Unintrusive multi-cancer detection by circulating cell-free DNA methylation sequencing (THUNDER): development and independent validation studies.

BACKGROUND: Early detection of cancer offers the opportunity to identify candidates when curative treatments are achievable. The THUNDER study (THe UNintrusive Detection of EaRly-stage cancers, NCT04820868) aimed to evaluate the performance of enhanced linear-splinter amplification sequencing, a previously described cell-free DNA (cfDNA) methylation-based technology, in the early detection and localization of six types of cancers in the colorectum, esophagus, liver, lung, ovary, and pancreas. PATIENTS AND METHODS: A customized panel of 161 984 CpG sites was constructed and validated by public and in-house (cancer: n = 249; non-cancer: n = 288) methylome data, respectively. The cfDNA samples from 1693 participants (cancer: n = 735; non-cancer: n = 958) were retrospectively collected to train and validate two multi-cancer detection blood test (MCDBT-1/2) models for different clinical scenarios. The models were validated on a prospective and independent cohort of age-matched 1010 participants (cancer: n = 505; non-cancer: n = 505). Simulation using the cancer incidence in China was applied to infer stage shift and survival benefits to demonstrate the potential utility of the models in the real world. RESULTS: MCDBT-1 yielded a sensitivity of 69.1% (64.8%-73.3%), a specificity of 98.9% (97.6%-99.7%), and tissue origin accuracy of 83.2% (78.7%-87.1%) in the independent validation set. For early-stage (I-III) patients, the sensitivity of MCDBT-1 was 59.8% (54.4%-65.0%). In the real-world simulation, MCDBT-1 achieved a sensitivity of 70.6% in detecting the six cancers, thus decreasing late-stage incidence by 38.7%-46.4%, and increasing 5-year survival rate by 33.1%-40.4%, respectively. In parallel, MCDBT-2 was generated at a slightly low specificity of 95.1% (92.8%-96.9%) but a higher sensitivity of 75.1% (71.9%-79.8%) than MCDBT-1 for populations at relatively high risk of cancers, and also had ideal performance. CONCLUSION: In this large-scale clinical validation study, MCDBT-1/2 models showed high sensitivity, specificity, and accuracy of predicted origin in detecting six types of cancers.

[Effects of cadmium chloride on testicular autophagy and blood-testis barrier integrity in prepubertal male rats].

Objective: To study the effects of cadmium chloride (CdCl(2)) exposure on testicular autophagy levels and blood-testis barrier integrity in prepubertal male SD rats and testicular sertoli (TM4) cells. Methods: In July 2021, 9 4-week-old male SD rats were randomly divided into 3 groups: control group (normal saline), low dose group (1 mg/kg·bw CdCl(2)) and high dose group (2 mg/kg·bw CdCl(2)), and were exposed with CdCl(2) by intrabitoneal injection. 24 h later, HE staining was used to observe the morphological changes of testis of rats, biological tracer was used to observe the integrity of blood-testis barrier, and the expression levels of microtubule-associated protein light chain 3 (LC3) -Ⅰ and LC3-Ⅱ in testicular tissue were detected. TM4 cells were treated with 0, 2.5, 5.0 and 10.0 µmol/L CdCl(2) for 24 h to detect the toxic effect of cadmium. The cells were divided into blank group (no exposure), exposure group (10.0 µmol/L CdCl(2)), experimental group[10.0 µmol/L CdCl(2)+60.0 µmol/L 3-methyladenine (3-MA) ] and inhibitor group (60.0 µmol/L 3-MA). After 24 h of treatment, Western blot analysis was used to detect the expression levels of LC3-Ⅱ, ubiquitin binding protein p62, tight junction protein ZO-1 and adhesion junction protein N-cadherin. Results: The morphology and structure of testicular tissue in the high dose group were obvious changed, including uneven distribution of seminiferous tubules, irregular shape, thinning of seminiferous epithelium, loose structure, disordered arrangement of cells, abnormal deep staining of nuclei and vacuoles of Sertoli cells. The results of biological tracer method showed that the integrity of blood-testis barrier was damaged in the low and high dose group. Western blot results showed that compared with control group, the expression levels of LC3-Ⅱ in testicular tissue of rats in low and high dose groups were increased, the differences were statistically significant (P<0.05). Compared with the 0 µmol/L, after exposure to 5.0, 10.0 µmol/L CdCl(2), the expression levels of ZO-1 and N-cadherin in TM4 cells were significantly decreased, and the expression level of p62 and LC3-Ⅱ/LC3-Ⅰ were significantly increased, the differences were statistically significant (P<0.05). Compared with the exposure group, the relative expression level of p62 and LC3-Ⅱ/LC3-Ⅰ in TM4 cells of the experimental group were significantly decreased, while the relative expression levels of ZO-1 and N-cadherin were significantly increased, the differences were statistically significant (P<0.05) . Conclusion: The mechanism of the toxic effect of cadmium on the reproductive system of male SD rats may be related to the effect of the autophagy level of testicular tissue and the destruction of the blood-testis barrier integrity.

[A randomized controlled study of erlotinib versus pemetrexed combined with cisplatin in neoadjuvant therapy of stage â ¢A EGFR-mutant lung adenocarcinoma].

Objective: To evaluate the feasibility, efficacy and safety of epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs) for neoadjuvant therapy. Methods: Eighty-six patients with stage ⅢA EGFR-mutant lung adenocarcinoma were assigned to 2 groups (n=43 in each group) according to the random number table method: neoadjuvant targeted therapy group (single oral dose of erlotinib 150 mg per day, for 9 weeks) and neoadjuvant chemotherapy group (2 cycles of pemetrexed combined with cisplatin chemotherapy followed by 3- week discontinuation). Surgical treatment was underwent after imaging efficacy evaluation. Results: In neoadjuvant targeted therapy group, 4 achieved complete response (CR), 25 achieved partial response (PR), giving an objective response rate (ORR) of 67.4%. In pathological response, 8 patients had grade Ⅰ, 20 patients had grade Ⅱ, giving a pathological response rate of 65.1%. The most frequent adverse events (AEs) were rash and diarrhea. In neoadjuvant chemotherapy group, 2 had CR and 17 had PR, giving an ORR of 44.2%. In pathological response, 3 patients had grade Ⅰ, 15 patients had grade Ⅱ, giving a pathological response rate of 41.9%. The main AEs were hematologic toxic effects. The ORR, histological efficacy and hematologic toxicity showed statistical significance between the two groups (P<0.05). The neoadjuvant targeted therapy group had 90.7% resection rate, (299.8±23.4) ml of hemorrhage volume during operation, (5.2±0.4) days of extubation time and 9.3% postoperative complication rate. Corresponding results were 83.7%, (308.9±22.7) ml, (5.4±0.6) days and 11.6% in neoadjuvant chemotherapy group, which showed no statistical significance (P>0.05). Conclusions: Neoadjuvant targeted treatment for stage ⅢA lung adenocarcinoma harboring EGFR mutations. The regimen could be considered as a choice of neoadjuvant treatment for patients with stage ⅢA EGFR-mutant lung adenocarcinoma.

Association of the LMNA gene single nucleotide polymorphism rs4641 with bdilated cardiomyopathy.

Recently, studies on the pathogenesis of dilated cardiomyopathy (DCM) have focused on the underlying molecular biology and the association between single nucleotide polymorphisms (SNPs) and disease. This study was designed to explore the association between the rs4641 SNP of the LMNA gene and DCM in order to identify a new gene locus related to DCM. Polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing were employed to detect and genotype rs4641 in 198 patients with DCM and 160 healthy controls. Genotype and allele frequencies were compared to discover their relationship and logistic regression was used to assess the risk of DCM associated with the polymorphic variants. In the DCM group, the frequencies of the TC and TT genotypes and the T allele of rs4641 were remarkably higher than those in the control group (P < 0.01). According to risk analysis, taking the CC genotype as a reference, both the TC and TT genotypes increased the risk of DCM pathogenesis, with OR (95%CI) values of 5.957 (2.903- 12.222) and 6.424 (2.156-19.141), respectively. Taking the C allele as the reference, presence of the T allele was found to increase DCM risk, with OR (95%CI) of 5.295 (3.121-8.983). These results suggested that the C to T mutation at the rs4641 locus of LMNA could enhance the risk of DCM, and that rs4641 represented a genetic susceptibility locus. Therefore, it was concluded that the LMNA rs4641 SNP was associated with DCM risk, which indicated that LMNA is a susceptibility gene for DCM.

Serum secretory phospholipase A2-IIa (sPLA2-IIA) levels in patients surviving acute myocardial infarction.

BACKGROUND: The increase of secretory phospholipase A2-IIa (sPLA2-IIa) in culprit coronary lesions is associated with myocardial infarction, and the increase of sPLA2-IIa in peripheral plasma levels has a significant risk and prognostic value in patients with coronary artery disease. Little is known about the prognostic significance of elevated serum sPLA2-IIa in post-acute myocardial infarction (post-AMI) patients. OBJECTIVES: The present study is designed to investigate the potential association between serum sPLA2-IIa and prognosis in post-acute myocardial infarction (post-AMI) patients. PATIENTS AND METHODS: From Oct 1998 to Sep 2008, a total of 964 post-AMI patients with serum samples collected in the convalescent stage were studied. Serum levels of sPLA2-IIa were measured by ELISA. According to the optimal cut-off value for sPLA2-IIa concentration, patients were then divided into 2 groups. Categorical variables were compared between the 2 groups using the χ2 test. All continuous variables are described as mean ± SD and were compared using Student's t-test. Statistical associations between clinicopathological observations and sPLA2-IIa levels were determined using the Mann-Whitney U test. The clinical value of sPLA2-IIa level as a prognostic parameter was evaluated using the Cox's proportional hazards model. RESULTS: During a median follow-up period of 1,462 days, 123 patients (12.7%) had adverse events (all-cause mortality, n=52; non-fatal MI, n=31; readmission for heart failure [HF], n=40). Patients were divided into 2 groups according to a serum sPLA2-IIa level of 360 ng/dl, which was determined to be the optimal cut-off for discriminating all-cause mortality based on the maximum value of the area under the receiver operating characteristic curve. Patients with elevated sPLA2-IIa (> 360 ng/dl, n=164) had a significantly higher prevalence of death (18.3% [30/164] vs. 2.75% [22/800] p < 0.001) and readmission for HF (14% [23/164/ vs. 2.1% [17/800], p < 0.0001), but not of non-fatal MI (4.88% [8/164]vs. 2.87% [23/800], p = 0.096), compared to those with sPLA2-IIa < 360 ng/dl. Multivariate Cox regression analysis indicated that elevated serum sPLA2-IIa was associated with an increased risk of mortality and readmission for HF. CONCLUSIONS: Elevated serum sPLA2-IIa during the convalescent stage of AMI predicted long-term mortality and readmission for HF after survival discharge in the post-AMI patients.

[Application of dental floss traction-assisted endoscopic submucosa dissection to rectal neuroendocrine neoplasm].

Objective: To evaluate the safety and efficacy of dental floss traction-assisted endoscopic submucosal dissection (DFS-ESD) for rectal neuroendocrine neoplasm (NEN). Methods: A retrospective cohort study was performed. Clinical data of rectal NEN patients undergoing ESD at Endoscopy Center of Zhongshan Hospital, Fudan University from January 2016 to December 2017 were retrospectively analyzed. Inclusion criteria: 1) age of 18 to 80 years old; 2) maximal diameter of lesions <1.5 cm; 3) tumor locating in the submucosa without invasion into the muscularis propria; 4) no enlarged lymph nodes around bowel and in abdominal cavity; 5) ESD requested actively by patients. A total of 37 patients were enrolled, including 23 male and 14 female cases with mean age of (56.0±11.3) years. All the lesions were single tumor of stage T1, and the mean size was 0.8±0.2(0.5-1.2) cm. Postoperative pathology revealed all samples as neuroendocrine tumors (NET). Seventeen patients received DFS-ESD treatment (DFS-ESD group) and 20 patient received conventional ESD treatment (conventional ESD group). In DFS-ESD group, after the mucosa was partly incised along the marker dots, the endoscopy was extracted, and the dental floss was tied to one arm of the metallic clip. When the endoscope was reinserted, the hemoclip was attached onto the incised mucosa; another hemoclip was attached onto normal mucosa opposite to the lesion in the same way. The submucosa was clearly exposed with the traction of dental floss and the resection could proceed. The conventional ESD group received the traditional ESD operation procedure. The operation time, modified operation time (remaining time after excluding the assembly time of dental floss traction in DFS-ESD group), en bloc resection rate, R0 resection rate, morbidity of operative complication, recurrence and metastasis were compared between two groups. Results: The average tumor size was (0.8±0.2) cm in DFS-ESD group and (0.7±0.2) cm in conventional ESD group (t=0.425, P=0.673). According to postoperative pathological grading of rectal neuroendocrine neoplasm, 13 were G1 and 4 were G2 in DFS-ESD group, while 17 cases were G1 and 3 cases were G2 in conventional ESD group without significant difference (P=0.680). There were no significant differences in baseline data between in the two groups (all P>0.05). All the basal resection margins were negative, the en bloc resection rate was 100% and the R0 resection rate was 100%. Pathological results showed tumor tissue close to the burning margin in 5 cases of conventional ESD group and in 2 cases of DFS-ESD group (P=0.416). The operation time was (17.9±6.6) minutes in conventional ESD group and (14.7±3.3) minutes in DFS-ESD group (t=1.776, P=0.084). The modified operation time of DFS-ESD group was (11.9±2.8) minutes, which was significantly shorter than (17.9±6.6) minutes in conventional ESD group (t=3.425, P=0.002). The hospital stay was (2.3±0.6) days and (2.0±0.5) days in conventional ESD group and DFS-ESD group, respectively, without significant difference (t=1.436, P=0.160). No patient was transferred to surgery, and no delayed bleeding or perforation occurred in either group. There was no recurrence or primary tumor-related death, and all the patients recovered well during a follow-up period of 14(1-24) months. Conclusion: Dental floss traction-assisted ESD for rectal neuroendocrine neoplasm can simplify operation and ensure negative basal margin.

Relationship of platelet microparticle CD62P and activated GP IIb/IIIa with hypercoagulable state after atrial fibrillation radiofrequency catheter ablation.

OBJECTIVE: The morbidity of atrial fibrillation (AF) is 1%-2% in clinic. Radiofrequency catheter ablation (RFCA) is a type of radical interventional therapy for AF, whereas it may lead to a hypercoagulable state. This study evaluated platelet particle CD62P and platelet activation biomarker GP IIb/IIIa expressions in AF patients treated by RFCA, and aimed to analyze their relationships with the hypercoagulable state after RFCA. PATIENTS AND METHODS: A total of 60 AF patients received RFCA in our hospital were enrolled. The patients were divided into group A as hypercoagulable state group and group B as non-hypercoagulable group. Healthy volunteers were selected as normal control. Serum D-Dimer, parathyroid activity index 1 (PAI-1), and tissue plasminogen activator (t-PA) content were tested by using enzyme-linked immunosorbent assay (ELISA), while peripheral CD62P and GP IIb/IIIa expressions were detected by using flow cytometry before, after, and seven days after RFCA. RESULTS: D-Dimer and PAI-1 levels increased, while t-PA reduced in group A compared with that in group B and control (p<0.05). D-Dimer and t-PA contents gradually elevated, whereas t-PA level gradually declined in group A before, after, and seven days after RFCA (p<0.05). Serum CD62P and GP IIb/IIIa expressions in group A were significantly higher compared to that in group B and control (p<0.05). CD62P and GP IIb/IIIa levels were significantly higher seven days after RFCA compared with immediate after RFCA in group A (p<0.05). CD62P showed a positive correlation with GP IIb/IIIa in hypercoagulable state patients after RFCA (p<0.05). CONCLUSIONS: AF patient may appear in hypercoagulable state after RFCA. CD62P and GP IIb/IIIa significantly increased and exhibited a positive correlation.

Multiple factor analysis of metachronous upper urinary tract transitional cell carcinoma after radical cystectomy.

Transitional cell carcinoma (TCC) of the urothelium is often multifocal and subsequent tumors may occur anywhere in the urinary tract after the treatment of a primary carcinoma. Patients initially presenting a bladder cancer are at significant risk of developing metachronous tumors in the upper urinary tract (UUT). We evaluated the prognostic factors of primary invasive bladder cancer that may predict a metachronous UUT TCC after radical cystectomy. The records of 476 patients who underwent radical cystectomy for primary invasive bladder TCC from 1989 to 2001 were reviewed retrospectively. The prognostic factors of UUT TCC were determined by multivariate analysis using the COX proportional hazards regression model. Kaplan-Meier analysis was also used to assess the variable incidence of UUT TCC according to different risk factors. Twenty-two patients (4.6%). developed metachronous UUT TCC. Multiplicity, prostatic urethral involvement by the bladder cancer and the associated carcinoma in situ (CIS) were significant and independent factors affecting the occurrence of metachronous UUT TCC (P = 0.0425, 0.0082, and 0.0006, respectively). These results were supported, to some extent, by analysis of the UUT TCC disease-free rate by the Kaplan-Meier method, whereby patients with prostatic urethral involvement or with associated CIS demonstrated a significantly lower metachronous UUT TCC disease-free rate than patients without prostatic urethral involvement or without associated CIS (log-rank test, P = 0.0116 and 0.0075, respectively). Multiple tumors, prostatic urethral involvement and associated CIS were risk factors for metachronous UUT TCC, a conclusion that may be useful for designing follow-up strategies for primary invasive bladder cancer after radical cystectomy.

Study on the effect of different doses of rosuvastatin on ventricular remodeling in patients with acute coronary syndrome after emergency percutaneous coronary intervention.

OBJECTIVE: The objective of the present study was to observe the effects of different doses of rosuvastatin on cardiac protection in patients with acute coronary syndrome (ACS) after stent implantation. PATIENTS AND METHODS: A total of 137 patients with ACS were selected from March 2014 to January 2015 and randomly divided into: 1. The conventional treatment group: 45 patients were treated with conventional drugs such as aspirin, clopidogrel, nitrates, and a ß-blocker; 2. The conventional rosuvastatin dose group: 45 patients received 10 mg/d rosuvastatin before sleep in addition to routine therapy; 3. The large rosuvastatin dose group: 47 patients received 20 mg/d rosuvastatin before sleep in addition to routine therapy. The course of treatment was 12 weeks. At 1, 6, and 12 week, ultrasound echocardiography, electrocardiogram (ECG), high-sensitivity C-reactive protein (hs-CRP), and pro-brain natriuretic peptide (pro-BNP) levels were tested to evaluate the therapeutic effects. The ultrasonic imaging criteria included left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), and left ventricular ejection fraction (LVEF). RESULTS: After 1 week, hs-CRP, pro-BNP, and echocardiography of the patients in the three groups showed no significant differences (p>0.05); after 6 and 12 weeks, the levels of hs-CRP, MMP-9, and pro-BNP in the large rosuvastatin dose group were significantly lower than in the conventional rosuvastatin dose group and conventional treatment group (p<0.05), and ultrasonic indexes changed significantly after 12 weeks (p<0.05). There were no significant differences in ultrasonic indexes after 6 weeks (p>0.05). No thrombosis or restenosis occurred during the follow-up period in each group. CONCLUSIONS: Three months after emergency percutaneous coronary intervention, a high-dose of rosuvastatin can delay ventricular remodeling, effectively inhibit malignant remodeling of the heart, improve left ventricular systolic function, reduce the prevalence of adverse events, and significantly improve the long-term prognosis.

Cellular origin of renal cell carcinoma--an immunohistological study on monoclonal antibodies.

It has been recognized in recent years that renal cell carcinoma possesses a proximal convoluted tubules origin. In an attempt of further intensive evaluation we assayed 51 renal cell carcinomas and 38 additional normal kidney specimens with 4 kidney segment-specific antibodies (Uro-2/S4, Uro-5/T16, Uro-10/T43, Anti Tamm-Horsfall Protein) directed against the nephrotic cells by indirect immunohistological techniques. Consistently stable staining was developed in the tests. The results showed that the majority of renal clear cell carcinomas expressed nephrogenous properties of proximal convoluted tubules but in part also of distal tubules and collecting ducts. Granular cell carcinoma mostly demonstrated histogenic appearance of distal convoluted tubules.

Laparoscopic excision of extraprostatic ejaculatory duct cyst.

Our experiences with laparoscopic excisions of symptomatic extraprostatic ejaculatory duct cysts (EDCs) are reported. Three laparoscopic excisions of extraprostatic EDCs performed by one urologist in 2003 and 2004 were retrospectively reviewed. Investigations included history, physical examination, image analysis, semen analysis, operation time, estimated blood loss, time of post-operative hospital stay, recovery time for regular activities, sexual function and complications. The laparoscopic excisions of EDCs were successful. The mean operation time was 105 min, and the mean estimated blood loss was 65 ml. The average post-operative hospital stay was 3.0 days. All patients exhibited normal erection and normal ejaculation. Improvement in semen quality was observed in two patients. All patients remained free of symptoms, and recurrence of EDCs was not detected on transrectal ultrasonography over a mean 32-month follow-up period. It is concluded that laparoscopic excision of an EDC is feasible and effective. Due to minimal invasiveness, short post-operative hospitalisation and rapid recovery, laparoscopic surgery is an attractive approach to managing symptomatic EDCs, especially for sizeable cysts or those including calculus.

Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells.

The members of MAGE gene family are highly expressed in human hepatocellular carcinoma (HCC). In the present study, we tested the tumour-specific MAGE-1 and MAGE-3 transcripts in the peripheral blood of HCC patients by nested RT-PCR to detect the circulating tumour cells and evaluate their potential clinical implication. Of 30 HCC patients, the positive rate of MAGE-1 and MAGE-3 transcripts was 43.3% (13 out of 30) and 33.3% (10 out of 30) in PBMC samples, whilst the positive rate was 70% (21 out of 30) and 53.3% (16 out of 30) in the resected HCC tissue samples, respectively. The positivity for at least one MAGE gene transcript was 63.3% (19 out of 30) in PBMC samples of HCC patients and 83.3% (25 out of 30) in the resected HCC tissue samples. MAGE-1 and/or MAGE-3 mRNA were not detected in the PBMC of those patients from whom the resected HCC tissues were MAGE-1 or MAGE-3 mRNA negative, nor in the 25 PBMC samples from healthy donors. The detection of MAGE transcripts in PBMC was correlated with the advanced stages and tumour size of the HCC, being 82.4% (14 out of 17) in tumour stages III and IVa, 56.6% (five out of nine) in stage II, and null (nought out of four) in stage I. The serum alpha-FP in 33.3% (10 out of 30) of HCC patients was normal or slightly elevated (< 40 ng ml(-1)). However, six of these 10 patients (alpha-FP < 40 ng ml(-1)) were MAGE-1 and /or MAGE-3 mRNA positive in their PBMC. The follow-up survey of MAGE mRNA in PBMC was performed in 12 patients. Seven patients with persistent MAGE-1 and/or MAGE-3 mRNA positive or from negative turned to positive died because of metastasis and/or recurrence. In striking contrast, all four patients with MAGE-1 and/or MAGE-3 mRNA from positive turned to negative and one patient with persistent MAGE-3 transcript negative are alive after last test. Collectively, detection of MAGE transcripts with follow-up survey in PBMC is a feasible and reliable assay for the early prediction of the relapse and prognosis of the HCC patients.

Multiple factor analysis of metachronous upper urinary tract transitional cell carcinoma after radical cystectomy

Transitional cell carcinoma (TCC) of the urothelium is often multifocal and subsequent tumors may occur anywhere in the urinary tract after the treatment of a primary carcinoma. Patients initially presenting a bladder cancer are at significant risk of developing metachronous tumors in the upper urinary tract (UUT). We evaluated the prognostic factors of primary invasive bladder cancer that may predict a metachronous UUT TCC after radical cystectomy. The records of 476 patients who underwent radical cystectomy for primary invasive bladder TCC from 1989 to 2001 were reviewed retrospectively. The prognostic factors of UUT TCC were determined by multivariate analysis using the COX proportional hazards regression model. Kaplan-Meier analysis was also used to assess the variable incidence of UUT TCC according to different risk factors. Twenty-two patients (4.6 percent). developed metachronous UUT TCC. Multiplicity, prostatic urethral involvement by the bladder cancer and the associated carcinoma in situ (CIS) were significant and independent factors affecting the occurrence of metachronous UUT TCC (P = 0.0425, 0.0082, and 0.0006, respectively). These results were supported, to some extent, by analysis of the UUT TCC disease-free rate by the Kaplan-Meier method, whereby patients with prostatic urethral involvement or with associated CIS demonstrated a significantly lower metachronous UUT TCC disease-free rate than patients without prostatic urethral involvement or without associated CIS (log-rank test, P = 0.0116 and 0.0075, respectively). Multiple tumors, prostatic urethral involvement and associated CIS were risk factors for metachronous UUT TCC, a conclusion that may be useful for designing follow-up strategies for primary invasive bladder cancer after radical cystectomy.