Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Beyond basics: Key mutation selection features for successful tumor-informed ctDNA detection.

Tumor-informed mutation-based approaches are frequently used for detection of circulating tumor DNA (ctDNA). Not all mutations make equally effective ctDNA markers. The objective was to explore if prioritizing mutations using mutational features-such as cancer cell fraction (CCF), multiplicity, and error rate-would improve the success rate of tumor-informed ctDNA analysis. Additionally, we aimed to develop a practical and easily implementable analysis pipeline for identifying and prioritizing candidate mutations from whole-exome sequencing (WES) data. We analyzed WES and ctDNA data from three tumor-informed ctDNA studies, one on bladder cancer (Cohort A) and two on colorectal cancer (Cohorts I and N). The studies included 390 patients. For each patient, a unique set of mutations (median mutations/patient: 6, interquartile 13, range: 1-46, total n = 4023) were used as markers of ctDNA. The tool PureCN was used to assess the CCF and multiplicity of each mutation. High-CCF mutations were detected more frequently than low-CCF mutations (Cohort A: odds ratio [OR] 20.6, 95% confidence interval [CI] 5.72-173, p = 1.73e-12; Cohort I: OR 2.24, 95% CI 1.44-3.52, p = 1.66e-04; and Cohort N: OR 1.78, 95% CI 1.14-2.79, p = 7.86e-03). The detection-likelihood was additionally improved by selecting mutations with multiplicity of two or above (Cohort A: OR 1.55, 95% CI 1. 14-2.11, p = 3.85e-03; Cohort I: OR 1.78, 95% CI 1.23-2.56, p = 1.34e-03; and Cohort N: OR 1.94, 95% CI 1.63-2.31, p = 2.83e-14). Furthermore, selecting the mutations for which the ctDNA detection method had the lowest error rates, additionally improved the detection-likelihood, particularly evident when plasma cell-free DNA tumor fractions were below 0.1% (p = 2.1e-07). Selecting mutational markers with high CCF, high multiplicity, and low error rate significantly improve ctDNA detection likelihood. We provide free access to the analysis pipeline enabling others to perform qualified prioritization of mutations for tumor-informed ctDNA analysis.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Race reporting and disparities regarding clinical trials in bladder cancer: a systematic review.

PURPOSE: To systematically review the literature to investigate racial disparities among bladder cancer clinical trial enrollees. METHODS: A systematic review was conducted using Ovid, MEDLINE® to identify clinical trials between 1970 and 2020. Articles were reviewed and were included if they assessed race in their outcomes reporting among bladder cancer patients enrolled in clinical trials. The review was conducted in accordance with the PRISMA statement. RESULTS: We identified 544 clinical trials meeting our initial search criteria, with only 24 (4.4%) studies reporting racial demographic data. Enrollees were largely Caucasian (81-98%), with a strikingly small proportion of enrolled patients consisting of African-Americans (2-8%) and Hispanics (2-5%). Only one of the studies reported results on the efficacy and safety/tolerability of the tested treatment separately for racial groups and performed analyses stratified by race. CONCLUSION: Race is poorly studied in bladder cancer clinical trials. Trial cohorts may not reflect multicultural populations. The potential association between race and efficacy, safety or tolerability of the tested interventions is unknown. Given the up to twofold increase in bladder cancer-specific death among African-Americans, further research is needed to address the impact of race in clinical trials, while encompassing socioeconomic factors and disease risk factor exposures.

A miRNA signature predicts benefit from addition of hypoxia-modifying therapy to radiation treatment in invasive bladder cancer.

BACKGROUND: miRNAs are promising biomarkers in oncology as their small size makes them less susceptible to degradation than mRNA in FFPE tissue. We aimed to derive a hypoxia-associated miRNA signature for bladder cancer. METHODS: Taqman miRNA array cards identified miRNA seed genes induced under hypoxia in bladder cancer cell lines. A signature was derived using feature selection methods in a TCGA BLCA training data set. miRNA expression data were generated for 190 tumours from the BCON Phase 3 trial and used for independent validation. RESULTS: A 14-miRNA hypoxia signature was derived, which was prognostic for poorer overall survival in the TCGA BLCA cohort (n = 403, p = 0.001). Univariable analysis showed that the miRNA signature predicted an overall survival benefit from having carbogen-nicotinamide with radiotherapy (HR = 0.30, 95% CI 0.094-0.95, p = 0.030) and performed similarly to a 24-gene mRNA signature (HR = 0.47, 95% CI 0.24-0.92, p = 0.025). Combining the signatures improved performance (HR = 0.26, 95% CI 0.08-0.82, p = 0.014) with borderline significance for an interaction test (p = 0.065). The interaction test was significant for local relapse-free survival LRFS (p = 0.033). CONCLUSION: A 14-miRNA hypoxia signature can be used with an mRNA hypoxia signature to identify bladder cancer patients benefitting most from having carbogen and nicotinamide with radiotherapy.

The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Identification of ADAM12 as a Novel Basigin Sheddase.

The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.

Discordant molecular subtype classification in the basal-squamous subtype of bladder tumors and matched lymph-node metastases.

Molecular subtypes of muscle-invasive bladder tumors have emerged as a promising research tool with potential to stratify patients for neoadjuvant treatment. Prior to radical cystectomy, the utility of molecular classification and biomarkers depend on concordance between tissue from transurethrally resected specimens and disseminated disease. We assess the concordance of molecular subtypes and a large number of potential biomarkers in 67 pairs of muscle-invasive bladder tumors and synchronous lymph-node metastases. Tissue cores were stained for 29 immunohistochemistry markers and immunohistochemistry-based molecular subtype classification was performed. Molecular subtype was determined by mRNA profiling for 57 bladder tumors and 28 matched lymph-node metastases. Full section immunohistochemistry was performed to assess intra-tumor subtype heterogeneity in discordant cases, and exome sequencing was performed for 20 sample pairs. Discordant subtype classification between the bladder tumor and lymph-node metastasis was generally rare (12/67, 18%), but most (7/12, 58%) involved the Basal/Squamous-like subtype. Discordant Basal/Squamous-like tumors showed either Urothelial-like or Genomically Unstable, luminal-like phenotype in the lymph-node metastasis. Full section immunohistochemistry revealed intra-tumor subtype heterogeneity for six discordant cases including four involving the Basal/Squamous-like subtype. Subtype concordance for non- Basal/Squamous-like tumors was 91%. RNA-based classification agreed with immunohistochemistry classification but quantitative assessment is necessary to avoid false detection of subtype shifts. Most high confidence cancer mutations were shared between samples (n = 93, 78%), and bladder tumor private mutations (n = 20, 17%) were more frequent than those private to the lymph-node metastasis (n = 7, 6%). We conclude that bladder tumors and lymph-node metastases have overall similar molecular subtype, biomarker expression, and cancer mutations. The main exception was tumors of the Basal/Squamous-like subtype where most cases showed discordant classification, some with evidence of intra-tumor heterogeneity. The data are of relevance for neoadjuvant treatment stratification and raises questions on the dynamics of molecular subtypes during bladder cancer progression.

Biology of nonmuscle-invasive bladder cancer: pathology, genomic implications, and immunology.

PURPOSE OF REVIEW: Despite that nearly 75% of bladder cancer patients are diagnosed with nonmuscle-invasive disease, our understanding of the biological landscape in bladder cancer is primarily within the context of muscle-invasive bladder cancer. More recent studies addressing the genomic changes and immunology of nonmuscle-invasive bladder cancer (NMIBC) have helped to extend our understanding of this prevalent disease. RECENT FINDINGS: Genomic studies reveal that NMIBC possesses complexity that can be defined by specific gene expression signatures and has helped to define subsets within this disease. These subsets possess different risk profiles that may impact treatment decisions. In addition, the baseline or posttreatment immunological response to the growing tumor may help to inform whether a specific NMIBC subset is likely to progress. SUMMARY: Findings from studies addressing the molecular landscape of NMIBC may help to establish parameters for stratifying patient risk within this disease as well as establish novel or targeted treatment strategies. Inclusion of information about the immune response within tumors will likely contribute to defining the relative risk and treatment strategy for these patients.

FGFR3, TERT and OTX1 as a Urinary Biomarker Combination for Surveillance of Patients with Bladder Cancer in a Large Prospective Multicenter Study.

PURPOSE: Patients with nonmuscle invasive bladder cancer are followed with frequent cystoscopies. In this study FGFR3, TERT and OTX1 were investigated as a diagnostic urinary marker combination during followup of patients with primary nonmuscle invasive bladder cancer. MATERIALS AND METHODS: In this international, multicenter, prospective study 977 patients with nonmuscle invasive bladder cancer were included. A total of 2,496 urine samples were collected prior to cystoscopy during regular visits. Sensitivity was estimated to detect concomitant recurrences. Kaplan-Meier curves were used to estimate the development of future recurrences after urinalysis and a negative cystoscopy. RESULTS: Sensitivity of the assay combination for recurrence detection was 57% in patients with primary low grade, nonmuscle invasive bladder cancer. However, sensitivity was 83% for recurrences that were pT1 or muscle invasive bladder cancer. Of the cases 2% progressed to muscle invasive bladder cancer. Sensitivity for recurrence detection in patients with primary high grade disease was 72% and 7% of them had progression to muscle invasive bladder cancer. When no concomitant tumor was found by cystoscopy, positive urine samples were more frequently followed by a recurrence over time compared to a negative urine sample (58% vs 36%, p <0.001). High stage recurrences were identified within 1 year after a positive urine test and a negative cystoscopy. CONCLUSIONS: Recurrences in patients with primary nonmuscle invasive bladder cancer can be detected by a combination of urine assays. This study supports the value of urinalysis as an alternative diagnostic tool in patients presenting with low grade tumors and as a means to identify high stage tumors earlier.

Validation of a DNA Methylation-Mutation Urine Assay to Select Patients with Hematuria for Cystoscopy.

PURPOSE: Only 3% to 28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer. Cystoscopy leads to high diagnostic costs and a high patient burden. Therefore, to improve the selection of patients for cystoscopy and reduce costs and over testing we aimed to validate a recently developed diagnostic urine assay. MATERIALS AND METHODS: Included in study were 200 patients from a total of 3 European countries who underwent cystoscopy for hematuria, including 97 with bladder cancer and 103 with nonmalignant findings. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and HRAS, and methylation of OTX1, ONECUT2 and TWIST1. Logistic regression was used to analyze the association between predictor variables and bladder cancer. RESULTS: Combining the methylation and mutation markers with age led to an AUC of 0.96 (95% CI 0.92-0.99) with 93% sensitivity and 86% specificity, and an optimism corrected AUC of 0.95. The AUC was higher for T1 or greater tumors compared to Ta tumors (0.99 vs 0.93). The AUC was also higher for high grade tumors compared to low grade tumors (1.00 vs 0.93). Overall negative predictive value was 99% based on the 5% to 10% prevalence of bladder cancer in patients with hematuria. This would lead to a 77% reduction in diagnostic cystoscopy. CONCLUSIONS: Analyzing hematuria patients for the risk of bladder cancer using novel molecular markers may lead to a reduction in diagnostic cystoscopy. Combining methylation analysis (OTX1, ONECUT2 and TWIST1) with mutation analysis (FGFR3, TERT and HRAS) and patient age resulted in a validated accurate prediction model.

Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors.

Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13- to 16-fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial-mesenchymal transition, including increased abundance of vimentin and hepatoma-derived growth factor in the membrane, and casein kinase II α and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process.

Multicenter validation of cyclin D1, MCM7, TRIM29, and UBE2C as prognostic protein markers in non-muscle-invasive bladder cancer.

Transcripts from the four genes encoding cyclin D1, MCM7, TRIM29, and UBE2C have previously been included in gene expression signatures for outcome prediction in stage Ta/T1 urothelial carcinomas. We investigated the prognostic value of the protein expressions in Ta/T1 urothelial carcinomas patients. We used four different tissue microarrays (TMAs) with a total of 859 Ta/T1 urothelial carcinomas from Danish, Swedish, Spanish, and Taiwanese patient cohorts with long-term follow-up. Protein expression was measured by IHC, and antibody specificity was validated by Western blotting. We found the expression of cyclin D1, MCM7, TRIM29, and UBE2C to be significantly associated with progression to muscle-invasive bladder cancer (log-rank test; P < 0.001) in the Danish training cohort (n = 283). Multivariate Cox regression analysis identified cyclin D1 (P = 0.003), TRIM29 (P = 0.001), and UBE2C (P < 0.001) as independent prognostic markers. The prognostic value of the four proteins was validated in a joint validation cohort from Sweden, Spain, and Taiwan (n = 576). Computer-assisted image analysis of the prognostic markers produced results comparable to those obtained by manual scoring. Finally, a four-protein maximum-likelihood classifier was trained on the Danish training cohort and applied to the validation cohort. The four protein markers may help optimize treatment of patients with Ta/T1 bladder cancer. Additional prospective studies are needed for further validation of their clinical relevance.

High miR-449b expression in prostate cancer is associated with biochemical recurrence after radical prostatectomy.

BACKGROUND: Prostate cancer is one of the leading causes of cancer death amongst men in economically advanced countries. The disease is characterized by a greatly varying clinical course, where some patients harbor non- or slowly-progressive disease, others highly aggressive disease. There is a great lack of markers to differentiate between aggressive and indolent disease. Markers that could help to identify patients needing curative treatment while sparing those who do not. METHODS: MicroRNA profiling of 672 microRNAs using multiplex RT-qPCR was performed using 36 prostate cancer samples to evaluate the association of microRNAs and biochemical recurrence after radical prostatectomy. RESULTS: Among 31 microRNAs associated with recurrence, we identified miR-449b, which was further validated in an independent cohort of 163 radical prostatectomy patients. Patients expressing miR-449b had a significantly higher risk of recurrence (HR = 1.57; p = 0.028), and miR-449b was shown to be an independent predictor of recurrence after prostatectomy (HR = 1.9; p = 0.003) when modeled with known risk factors of recurrent disease in multivariate analysis. CONCLUSION: High miR-449b expression was shown to be an independent predictor of biochemical recurrence after radical prostatectomy.

High expression of GEM and EDNRA is associated with metastasis and poor outcome in patients with advanced bladder cancer.

BACKGROUND: The standard treatment for non-metastatic muscle-invasive bladder cancer (stages T2-T4a) is radical cystectomy with lymphadenectomy. However, patients undergoing cystectomy show metastatic spread in 25% of cases and these patients will have limited benefit from surgery. Identification of patients with high risk of lymph node metastasis will help select patients that may benefit from neoadjuvant and/or adjuvant chemotherapy. METHODS: RNA was procured by laser micro dissection of primary bladder tumors and corresponding lymph node metastases for Affymetrix U133 Plus 2.0 Gene Chip expression profiling. A publically available dataset was used for identification of the best candidate markers, and these were validated using immunohistochemistry in an independent patient cohort of 368 patients. RESULTS: Gene Set Enrichment Analysis showed significant enrichment for e.g. metastatic signatures in the metastasizing tumors, and a set of 12 genes significantly associated with lymph node metastasis was identified. Tumors did not cluster according to their metastatic ability when analyzing gene expression profiles using hierarchical cluster analysis. However, half (6/12) of the primary tumor clustered together with matching lymph node metastases, indicating a large degree of intra-patient similarity in these patients. Immunohistochemical analysis of 368 tumors from cystectomized patients showed high expression of GEM (P = 0.033; HR = 1.46) and EDNRA (P = 0.046; HR = 1.60) was significantly associated with decreased cancer-specific survival. CONCLUSIONS: GEM and EDNRA were identified as promising prognostic markers for patients with advanced bladder cancer. The clinical relevance of GEM and EDNRA should be evaluated in independent prospective studies.

The Elusive Horizon: Biomarkers in Urothelial Carcinoma.

Urinary tests for circulating tumor DNA have potential for accurate discrimination of bladder cancer from other common inflammatory processes. Efforts are still needed to determine whether these tests can differentiate between cancer and field cancerization and to demonstrate clinical benefit in prospective trials.

Management of Renal Cell Carcinoma: Promising Biomarkers and the Challenges to Reach the Clinic.

The incidence of renal cell carcinoma (RCC) is increasing worldwide, yet research within this field is lagging behind other cancers. Despite increased detection of early disease as a consequence of the widespread use of diagnostic CT scans, 25% of patients have disseminated disease at diagnosis. Similarly, around 25% progress to metastatic disease following curatively intended surgery. Surgery is the cornerstone in the treatment of RCC; however, when the disease is disseminated, immunotherapy or immunotherapy in combination with a tyrosine kinase inhibitor is the patient's best option. Immunotherapy is a potent treatment, with durable treatment responses and potential to cure the patient, but only half of the patients benefit from the administered treatment, and there are currently no methods that can identify which patients will respond to immunotherapy. Moreover, there is a need to identify the patients in greatest risk of relapsing after surgery for localized disease and direct adjuvant treatment there. Even though several molecular biomarkers have been published to date, we are still lacking routinely used biomarkers to guide optimal clinical management. The purpose of this review is to highlight some of the most promising biomarkers, discuss the efforts made within this field to date, and describe the barriers needed to be overcome to have reliable and robust predictive and prognostic biomarkers in the clinic for renal cancer.

Circulating tumor DNA as a Predictive and Prognostic Biomarker in the Perioperative Treatment of Muscle-invasive Bladder Cancer: A Systematic Review.

CONTEXT: Predictive and prognostic biomarkers in the perioperative treatment of muscle-invasive bladder cancer (MIBC) are an unmet need. Circulating tumor DNA (ctDNA) holds promise as a biomarker in this setting. OBJECTIVE: To review the evidence of ctDNA as a prognostic and predictive biomarker in the perioperative treatment of MIBC. EVIDENCE ACQUISITION: We systematically reviewed the literature using PubMed, MEDLINE, and Embase databases according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. We included prospective studies investigating neoadjuvant and/or adjuvant chemotherapy and/or immunotherapy for MIBC (T2-T4a, any N, and M0) treated with radical cystectomy. We reported ctDNA results to monitor and/or predict disease status, relapse, and progression. The research retrieved 223 records. Six papers were considered for this review based on prespecified inclusion criteria. EVIDENCE SYNTHESIS: Our review confirms the prognostic role of ctDNA after cystectomy and shows a potential predictive benefit in using neoadjuvant chemotherapy and preoperative immunotherapy. Circulating tumor DNA was used to monitor recurrence, and changes in ctDNA status anticipated radiological progression with a median difference of time from 101 to 932 d. A subgroup analysis of the phase 3 Imvigor010 trial showed that only ctDNA-positive patients treated with atezolizumab had an improvement in disease-free survival (DFS; hazard ratio [HR] = 3.36, 95% confidence interval [CI]: 2.44-4.62). Clearance of ctDNA after two cycles of adjuvant atezolizumab was associated with improved outcomes (DFS HR = 0.26, 95% CI: 0.12-0.56, p = 0.0014; overall survival HR = 0.14, 95% CI: 0.03-0.59). CONCLUSIONS: Circulating tumor DNA is a prognostic factor after cystectomy and may be used to monitor recurrence. In the adjuvant immunotherapy setting, ctDNA might select patients who benefit the most from this strategy. PATIENT SUMMARY: In the perioperative treatment of muscle-invasive bladder cancer, circulating tumor DNA (ctDNA) positivity correlates with the outcomes after cystectomy and might select patients who may benefit from neoadjuvant chemotherapy and/or immunotherapy. Changes in ctDNA status anticipated radiological progression.

Field Cancerization Is Associated with Tumor Development, T-cell Exhaustion, and Clinical Outcomes in Bladder Cancer.

BACKGROUND: Field cancerization is characterized by areas of normal tissue affected by mutated clones. Bladder field cancerization may explain the development and recurrence of bladder cancer and may be associated with treatment outcomes. OBJECTIVE: To investigate the predictive and prognostic roles of field cancerization in patients with high-risk non-muscle-invasive bladder cancer (NMIBC) treated with bacillus Calmette-Guérin (BCG). DESIGN, SETTING, AND PARTICIPANTS: We conducted comprehensive genomic and proteomic analyses for 751 bladder biopsies and 234 urine samples from 136 patients with NMIBC. The samples were collected at multiple time points during the disease course. Field cancerization in normal-appearing bladder biopsies was measured using deep-targeted sequencing and error correction models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Endpoints included the rates of recurrence and progression. Cox regression and Wilcoxon rank-sum and Fisher's exact tests were used. RESULTS AND LIMITATIONS: A high level of field cancerization was associated with high tumor mutational burden (p = 0.007), high tumor neoantigen load (p = 0.029), and high tumor-associated CD8 T-cell exhaustion (p = 0.017). In addition, high field cancerization was associated with worse short-term outcomes (p = 0.029). Nonsynonymous mutations in bladder cancer-associated genes such as KDM6A, ARID1A, and TP53 were identified as early disease drivers already found in normal-appearing bladder biopsies. Urinary tumor DNA (utDNA) levels reflected the bladder tumor burden and originated from tumors and field cancerization. High levels of utDNA after BCG were associated with worse clinical outcomes (p = 0.027) and with disease progression (p = 0.003). High field cancerization resulted in high urinary levels of proteins associated with angiogenesis and proliferation. Limitations include variation in the number of biopsies and time points analyzed. CONCLUSIONS: Field cancerization levels are associated with tumor development, immune responses, and clinical outcomes. utDNA measurements can be used to monitor disease status and treatment response. PATIENT SUMMARY: Molecular changes in the tissue lining the bladder result in tumor recurrence. Urinary measurements may be used to monitor bladder cancer status and treatment responses.

Optimizing the Use of Next-Generation Sequencing Assays in Patients With Urothelial Carcinoma: Recommendations by the 2023 San Raffaele Retreat Panel.

BACKGROUND: The application of precision medicine in clinical practice implies a thorough evaluation of actionable genomic alterations to streamline therapeutic decision making. Comprehensive genomic profiling of tumor via next-generation sequencing (NGS) represents a great opportunity but also several challenges. During the 2023 San Raffaele Retreat, we aimed to provide expert recommendations for the optimal use of NGS in urothelial carcinoma (UC). MATERIALS AND METHODS: A modified Delphi method was utilized, involving a panel of 12 experts in UC from European and United States centers, including oncologists, urologists, pathologists, and translational scientists. An initial survey, conducted before the meeting, delivered 15 statements to the panel. A consensus was defined when ≥70% agreement was reached for each statement. Statements not meeting the consensus threshold were discussed during the meeting. RESULTS: Nine of the 15 statements covering patient selection, cancer characteristics, and type of NGS assay, achieved a consensus during the survey. The remaining six statements addressing the optimal timing of NGS use, the ideal source of tumor biospecimen for NGS testing, and the subsequent need to evaluate the germline nature of certain genomic findings were discussed during the meeting, leading to unanimous agreement at the end of the conference. CONCLUSION: This consensus-building effort addressed multiple unanswered questions regarding the use of NGS in UC. The opinion of experts was in favor of broader use of NGS. In a setting where recommendations/guidelines may be limited, these insights may aid clinicians to provide informed counselling and raise the bar of precision and personalized therapy.