one publications were included in the review.Twenty-nine studies showed that this technology can identify cell subtypes in the tumor microenvironment that may predict prognosis or response to immune checkpoint inhibition therapy.Two studies were able to diagnose BC by identifying neoplastic cells through single-cell sequencing urine samples.The remaining studies were mainly a preclinical exploration of tumor microenvironment at single cell level.Single-cell sequencing technology can discriminate heterogeneity in bladder tumor cells and determine the key molecular properties that can lead to the discovery of novel perspectives on cancer management.This nascent tool can advance the early diagnosis,prognosis judgment,and targeted therapy of bladder cancer.

Effects of Combined siRNA-TR and-TERT on Telomerase Activity and Growth of Bladder Transitional Cell Cancer BIU-87 Cells

The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.

RECURRENCE RISK FACTORS IN PATIENTS WITH TRANSITIONAL CELL CARCINOMA OF BLADDER

Objective： To study recurrence factors and set up a model to evaluate the prognosis of patients with bladder cancer. Methods： An analysis on recurrence-related factors was made by Cox＇s proportional hazards model analysis and logistic multiple linear regression model analysis in 212 patients with transitional cell carcinoma treated surgically from 1995-2001. These factors included clinical and pathologic figures. Results： The most important factor is metastasis to the regional lymph nodes, the Hazards ratio is 6.6 （P=0.0004）, followed by multiple tumors （Hr=2.255, P〈0.0001）, tumor in trigone and bladder neck （Hr=2.053, P〈0.0001）, stage （Hr=2.057, P〈0.0001）, grade （Hr=1.569, P=0.0081）, intravesical chemotherapeutic instillations （Hr-0.559, P=0.0011） and hematuria （Hr=0.762, P=0.0076）. A predicting equation was established, and the predicting values were calculated according to the individual features of patients. The predicting and actual values were compared, and the sensitivity, specificity and overall concordance were 83.5%, 67.6% and 80.1% respectively. Conelusion：The evaluation of prognosis could be made quite accurately based on these factors.

CLINICAL SIGNIFICANCES OF THE EXPRESSION OF METALLO- THIONEIN IN FIVE TYPES EPITHELIUM CELL CANCER

Objective： To study the expressions of （CSC）, bladder transitional cell cancer metallothionein and the significances in cervical squamous cell cancer （BTC）, esophageal squamous cell cancer （ESC）, gastral tubular adenocarcinoma （GC） and large intestinal tubular adenocarcinoma （LIC）. Methods： lmmunohistochemical method was used to examine the expression rates of MT in five types of cancer tissue. Results： The expression rates of MT were 75.00% （24/32） in ESC, 52.27% （46/88） in GTC, 59.46% （44/74） in LIC, 64.86% （48/74） in BTC and 58.57% （41/70） in CSC respectively. The positive rates of MT expression were higher in low differentiation and deep muscular group than those in medium or high differentiation and superficial muscular invasion group （P〈0.05）. Conclusion： The expression of MT is related to differentiation degree and invasion degree.

Vascular endothelial growth factor,p53,and the H-ras oncogene in Egyptian patients with bladder cancer

AIM:To evaluate the relationship between vascular endothelial growth factor(VEGF),p53,and the H-ras oncogene and different clinicopathological parameters in Egyptian patients with Schistosoma-associated transitional cell carcinoma of the bladder.METHODS:The study included 50 patients with transitional cell carcinoma for whom radical cystectomy and urinary diversions were carried out.VEGF and p53 protein expressions were evaluated with an immunohistochemical staining method,and H-ras oncogene mutations were analyzed with a polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique.RESULTS:High grade tumors revealed higher p53 immunostaining than low grade tumors(P = 0.016).p53 and VEGF protein expressions,as well as H-ras oncogene mutations,had an insignificant impact on patient outcomes(P = 0.962,P = 0.791,and P = 967,respectively).Cancer extension to regional lymph nodes was associated with poor outcomes(P = 0.008).CONCLUSION:VEGF,p53 and the H-ras oncogene have no relation to patient survival and outcome in Schistosoma-associated transitional cell carcinoma.

μ-opioid receptor agonist facilitates circulating tumor cell formation in bladder cancer via the MOR/AKT/Slug pathway:a comprehensive study including randomized controlled trial

Background:μ-opioid receptor agonists(MORAs)are indispensable for analgesia in bladder cancer(BC)patients,both during surgery and for chronic pain treatment.Whether MORAs affect BC progression and metastasis remains largely unknown.This study focused on the effects of MORAs on the formation of circulating tumor cells(CTCs)in BC and aimed to provide potential therapeutic targets,which would retain the pain-relieving effects of MORAs in BC patients without sacrificing their long-term prognosis.Methods:Different preclinical models were used to identify the effects of MORAs on the progression of BC.A novel immunocapture microfluidic chip was utilized to analyze whether MORAs affected the number of CTCs in mouse models and clinical BC patients.Bioinformatic analyses,total transcriptome sequencing,and molecular biology methods were then used to investigate the underlying mechanisms in these models and in BC cell lines.Results:Mouse models of hematogenous metastasis and in situ BC demonstrated that tumor metastasis was significantly increased after MORA treatment.A significant increase in the number of mesenchymal and/or epithelial CTCs was detected after MORA treatment in both the mouse models and clinical trial patients.Mechanistically,MORAs facilitated the formation of CTCs by activating the MOR/PI3K/AKT/Slug signaling pathway,hereby promoting the epithelialmesenchymal transition(EMT)of BC cells,as knockdown of MOR,Slug or blockade of PI3K inhibited the EMT process and CTC formation.Conclusion:MORAs promoted BC metastasis by facilitating CTC formation.The EMT-CTC axis could be targeted for preventive measures during MORA treatment to inhibit the associated tumormetastasis or recurrence in BC patients.

Clinical signification on the expressions of metallothionein in three types cancer of woman

Objective: To study the expressions and significations of metallothionein (MT) in cervical squamous cell cancer (CSC), bladder transitional cell cancer (BTC) and breast cancer (BC) of woman. Methods: Immunohistochemical method was used to examine the expresses rate of MT in three types of woman cancer tissue. Results: The expressions rates of MT were 54.35% (29/46) in BTC, 67.05% (59/88) in BC and 57.14% (40/70) in CSC. The positive rate of MT expression was higher in low differentiation group than well differentiation group in BTC and CSC (P < 0.05). Positive of MT in lobular cancer was significance higher than medullary and duct cancers (P < 0.05). Conclusion: The expression of MT is related to differentiation degree, and it is a guidance for clinical choice of chemotherapy project.

Bladder urothelial carcinoma extending to rectal mucosa and presenting with rectal bleeding

An 87-year-old-man with prostate-cancer-stage-T1cGleason-6 treated with radiotherapy in 1996, recurrent prostate cancer treated with leuprolide hormonal therapy in 2009, and bladder-urothelial-carcinoma in situ treated with Bacillus-Calmette-Guerin and adriamycin in 2010, presented in 2015 with painless, bright red blood per rectum coating stools daily for 5 mo. Rectal examination revealed bright red blood per rectum; and a hard, fixed, 2.5 cm × 2.5 cm mass at the normal prostate location. The hemoglobin was 7.6 g/d L(iron saturation = 8.4%,indicating iron-deficiency-anemia). AbdominopelvicCT-angiography revealed focal wall thickening at the bladder neck; a mass containing an air cavity replacing the normal prostate; and adjacent rectal invasion. Colonoscopy demonstrated an ulcerated, oozing, multinodular, friable, 2.5 cm × 2.5 cm mass in anterior rectal wall, at the usual prostate location. Histologic and immunohistochemical analysis of colonoscopic biopsies of the mass revealed poorly-differentiatedcarcinoma of urothelial origin. At visceral angiography, the right-superior-rectal-artery was embolized to achieve hemostasis. The patient subsequently developed multiple new metastases and expired 13 mo postembolization. Comprehensive literature review revealed 16 previously reported cases of rectal involvement from bladder urothelial carcinoma, including 11 cases from direct extension and 5 cases from metastases. Patient age averaged 63.7 ± 9.6 years(all patients male). Rectal involvement was diagnosed on average 13.5 ± 11.8 mo after initial diagnosis of bladder urothelial carcinoma. Symptoms included constipation/gastrointestinal obstruction-6, weight loss-5, diarrhea-3, anorexia-3, pencil thin stools-3, tenesmus-2, anorectal pain-2, and other-5. Rectal examination in 9 patients revealed annular rectal constriction-6, and rectal mass-3. The current patient had the novel presentation of daily bright red blood per rectum coating the stools simulating hemorrhoidal bleeding; the novel mechanism of direct bladder urothelial carcinoma extension into rectal mucosa via the prostate; and the novel aforementioned colonoscopic findings underlying the clinical presentation.

浅表性和浸润性膀胱癌微环境中树突状细胞的变化及意义

目的研究树突状细胞(Dc)在不同类型膀胱移行细胞癌及癌旁组织中的变化以及与肿瘤病理分级的关系,探讨恶性肿瘤免疫逃避的可能机制。方法将133例膀胱移行细胞癌病理标本按WHO标准进行病理分级,按1987年国际抗癌协会(UICC)标准进行临床分型(浅表和浸润),应用免疫组化法检测病理标本中Dc。结果133例标本中肿瘤Dc数目明显少于瘤旁组织(G116.85±1.1,G29.45±2.17,G32.99±1.19vsG121.8±4.78,G221.71±4.72,G320.00±5.49,P<0.01)并随肿瘤病理分级的增加而减少,相同病理分级浅表性膀胱癌实质内Dc数目(G210.79±1.69;G34.79±0.67)明显多于浸润性膀胱癌实质内Dc数目(G27.52±1.0;G32.46±0.66),其差别具有统计学意义(P<0.01);肿瘤旁组织Dc数目在不同肿瘤病理分级、临床分型的标本中差别无统计学意义(P>0.05)。结论Dc仅在机体有炎症或肿瘤时作为抗原递呈者出现;肿瘤内Dc数目的减少是恶性肿瘤逃避机体的免疫监视和排斥的一个可能机制;浸润性膀胱癌容易转移可能与瘤内Dc数目及活性过度下调和Dc过早凋亡有关。

保留膀胱手术联合动脉化疗治疗浸润性膀胱癌的临床研究

目的评价保留膀胱手术联合动脉化疗治疗浸润性膀胱癌的临床疗效。方法2003年4月-2006年12月,对35例浸润性膀胱癌患者采用经尿道膀胱肿瘤电切或膀胱部分切除术联合GC(吉西他滨+顺铂)方案动脉化疗治疗,总结分析肿瘤控制情况、膀胱保存率和患者的生存率。结果33例患者获随访,2例失访,平均随访24.3个月(3-45个月)。27例无瘤生存,2例带瘤生存,4例死于肿瘤转移,2年生存率为88.8%;19例无复发及转移,5例浅表性复发,3例浸润性复发,6例转移;25例保留膀胱生存,4例行挽救性全膀胱切除,4例死亡,2年膀胱保存率为74.1%。全部患者对动脉化疗耐受良好,无严重全身和局部不良反应。结论保留膀胱手术联合GC方案动脉化疗治疗浸润性膀胱移行细胞癌近期疗效满意,毒副作用轻,值得临床进一步观察研究。

尿膀胱癌抗原作为膀胱肿瘤标志物的临床评价

为评价尿膀胱癌抗原(urinary bladder cancer antigen,UBC)在膀胱移行细胞癌(BTCC)中的诊断价值,采用ELISA法对53例BTCC患者、25例泌尿系统良性疾病患者和13例健康志愿者进行UBC检测,并同时行尿细胞学检查。结果显示:(1)BTCC患者UBC平均含量为26.26±28.49μg/L,与泌尿系统良性疾病患者和健康志愿者相比(9.41±9.63μg/L、1.73±0.79μg/L),均有显著性差异(P均<0.01)。(2)以7.5μg/L为最适临界值时,UBC诊断BTCC的敏感性和特异性分别为86.8%、76.3%,与尿细胞学检查(32.1%、97.4%)相比,均有显著性差异(P均<0.01)。结论:UBC具有简便、敏感和无创的特点,可作为辅助诊断BTCC的尿肿瘤标志物。

膀胱移行细胞癌中肿瘤-睾丸抗原基因的表达情况

目的:研究4种肿瘤-睾丸抗原(CT)基因在膀胱移行细胞癌中的表达及其临床意义。方法:采用反转录聚合酶链反应(RT-PCR)技术检测49例膀胱移行细胞癌患者癌组织(新鲜标本,Ta-T1期28例,T2-T4期21例;G120例,G216例,G313例)及其中15例患者癌旁组织的cTAGE-1、cTAGE-2、MAGE-A1及NY-ESO-1等4种CT基因mRNA的表达。结果:49例膀胱移行细胞癌组织中MAGE-A1表达最高,其次为cTAGE-1,cTAGE-2及NY-ESO-1,分别为59%(29/49),55%(27/49),51%(25/49)及47%(23/49)。15例癌旁组织表达均阴性。膀胱移行细胞癌组织中肿瘤不同分期、不同分级之间4种CT基因表达的差异均无统计学意义(Pearsonχ2检验法,P>0.05)。结论:CT基因在膀胱移行细胞癌组织中有较高表达,而在癌旁组织无表达,可以进一步研究作为膀胱移行细胞癌特异性免疫治疗靶基因的可行性。

CD44v6和C-myc在膀胱移行细胞癌中的表达与临床意义

目的 探讨CD4 4v6和C myc在膀胱移行细胞癌中的表达及其临床意义。方法 应用免疫组化法检测 5 0例膀胱移行细胞癌标本中CD4 4v6和C myc的表达情况、并与膀胱癌的病理分级及随访结果相比较。 结果 2 8例CD4 4 v6表达阳性 ,阳性率 5 6 % ,且膀胱癌分化越好 ,CD4 4v6表达率越高 ,临床预后亦较好 ,这与其它肿瘤具有很大区别。 36例C myc表达阳性 ,阳性率 72 % ,膀胱癌分化越差 ,表达率越高 ,临床预后也较差。结论 CD4 4v6和C myc表达可以作为监测膀胱移行细胞癌恶性程度及预后的重要指标。

AMPKα过表达对膀胱癌T24细胞增殖、侵袭和EMT的抑制作用及其机制

目的:探讨AMP依赖的蛋白激酶α(AMP-activated protein kinaseα,AMPKα)过表达对膀胱癌T24细胞增殖、迁移、侵袭和EMT的作用及其机制。方法:建立AMPKα过表达的膀胱癌T24细胞株,依据转染质粒的不同分为T24空白组、pc-DNA空载组和pc-AMPKα组。用Wb检测T24细胞AMPKα、EMT相关蛋白及EMT通路相关分子的表达水平,用Hoechst染色法检测转染后T24细胞的凋亡,CCK-8法检测T24细胞的增殖,细胞划痕愈合实验检测T24细胞的迁移,Transwell实验检测细胞的侵袭。结果:成功构建AMPKα过表达的膀胱癌T24细胞株。与T24空白组和pc-DNA空载组比较,pc-AMPKα组T24细胞上皮钙黏蛋白水平显著升高(P<0.01)、波形蛋白和神经钙黏蛋白表达水平显著降低(均P<0.01),EMT通路相关信号分子P38、STAT3活性受到显著抑制(均P<0.01),细胞发生明显凋亡、增殖能力显著减弱(均P<0.01);T24细胞迁移和侵袭能力显著降低(均P<0.01)。结论:AMPKα过表达可使EMT通路相关分子活性受到抑制,使得膀胱癌T24细胞发生明显凋亡、增殖受限并使其侵袭和迁移能力降低及伴随EMT的逆转。

α-2b干扰素加顺铂预防膀胱癌术后复发的远期疗效

目的 观察α-2b干扰素联合顺铂膀胱粘膜下注射及术后膀胱灌注对预防膀胱移行细胞癌复发的远期临床疗效。方法 106例诊断为膀胱移行细胞癌的病人,随机分为两组。A组56例,采用术前60-90 min行膀胱内灌注顺铂50mg,术中用α-2b干扰素300万U加顺铂30mg,生理盐水稀释60ml-80ml行瘤体周围粘膜下广泛注射,并于术后应用两药联合灌 注(α-2b干扰素300万U+顺铂50mg)。B组50例仅用顺铂,方法和剂量同A组。结果 101例获得随访5-10年,平均7年。A组和B组存活率分别为73.5％(36/53)和67.4％(31/48),差异元显著性(P>0.05)。但复发率和无瘤存活率分别为26.5％(13/53)和62.3％(313/53)及45.8％和41.7％(20/48),两组差异均有显著性(P<0.05)。结论α-2b干扰素加顺铂粘膜下注射及术后灌注可有效的预防膀胱移行细胞癌术后复发,降低复发率、延迟复发时间,效果优于单用顺铂或干扰素,两药联合有相加和合成作用。

吉西他滨联合顺铂的新辅助化疗治疗局部晚期膀胱移行细胞癌

目的探讨吉西他滨联合顺铂(GC方案)的新辅助化疗方案治疗局部晚期膀胱移行细胞癌的临床疗效及毒性反应。方法 12例局部晚期膀胱移行细胞癌患者接受4个周期GC方案的新辅助化疗(吉西他滨1000mg/m2,第1、8天静脉滴注;顺铂20mg,第1～5天静脉滴注)。结果 12例均完成4个周期的GC方案化疗,总有效率58%,其中完全缓解2例,部分缓解5例,稳定3例,进展2例。6例患者化疗后行全膀胱切除术+回肠代膀胱术,3例经剖腹探查肿块无法切除而仅行放疗,2例患者因病情进展行姑息性化疗,1例患者拒绝手术治疗后仅行姑息性化疗。GC方案化疗的主要毒性为骨髓抑制,91%白细胞减少。结论吉西他滨联合顺铂是目前治疗局部晚期膀胱移行细胞癌有效、安全的新辅助化疗方案,为不能手术的局部晚期的膀胱移行细胞癌患者提供了新的治疗模式。

膀胱移行细胞癌增殖细胞核抗原与肿瘤间质微血管密度的表达与临床及预后的意义

目的 :探讨膀胱移行细胞癌组织中增殖细胞核抗原 (PCNA)与肿瘤间质微血管密度 (MVD)的表达及其临床与预后的意义。方法 :对 72例膀胱移行细胞癌进行PCNA及第Ⅷ因子相关抗原 (VWF :Ag)单克隆抗体免疫组化染色。结果 :PCNA增殖指数与肿瘤间质微血管密度之间存在正相关性 ,二者的表达皆与膀胱移行细胞癌的病理分级显著相关 ,Ⅰ级与Ⅱ级、Ⅱ级与Ⅲ级之间存在显著性差异 (P <0 .0 1) ;侵袭性肿瘤明显高于浅表性肿瘤 (P <0 .0 1)。手术 2年后随访 ,复发组明显高于未复发组 (P <0 .0 1)。结论 :PCNA的表达为膀胱移行细胞癌的恶性表型 ,且与膀胱肿瘤间质微血管形成有关 ,上述二项指标对评估膀胱移行细胞癌的预后有重要意义。

原发性膀胱移行细胞癌 D17S5 VNTR 位点的遗传多态性研究

Ｄ１７Ｓ５ＶＮＴＲ定位于人类第１７号染色体的短臂１区３带，与Ｐ５３基因紧密连锁，在Ｍｉｌｅｒ—Ｄｉｃｋｅｒ综合征中发现有Ｄ１７Ｓ５ＶＮＴＲ的信息丢失。为了进一步了解此基因的功能，本文采用聚合酶链反应结合聚丙烯酰胺凝胶电泳的方法对膀胱移行细胞癌的Ｄ１７Ｓ５ＶＮＴＲ的遗传多态性进行了研究。结果显示：膀胱移行细胞癌中Ｄ１７Ｓ５ＶＮＴＲ位点的频率不符合Ｈａｒｄｙ—Ｗｅｉｂｅｒｇ平衡；且ＰＹＮＺ２２ＶＮＴＲ位点的频率分布与正常人群之间存在显著性差异，肿瘤中纯合子的频率高于正常人群。结果表明：Ｄ１７Ｓ５ＶＮＴＲ可作为膀胱癌基因诊断的一个有用指标。

膀胱移行细胞癌中凋亡相关蛋白survivin及caspase3的表达及相关性

目的探讨凋亡相关蛋白survivin及caspase3的表达与膀胱移行细胞癌发生及发展的关系。方法应用SP免疫组织化学法检测69例膀胱移行细胞癌石蜡切片,应用免疫印迹法(Western blotting)检测33例膀胱移行细胞癌新鲜组织中survivin和caspase3表达的情况,结合临床资料进行分析。结果免疫组化结果显示,survivin在膀胱移行细胞癌标本中的表达阳性率为88.4%(61/69),而正常对照组均呈阴性;caspase3在膀胱移行细胞癌标本中的表达阳性率为59.4%(41/69),但是与对照组相比无明显差异。Western blotting结果显示,93.9%(31/33)的肿瘤组织可见survivin蛋白表达,而对照组全部阴性表达;81.8%(27/33)的肿瘤标本可见caspase3蛋白表达,对照组中阳性表达率为80%(8/10),实验组与对照组间无明显差异。在Ⅲ级膀胱移行细胞癌中survivin的表达强度较Ⅰ级为高,二者之间差异有显著性(P<0.05)。survivin和caspase3的表达与肿瘤的临床分期无关(P>0.05),而且survivin的表达与caspase3的表达之间也没有相关性。结论survivin蛋白在膀胱移行细胞癌中特异性表达,其表达的上调可能提示肿瘤分化不良。Caspase3蛋白的表达情况与膀胱移行细胞癌关系不密切。