Plasma ctDNA as a treatment response biomarker in metastatic cancers: evaluation by the RECIST working group.

Early indicators of metastatic cancer response to therapy are important for evaluating new drugs and stopping ineffective treatment. The Response Evaluation Criteria in Solid Tumors (RECIST) based on repeat cancer imaging are widely adopted in clinical trials, are used to identify active regimens that may change practice, and contribute to regulatory approvals. However, these criteria do not provide insight before 6 - 12 weeks of treatment and typically require that patients have measurable disease. Recent data suggests that measuring on-treatment changes in the amount or proportion of circulating tumor DNA (ctDNA) in peripheral blood plasma may accurately identify responding and non-responding cancers at earlier time points. Over the past year, the RECIST working group has evaluated current evidence for plasma ctDNA kinetics as a treatment response biomarker in metastatic cancers and early endpoint in clinical trials, to identify areas of focus for future research and validation. Here, we outline the requirement for large standardized trial datasets, greater scrutiny of optimal ctDNA collection time points and assay thresholds, and consideration of regulatory body guidelines and patient opinions. In particular, clinically-meaningful changes in plasma ctDNA abundance are likely to differ by cancer type and therapy class, and must be assessed before ctDNA can be considered as a potential pan-cancer response evaluation biomarker. Despite the need for additional data, minimally-invasive on-treatment ctDNA measurements hold promise to build upon existing response assessments such as RECIST, and offer opportunities for developing novel early endpoints for modern clinical trials.

[15O]H2O PET/MRI for Assessment of Complete Response to Neoadjuvant or Induction Chemotherapy in Patients with Muscle-Invasive Bladder Cancer: A Pilot Study.

Background: Accurate assessment of therapy response to chemotherapy could possibly offer a bladder-sparing approach in selected patients with localized muscle-invasive bladder cancer (MIBC). The aim of this study was to evaluate whether [15O]H2O PET/MRI can be used for assessment of complete local pathological response to preoperative chemotherapy in patients with MIBC. Methods: This prospective pilot study included 13 patients with MIBC treated with neoadjuvant or induction chemotherapy and subsequent radical cystectomy. Patients underwent a [15O]H2O PET/MRI scan before chemotherapy and another scan after chemotherapy before radical cystectomy. Volumes of interest were delineated on T2-weighted MRI and transferred to parametric images for dynamic analysis. Tumor blood flow (TBF) was estimated by [15O]H2O PET. Changes in TBF were compared with histopathology. The Wilcoxon matched-pairs signed-ranks test was used for comparing pre- and post-chemotherapy measurements. Results: Mean TBF decreased by 49%. Mean TBF in complete responders (ypT0N0/ypTis) was not significantly different from non-complete responders (≥ypT1) (p = 0.52). Conclusions: Despite a measurable decrease in TBF after chemotherapy treatment, we were not able to estimate a TBF threshold for identifying complete responders to chemotherapy for MIBC patients. Further studies are needed to elucidate the potential of [15O]H2O PET/MRI in assessing therapy response in MIBC.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

Whole-genome Mutational Analysis for Tumor-informed Detection of Circulating Tumor DNA in Patients with Urothelial Carcinoma.

BACKGROUND AND OBJECTIVE: Circulating tumor DNA (ctDNA) can be used for sensitive detection of minimal residual disease (MRD). However, the probability of detecting ctDNA in settings of low tumor burden is limited by the number of mutations analyzed and the plasma volume available. We used a whole-genome sequencing (WGS) approach for ctDNA detection in patients with urothelial carcinoma. METHODS: We used a tumor-informed WGS approach for ctDNA-based detection of MRD and evaluation of treatment responses. We analyzed 916 longitudinally collected plasma samples from 112 patients with localized muscle-invasive bladder cancer who received neoadjuvant chemotherapy (NAC) before radical cystectomy. Recurrence-free survival (primary endpoint), overall survival, and ctDNA dynamics during NAC were assessed. KEY FINDINGS AND LIMITATIONS: We found that WGS-based ctDNA detection is prognostic for patient outcomes with a median lead time of 131 d over radiographic imaging. WGS-based ctDNA assessment after radical cystectomy identified recurrence with sensitivity of 91% and specificity of 92%. In addition, genomic characterization of post-treatment plasma samples with a high ctDNA level revealed acquisition of platinum therapy-associated mutational signatures and copy number variations not present in the primary tumors. The sequencing depth is a limitation for studying tumor evolution. CONCLUSIONS AND CLINICAL IMPLICATIONS: Our results support the use of WGS for ultrasensitive ctDNA detection and highlight the possibility of plasma-based tracking of tumor evolution. WGS-based ctDNA detection represents a promising option for clinical use owing to the low volume of plasma needed and the ease of performing WGS, eliminating the need for personalized assay design.

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.

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.

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.

Multiomics profiling of urothelial carcinoma in situ reveals CIS-specific gene signature and immune characteristics.

Urothelial carcinoma in situ (CIS) is an aggressive phenotype of non-muscle-invasive bladder cancer. Molecular features unique to CIS compared to high-grade papillary tumors are underexplored. RNA sequencing of CIS, papillary tumors, and normal urothelium showed lower immune marker expression in CIS compared to papillary tumors. We identified a 46-gene expression signature in CIS samples including selectively upregulated known druggable targets MTOR, TYK2, AXIN1, CPT1B, GAK, and PIEZO1 and selectively downregulated BRD2 and NDUFB2. High expression of selected genes was significantly associated with CIS in an independent dataset. Mutation analysis of matched CIS and papillary tumors revealed shared mutations between samples across time points and mutational heterogeneity. CCDC138 was the most frequently mutated gene in CIS. The immunological landscape showed higher levels of PD-1-positive cells in CIS lesions compared to papillary tumors. We identified CIS lesions to have distinct characteristics compared to papillary tumors potentially contributing to the aggressive phenotype.

Association of Age with Non-muscle-invasive Bladder Cancer: Unearthing a Biological Basis for Epidemiological Disparities?

BACKGROUND: Age disparity in patients with non-muscle-invasive bladder cancer (NMIBC) exists. Whether this is due to differences in adequate cancer care or tumour biology is unclear. OBJECTIVE: To investigate age disparities in NMIBC using the Surveillance, Epidemiology, and End Results (SEER)-Medicare and UROMOL datasets. DESIGN, SETTING, AND PARTICIPANTS: The SEER-Medicare data were used to identify patients with clinical stage Ta, Tis, and T1 NMIBC between 2005 and 2017 (n = 32 225). Using the UROMOL cohort (n = 834), age disparities across transcriptomic, genomic, and spatial proteomic domains were assessed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For the SEER-Medicare data, multivariable competing-risk regression was used to examine the association between age and recurrence, progression, and bladder cancer-specific mortality (BCSM). For the UROMOL cohort, multivariable general linear model and multinomial logistic regression were performed to evaluate the association between age and tumour biology. RESULTS AND LIMITATIONS: An analysis of the SEER-Medicare cohort revealed 5-yr recurrence rates of 55.2%, 57.4%, and 58.9%; 5-yr progression rates of 25.6%, 29.2%, and 36.9%; and 5-yr BCSM rates of 3.9%, 5.8%, and 11.8% in patients aged 66-70, 71-80, and ≥81 yr, respectively. After multivariable adjustment, age ≥81 yr was associated with a higher risk of recurrence (hazard ratio [HR] 1.07, 95% confidence interval [CI] 1.03-1.12; p = 0.001), progression (HR 1.32, p < 0.001), and BCSM (HR 2.58, p < 0.001). UROMOL2021 transcriptomic class 2a was most frequently observed in patients with advanced age (34.0% in ≥76 yr vs 21.6% in ≤65 yr; p = 0.004), a finding confirmed on multivariable analysis (risk ratio [RR] 3.86, p = 0.002). UROMOL2021 genomic class 3 was observed more frequently in patients aged ≥76 yr (4.9% vs 24.2%; p = 0.001). Limitations include the definitions used for recurrence and progression, which may lead to under- or overestimation of true rates. CONCLUSIONS: Among SEER-Medicare patients with NMIBC, advanced age is associated with inferior oncological outcomes. These results reflect age-related molecular biological differences observed across transcriptomic and genomic domains, providing further evidence that innate tumour biology contributes to observed disparities in NMIBC outcomes. PATIENT SUMMARY: Older patients with non-muscle-invasive bladder cancer have worse oncological outcomes than younger patients. Some of this age disparity may be due to differences in tumour biology.

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.

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.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

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.

Immune Contexture Changes Following Blue Light Cystoscopy with Hexaminolevulinate in Bladder Cancer.

Background: Transurethral resection of bladder tumor (TURBT) is a central component in the diagnosis of non-muscle-invasive bladder cancer (NMIBC) and can be guided by several optical imaging techniques for better visualization of lesions. Objective: To investigate if a change in tumor microenvironment (TME) composition could be observed as an effect of hexaminolevulinate (HAL)-assisted blue light cystoscopy (BLC) in TURBT samples from patients with bladder cancer. Design setting and participants: This was a retrospective study of 40 patients with bladder cancer who underwent either BLC-guided TURBT (n = 20) or white light cystoscopy (WLC)-guided TURBT (n = 20) before radical cystectomy (RC). Tissue samples (n = 80) were collected from paired TURBT and RC specimens for all 40 patients. Tumor tissue was stained using multiplex immunofluorescence and immunohistochemistry. Outcome measurements and statistical analysis: Immune cell infiltration was assessed according to the proportions of each immune cell or immune evasion marker and the relative change from TURBT as baseline was calculated. Statistical comparisons between groups were performed using the Wilcoxon rank-sum test or the paired-sample Wilcoxon test. Results and limitations: Comparison of relative changes in the TME revealed a significant decrease in stromal infiltration of cytotoxic T cells (p = 0.024), B cells (p = 0.041), and stromal cells expressing PD-1 (p = 0.011) in patients treated with BLC-guided TURBT compared to WLC-guided TURBT. Conclusions: Our pilot study showed that HAL-BLC during TURBT in bladder cancer may influence the immune cell composition and TME. Patient summary: We investigated the potential therapeutic effect of blue light versus white light for guidance in removing bladder tumors via the urethra in patients with bladder cancer. For blue light guidance, a compound called hexaminolevulinate is used to visualize tumor tissue. We found changes in immune cell composition that may have been influenced by the blue light guidance.

Circulating Tumor DNA Analysis in Advanced Urothelial Carcinoma: Insights from Biological Analysis and Extended Clinical Follow-up.

PURPOSE: To investigate whether circulating tumor DNA (ctDNA) assessment in patients with muscle-invasive bladder cancer predicts treatment response and provides early detection of metastatic disease. EXPERIMENTAL DESIGN: We present full follow-up results (median follow-up: 68 months) from a previously described cohort of 68 neoadjuvant chemotherapy (NAC)-treated patients who underwent longitudinal ctDNA testing (712 plasma samples). In addition, we performed ctDNA evaluation of 153 plasma samples collected before and after radical cystectomy (RC) in a separate cohort of 102 NAC-naïve patients (median follow-up: 72 months). Total RNA sequencing of tumors was performed to investigate biological characteristics of ctDNA shedding tumors. RESULTS: Assessment of ctDNA after RC identified metastatic relapse with a sensitivity of 94% and specificity of 98% using the expanded follow-up data for the NAC-treated patients. ctDNA dynamics during NAC was independently associated with patient outcomes when adjusted for pathologic downstaging (HR = 4.7; P = 0.029). For the NAC-naïve patients, ctDNA was a prognostic predictor before (HR = 3.4; P = 0.0005) and after RC (HR = 17.8; P = 0.0002). No statistically significant difference in recurrence-free survival for patients without detectable ctDNA at diagnosis was observed between the cohorts. Baseline ctDNA positivity was associated with the Basal/Squamous (Ba/Sq) subtype and enrichment of epithelial-to-mesenchymal transition and cell cycle-associated gene sets. CONCLUSIONS: ctDNA is prognostic in NAC-treated and NAC-naïve patients with more than 5 years follow-up and outperforms pathologic downstaging in predicting treatment efficacy. Patients without detectable ctDNA at diagnosis may benefit significantly less from NAC, but additional studies are needed.

Bladder cancer.

Bladder cancer is a global health issue with sex differences in incidence and prognosis. Bladder cancer has distinct molecular subtypes with multiple pathogenic pathways depending on whether the disease is non-muscle invasive or muscle invasive. The mutational burden is higher in muscle-invasive than in non-muscle-invasive disease. Commonly mutated genes include TERT, FGFR3, TP53, PIK3CA, STAG2 and genes involved in chromatin modification. Subtyping of both forms of bladder cancer is likely to change considerably with the advent of single-cell analysis methods. Early detection signifies a better disease prognosis; thus, minimally invasive diagnostic options are needed to improve patient outcomes. Urine-based tests are available for disease diagnosis and surveillance, and analysis of blood-based cell-free DNA is a promising tool for the detection of minimal residual disease and metastatic relapse. Transurethral resection is the cornerstone treatment for non-muscle-invasive bladder cancer and intravesical therapy can further improve oncological outcomes. For muscle-invasive bladder cancer, radical cystectomy with neoadjuvant chemotherapy is the standard of care with evidence supporting trimodality therapy. Immune-checkpoint inhibitors have demonstrated benefit in non-muscle-invasive, muscle-invasive and metastatic bladder cancer. Effective management requires a multidisciplinary approach that considers patient characteristics and molecular disease characteristics.

Spatial transformation of multi-omics data unlocks novel insights into cancer biology.

The application of next-generation sequencing (NGS) has transformed cancer research. As costs have decreased, NGS has increasingly been applied to generate multiple layers of molecular data from the same samples, covering genomics, transcriptomics, and methylomics. Integrating these types of multi-omics data in a combined analysis is now becoming a common issue with no obvious solution, often handled on an ad hoc basis, with multi-omics data arriving in a tabular format and analyzed using computationally intensive statistical methods. These methods particularly ignore the spatial orientation of the genome and often apply stringent p-value corrections that likely result in the loss of true positive associations. Here, we present GENIUS (GEnome traNsformatIon and spatial representation of mUltiomicS data), a framework for integrating multi-omics data using deep learning models developed for advanced image analysis. The GENIUS framework is able to transform multi-omics data into images with genes displayed as spatially connected pixels and successfully extract relevant information with respect to the desired output. We demonstrate the utility of GENIUS by applying the framework to multi-omics datasets from the Cancer Genome Atlas. Our results are focused on predicting the development of metastatic cancer from primary tumors, and demonstrate how through model inference, we are able to extract the genes which are driving the model prediction and are likely associated with metastatic disease progression. We anticipate our framework to be a starting point and strong proof of concept for multi-omics data transformation and analysis without the need for statistical correction.

Circular stable intronic RNAs possess distinct biological features and are deregulated in bladder cancer.

Until recently, intronic lariats were regarded as short-lasting splicing byproducts with no apparent function; however, increasing evidence of stable derivatives suggests regulatory roles. Yet little is known about their characteristics, functions, distribution, and expression in healthy and tumor tissue. Here, we profiled and characterized circular stable intronic sequence RNAs (sisRNAs) using total RNA-Seq data from bladder cancer (BC; n = 457, UROMOL cohort), healthy tissue (n = 46), and fractionated cell lines (n = 5). We found that the recently-discovered full-length intronic circles and the stable lariats formed distinct subclasses, with a surprisingly high intronic circle fraction in BC (∼45%) compared to healthy tissues (0-20%). The stable lariats and their host introns were characterized by small transcript sizes, highly conserved BP regions, enriched BP motifs, and localization in multiple cell fractions. Additionally, circular sisRNAs showed tissue-specific expression patterns. We found nine circular sisRNAs as differentially expressed across early-stage BC patients with different prognoses, and sisHNRNPK expression correlated with progression-free survival. In conclusion, we identify distinguishing biological features of circular sisRNAs and point to specific candidates (incl. sisHNRNPK, sisWDR13 and sisMBNL1) that were highly expressed, had evolutionary conserved sequences, or had clinical correlations, which may facilitate future studies and further insights into their functional roles.