Mechanism Of Chromosomal Instability In Urothelial Carcinoma And Its Application In Non-invasive Diagnosis,Follow-up And Molecular Subtyping

BackgroundUrinary tract urothelial carcinoma primarily includes urothelial carcinoma of the bladder and upper urinary tract urothelial carcinoma,representing one of the most common tumors in the urogenital system.These cancers are characterized by diagnostic challenges,high recurrence rates,postoperative management difficulties,and substantial economic burden.Despite rapid advancements in medical technology,the diagnosis,treatment,and follow-up protocols for urinary tract urothelial carcinoma have not significantly progressed in recent decades.Cystoscopy serves as the gold standard for diagnosing bladder cancer and is the primary method for postoperative follow-up in upper urinary tract urothelial carcinoma.It offers the advantage of directly observing tumors and obtaining pathological biopsies.However,being an invasive procedure,cystoscopy causes significant discomfort and financial burden to patients.Additionally,it carries risks of complications such as infection,bleeding,perforation,and urethral stricture.Commonly used imaging studies and urine cytology for upper urinary tract urothelial carcinoma have relatively low accuracy.Due to the high postoperative recurrence rate of urinary tract urothelial carcinoma,frequent follow-up is necessary.The postoperative follow-up plan and prognosis assessment heavily rely on the tumor’s pathological characteristics.However,due to tumor heterogeneity,personalized follow-up plans are lacking,leading to inaccurate prognostic judgments.Although guidelines recommend some prognostic models for calculating postoperative recurrence rates,their accuracy remains suboptimal and has not been widely adopted in clinical practice.As our understanding of urinary tract urothelial carcinoma deepens,molecular subtyping based on gene expression becomes a feasible approach for precision medicine.However,transcriptome sequencing-based molecular subtyping methods are costly,and obtaining and preserving clinical samples pose challenges,limiting their clinical application.Therefore,there is an urgent need for a tumor marker that allows non-invasive diagnosis and follow-up of urinary tract urothelial carcinoma while stratifying patients’prognosis.Such a marker would reduce the frequency of cystoscopy and facilitate personalized treatment planning.Chromosomal instability,referring to the ongoing alteration of chrosome,is a major characteristic of tumors,has been widely reported in various malignancies.It can reflect the presence of tumors and their biological behavior.Investigating the role of chromosomal instability in urinary tract urothelial carcinoma can not only aid in early diagnosis,postoperative follow-up,and prognosis evaluation but also contribute to molecular subtyping,potentially reducing the frequency of cystoscopy and enabling individualized treatment.Although some research teams have explored the mechanisms underlying chromosomal instability,its specific mechanisms and potential translational value remain to be further investigated.Research Objectives1.Utilize low-coverage whole-genome sequencing to analyze chromosomal instability in cell-free DNA from urine sediment,achieving non-invasive preoperative diagnosis of urinary tract urothelial carcinoma.2.Employ urine sediment cell DNA chromosomal instability analysis to reflect the original tumor’s chromosomal instability status and establish follow-up and prognosis models.3.Combine immunohistochemistry-based luminal/basal subtyping with chromosomal instability analysis to achieve a cost-effective,highly accurate,and clinically feasible molecular subtyping of urinary tract urothelial carcinoma.4.Explore the mechanisms underlying chromosomal instability in urinary tract urothelial carcinoma to better understand the association between bladder cancer and chromosomal instability,laying the theoretical foundation for exploring novel bladder cancer therapies.Study Subjects and Methods1.Conduct a single-blind,dual-center,prospective clinical study by including preoperative urine samples from patients diagnosed with urinary tract urothelial carcinoma undergoing surgical treatment,urine samples from patients admitted to urology departments due to benign diseases,and urine samples from healthy volunteers.Analyze chromosomal instability and establish a non-invasive diagnostic model during the exploratory phase,followed by validation using an additional verification group.2.Compare urine samples from patients with corresponding tumor tissues,analyze their consistency,and explore whether urine chromosomal instability can reflect the tumor’s molecular features,thereby establishing a prognosis model to guide postoperative follow-up.3.Retrospectively collect patient tissue specimens and use immunohistochemistry to explore the feasibility of luminal/basal subtyping.Combine this with chromosomal instability testing to develop a clinically viable method for precise molecular subtyping.4.Screen driver genes associated with chromosomal instability in bladder cancer using database data and literature reports.Investigate the specific mechanisms in bladder cancer cell lines,aiming to explore potential therapeutic targets related to chromosomal instability.Results1.In this prospective study,a total of 337 patients were enrolled from two clinical centers.After quality control and excluding ineligible patients,the exploratory group included 126 urinary tract urothelial carcinoma patients and 64 control cases,while the validation group included 56 urothelial carcinoma patients and 39 control cases.During the exploratory phase,we established a diagnostic technique named UroCAD,based on low-coverage urine sediment DNA sequencing and analysis of chromosomal instability.UroCAD demonstrated high reliability,repeatability,stability,and cost-effectiveness.Its sensitivity for diagnosing urinary tract urothelial carcinoma was 82.5%,with a specificity of 96.9%.Subsequently,in an external validation group of 99 samples,UroCAD achieved similar diagnostic efficiency(sensitivity:80.4%,specificity:94.9%).Compared to urine cytology experiments,this model exhibited significantly higher sensitivity(80.4%vs.33.9%)while maintaining similar specificity(94.9%vs.100%).Furthermore,this model showed advantages in diagnosing urinary tract tumors.2.We further explored the role of UroCAD in postoperative prognosis evaluation.By collecting urine samples and corresponding tumor tissues for chromosomal instability testing,we found high consistency between urine and tumor tissue chromosomal instability levels.Therefore,we conducted postoperative follow-up on patients from the initial clinical trial,analyzing the correlation between chromosomal instability levels and patient prognosis.A total of 137 urothelial carcinoma patients were included,with a median follow-up time of 44.2 months.Among them,41.6%experienced recurrence or progression.Based on their chromosomal instability levels,patients were divided into "CIN-high" and "CINlow" groups.After transurethral resection of bladder tumors,the recurrence and progression rates were 57.9%for the "CIN-high" group and 34.29%for the "CINlow" group.Further multivariate Cox regression analysis confirmed that chromosomal instability was an independent prognostic factor for urothelial carcinoma.Additionally,when combined with internationally recognized prognostic models,chromosomal instability significantly improved the predictive value of the model.3.Given the significant heterogeneity of urinary tract urothelial carcinoma,which leads to varying prognoses despite similar pathological features,we explored a molecular subtyping method based on four immunohistochemical markers:CK5/6,CK14,CK20,and GATA3.We retrospectively included 236 UC patients and obtained their tumor tissues.After staining these four markers and scoring them,we classified tumors into luminal and basal subtypes.Basal-type tumors exhibited significantly worse prognosis.Multivariate Cox analysis revealed that basal subtyping was an independent prognostic marker(hazard ratio:1.91,p<0.01).Subsequently,we combined chromosomal instability testing with immunohistochemical subtyping in 30 patients.We found that luminal-type patients with high chromosomal instability had significantly worse prognosis.4.Recognizing the importance of chromosomal instability in urinary tract urothelial carcinoma,we explored its mechanisms in bladder cancer cell lines.Through literature review and database screening,we identified two genes,ACTL6A and IQGAP3,potentially related to chromosomal instability in bladder cancer.We conducted various experiments to investigate how these two genes contribute to chromosomal instability in bladder cancer cell lines.Further research revealed an association between ACTL6A and IQGAP3 through the Hippo pathway.We identified the ACTL6A-YAP1-IQGAP3/ANLN pathway as playing a crucial role in the formation of chromosomal instability in bladder cancer.Conclusions1.In urinary tract urothelial carcinoma,using low-coverage whole-genome sequencing to detect chromosomal instability in preoperative urine sediment can achieve non-invasive diagnosis with high sensitivity and specificity.2.Preoperative urine chromosomal instability testing can reflect the tumor’s chromosomal instability level,making it useful for prognosis evaluation in urinary tract urothelial carcinoma.3.Immunohistochemistry-based molecular subtyping is able to stratify bladder cancer into luminal and basal subtypes.Combining with chromosomal instability testing allows for cost-effective and clinically feasible prognosis prediction.4.The ACTL6A-YAP1-IQGAP3/ANLN pathway,associated with chromosomal instability,plays a critical role in bladder cancer cell lines,providing insights for exploring novel therapeutic targets related to chromosomal instability in bladder cancer.