Abstract Clear cell renal cell carcinoma (ccRCC) is characterized by transcriptional reprogramming driven by hypoxia signaling, metabolic rewiring, and immune modulation. While gene-level analyses have defined key features of ccRCC biology, they do not capture isoform-level variation arising from alternative splicing. Differential transcript usage (DTU) represents an additional regulatory layer that may influence protein function, pathway activity, and clinical outcomes, yet its role in ccRCC biology and prognosis remains incompletely understood. We assessed differential expression in 127 ccRCC tumors and 33 normal-adjacent tissues from the Dartmouth Cancer Center cohort, with external validation in 94 CPTAC tumors, adjusting for cell-type proportions. DTU was identified using DRIMSeq/stageR, followed by limmavoom modeling with clinical and tumor microenvironment covariates. Transcript-based consensus clustering defined tumor subgroups, and Cox proportional hazards modeling integrated transcript-level features with clinical variables. In tumor versus normal comparisons, 1,170 transcripts exhibited significant differential usage, mapping to canonical ccRCC pathways with distinct patterns across functional and non-functional transcript classes. Consensus clustering based on transcript us-age identified two subgroups with distinct angiogenic profiles and significant survival differences. Cluster-level analysis revealed DTU in genes involved in cytoskeletal organization ( ACTB ), immune processes ( B2M ), extracellular matrix organization ( FN1 , APLP2 ), and iron metabolism ( FTH1 ) with protein domain alterations, including the loss of actin-associated domains in ACTB and immunoglobulin-like domains in B2M . Prognostic modeling identified twelve transcripts consistently retained across bootstraps, improving risk stratification over clinical variables alone. External validation confirmed overlapping prognostic transcripts, including FGFR1 and NUCB1 . Isoform-level features may serve as biomarkers and therapeutic targets in ccRCC. Statement of significance Transcript-level analysis uncovers potential regulatory pathways in ccRCC missed by gene-level approaches, revealing isoform-specific alterations that define survival sub-groups and offer potential biomarkers and therapeutic targets.

Background:Retrotransposable elements (RE) comprise approximately 45% of the human genome and are typically repressed by DNA methylation to preserve genomic integrity. In cancer, global DNA hypomethylation can lead to RE derepression, resulting in genomic instability and activation of innate immune pathways through viral mimicry. While individual RE classes have been examined in clear cell renal cell carcinoma (ccRCC), the integrated epigenetic landscape of multiple RE families and their clinical relevance remain incompletely characterized. Methods:We performed a genome-wide prediction of DNA methylation across three major RE classes (Alu, LINE-1, and LTR elements) using a validated computational framework applied to Illumina methylation array data from two independent ccRCC tumor cohorts. Integrated unsupervised clustering of RE methylation profiles was used to define the epigenetic subtypes. Associations with clinicopathologic variables, tumor immune microenvironment composition (DNA Methylation-derived), hypoxia signaling, innate immune activation, and overall survival were evaluated. Prognostic relevance was assessed using multivariable Cox regression models adjusting for age, sex, AJCC stage or AUA risk group, and immune and angiogenic tumor microenvironment features. Key findings were then externally validated in CPTAC-ccRCC and independently replicated in an institutional Dartmouth Cancer Center (DCC) cohort with matched methylation and RNA-sequencing data. Results:Integrated clustering identified three reproducible RE methylation subtypes, Repressed, Transient, and Active. In the discovery cohort, the Active subtype showed significantly worse overall survival than the Repressed subtype, with a graded survival pattern across RE methylation states that persisted after multivariable adjustment. RE hypomethylation was associated with reduced EPAS1 (HIF2A) expression, increased immune infiltration, elevated PD-1 expression, and heightened cGAS-STING and interferon signaling, consistent with an immune-inflamed yet immunosuppressed tumor state. In the external CPTAC validation cohort, RE methylation subtypes recapitulated key molecular features and showed supportive survival trends. In the independent DCC replication cohort, an Active RE state was again associated with poorer survival, lower EPAS1 expression, increased PD-1 expression, greater CD8 T-cell and Treg infiltration, and elevated T-cell exhaustion signatures, supporting the reproducibility of the prognostic and immune-exhausted phenotype across cohorts. Conclusions:We identified RE methylation subtypes with distinct molecular, immunologic, and prognostic features in ccRCC. External validation in CPTAC and independent replication in DCC support the robustness of this RE methylation framework across large-scale and institutional cohorts. These findings highlight the prognostic potential of RE methylation profiles and support their integration into molecular classification strategies to improve risk stratification in ccRCC.

Intravesical instillation of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was first used to treat bladder cancer in 1976. Since then, it has become a common therapy for non-muscle invasive bladder cancer. A live, attenuated mycobacterium, M. bovis BCG causes infectious complications in approximately 1% of patients. These infections can involve various anatomic sites and have a range of presentations. Diagnosis may require several testing modalities, and treatment typically involves multiple antimicrobials for a long duration. We report three cases of M. bovis BCG infection following intravesical BCG instillation for the treatment of bladder cancer. The patients are men ranging in age from 71 to 83 years old. One had a ruptured mycotic aneurysm of the aorta, another had a presumed anaphylactic reaction, and the final had a bone marrow infection. Isolation of BCG was achieved through mycobacterial blood culture in two cases and also through mycobacterial culture of bone marrow and hematoma aspirate. Nucleic acid amplification testing for Mycobacterium tuberculosis complex (of which BCG is a member) was utilized in two cases, and histologic examination identified granulomas in one case. Two of the patients received mycobacterial treatment, including rifampin, isoniazid, ethambutol, and linezolid. One patient remains living over 3 years after infection, one died despite treatment, and one died from unrelated causes. These cases highlight the diversity of BCG infection symptoms and sites and the various laboratory testing modalities that can inform diagnosis and treatment.

Supplementary Figure S2: Blood immune cell profiles, age and age acceleration distribution of three groups assigned by partDSA algorithm in NMIBC patients

Supplementary Figure S3: Semi-Supervised Recursively Partitioned Mixture Model (SS-RPMM) for 10-year overall survival (OS) in NMIBC patients [For Hannum age acceleration]

Supplementary Figure S5: Kaplan-Meier analysis of 10-year overall survival based on the grouping results from both partDSA and SS-RPMM in all NMIBC patients [For Hannum age acceleration]

Supplementary Table S5: The information of optimal CpGs selected by SS-RPMM using the model controlling for Pheno and Hannum age acceleration respectively

Supplementary Figure S4: Blood immune cell profiles, age and age acceleration distribution of two clusters assigned by the SS-RPMM approach in NMIBC patients

Supplementary Figure S1: The distribution of chronological age, methylation age, age acceleration, and each immune cell profile

Supplementary Table S3: Cox proportional hazards models of immune cell proportions and NMIBC patient outcomes (For Hannum Age Acceleration)

Supplementary Table S4: Cox proportional hazards models of immune cell proportions and NMIBC patient outcomes (For Pheno Age Acceleration; NMIBC patients without BCG treatment; N = 512)

Supplementary Table S1: Cox proportional hazards multivariable models for age acceleration of 601 NMIBC patients

Supplementary Table S6: Characteristics of subjects of each group based on the grouping results from both partDSA and SS-RPMM in all NMIBC patients

OBJECTIVE:To characterize patient symptoms and toxicity associated with present-day ambulatory transurethral resection of bladder tumor (TURBT). METHODS:A multicenter prospective cohort study of participants with bladder cancer undergoing TURBT was performed from January 2023 to August 2023. Visual analog scale (VAS) pain scores and open-ended feedback responses were collected preoperatively, immediately postop, 6-10 hours, 2-3 days, and 7-9 days after surgery. Linear mixed-effects modeling with random intercepts to account for repeated measures was used to assess the relationship between patient characteristics and study outcomes. RESULTS:One hundred fifty-nine patients underwent TURBT during the study period. Most patients were undergoing a TURBT for the first time (46.5%), were male (77.4%), and had a median age of 71 years. Mean suprapubic pain, dysuria, and penile or vaginal pain among patients in the third tertile of symptom burden were 5.6, 2.4, and 2.4 times higher than patients in the second tertile, respectively. Most patients experienced post-procedural hematuria (79.3%), while a third of patients experienced bladder spasms and incontinence. Issues with constipation were self-reported by 24.7% of patients, while 19.7% and 6.8% struggled with lack of sleep and depression/anxiety. An unplanned emergency room (ED) or clinic visit occurred in 10.1% of patients. After adjustment, female sex (β 0.96, 95% CI [0.47, 1.45]) and having stage ≥T2 bladder cancer on final pathology (β 1.04, 95% CI [0.32, 1.76]) were significantly associated with greater suprapubic pain during the study period. A Foley catheter at discharge (β -1.36, 95% CI [-1.86, -0.87]) and increasing patient age (β -0.02, 95% CI [-0.05, -0.002]) were inversely associated with dysuria. CONCLUSION:The degree of distress and discomfort experienced by some patients after TURBT is likely underappreciated. To embrace a model of survivorship in bladder cancer care, symptom reduction and quality of recovery after TURBT must be optimized.

Supplementary Table S2: Cox proportional hazards multivariable models for demographic and tumor characteristics of 601 NMIBC patients (For Hannum Age Acceleration)

Background: Bladder cancer and therapy responses hinge on immune profiles in the tumor microenvironment (TME) and blood, yet studies linking tumor-infiltrating immune cells to peripheral immune profiles are limited. Methods: DNA methylation cytometry quantified TME and matched peripheral blood immune cell proportions. With tumor immune profile data as the input, subjects were grouped by immune infiltration status and consensus clustering. Results: Immune hot and cold groups had different immune compositions in the TME but not in circulating blood. Two clusters of patients identified with consensus clustering had different immune compositions not only in the TME but also in blood. Conclusion: Detailed immune profiling via methylation cytometry reveals the significance of understanding tumor and systemic immune relationships in cancer patients.

Immune cell profiles in peripheral blood have been associated with bladder cancer outcomes, however, their association with response to immunotherapy and the tumor microenvironment is a major unresolved issue. Although tumor growth can be attenuated via the activation of tumor-infiltrating effector T cells, the relationship between tumor infiltration and immune activation remains unclear. This study explored the interaction between bladder cancer outcomes and immune profiles within peripheral blood and a tumor microenvironment (TME) based on DNA methylation profiles. Peripheral blood and the matched tumor FFPE DNA methylation profiles of 60 non-muscle-invasive bladder cancer (NMIBC) and 12 muscle-invasive bladder cancer (MIBC) patients. Cell-type deconvolution approaches were applied to estimate 12 peripheral immune cell-type proportions and 17 cell-type proportions within TME. We found a positive correlation between dendritic cell proportions in the TME with peripheral CD8T memory cell proportions (r = 0.35, P = 0.003) and a negative correlation between dendritic cell proportions in the TME with peripheral regulatory T cell proportions (r = -0.28, P = 0.021). In addition, monocyte cell proportions in TME had a positive correlation with peripheral B memory (r = 0.37, P = 0.002) and CD8T memory cell proportions (r = 0.43, P = 0.0002). To investigate associations of bladder cancer outcomes with immune cell profiles, using Cox proportional hazard models, we observed an association between the fraction of dendritic cells and the hazard of death (HR = 1.27, 95% CI = 1.06-1.53). Further, a high endothelial cell proportion was significantly associated with an increased hazard of death and tumor recurrence (HR = 1.06, 95% CI = 1.01-1.13) in TME. In addition, the peripheral neutrophil-to-lymphocyte ratio (HR = 1.49, 95% CI = 1.01-2.22), monocyte (HR = 1.17, 95% CI = 1.05-1.31), neutrophil (HR = 1.04, 95% CI = 1.01-1.07), and basophil (HR = 1.35, 95% CI = 1.01-1.81) cell proportions were associated with an increased hazard of death and tumor recurrence. Our results integrated the information on bladder cancer outcomes and cell profiles in TME and peripheral blood, providing biomarkers for estimating bladder cancer prognosis using genome-scale DNA methylation measures. Citation Format: Ji-Qing Chen, Lucas A. Salas, John K. Wiencke, Devin C. Koestler, Annette M. Molinaro, Angeline S. Andrew, John D. Seigne, Margaret R. Karagas, Karl T. Kelsey, Brock C. Christensen. Integration of associations of immune profiles in peripheral blood and tumor microenvironment with bladder cancer outcomes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6675.

AbstractBackground: Immune profiles have been associated with bladder cancer outcomes and may have clinical applications for prognosis. However, associations of detailed immune cell subtypes with patient outcomes remain underexplored and may contribute crucial prognostic information for better managing bladder cancer recurrence and survival. Methods: Bladder cancer case peripheral blood DNA methylation was measured using the Illumina HumanMethylationEPIC array. Extended cell-type deconvolution quantified 12 immune cell-type proportions, including memory, naïve T and B cells, and granulocyte subtypes. DNA methylation clocks determined biological age. Cox proportional hazards models tested associations of immune cell profiles and age acceleration with bladder cancer outcomes. The partDSA algorithm discriminated 10-year overall survival groups from clinical variables and immune cell profiles, and a semi-supervised recursively partitioned mixture model (SS-RPMM) with DNA methylation data was applied to identify a classifier for 10-year overall survival. Results: Higher CD8T memory cell proportions were associated with better overall survival [HR = 0.95, 95% confidence interval (CI) = 0.93–0.98], while higher neutrophil-to-lymphocyte ratio (HR = 1.36, 95% CI = 1.23–1.50), CD8T naïve (HR = 1.21, 95% CI = 1.04–1.41), neutrophil (HR = 1.04, 95% CI = 1.03–1.06) proportions, and age acceleration (HR = 1.06, 95% CI = 1.03–1.08) were associated with worse overall survival in patient with bladder cancer. partDSA and SS-RPMM classified five groups of subjects with significant differences in overall survival. Conclusions: We identified associations between immune cell subtypes and age acceleration with bladder cancer outcomes. Impact: The findings of this study suggest that bladder cancer outcomes are associated with specific methylation-derived immune cell-type proportions and age acceleration, and these factors could be potential prognostic biomarkers.

OBJECTIVE:To quantify the short-term burden associated with continent diversion relative to ileal conduit creation.METHODS:Bladder cancer patients who underwent radical cystectomy in 2019 and 2020 were identified in the American College of Surgeons National Surgical Improvement Program database using current procedural terminology codes and pathology reports. Patients were grouped by urinary diversion performed: ileal conduit versus continent diversion (neobladder or cutaneous reservoir). Multiple logistic regression was used to examine the association between type of urinary diversion and 30-day outcomes, including postoperative complications, all-cause readmissions, and mortality, adjusting for baseline differences.RESULTS:Of 4,755 patients who underwent radical cystectomy, 677 underwent continent diversion (14.2%). These patients were significantly younger (median 62 vs 71 years, P <.01) and less likely to have diabetes (13.6% vs 20.1%, P <.01), COPD (3.7% vs 7.1%, P<0.01), and prior pelvic radiation (5.5% vs 13.1%, P <.01). A greater proportion of continent diversion patients experienced a postoperative complication (56.0% vs 48.9%, P <.01) and all-cause readmission (30.3% vs 20.4%, P <.0). After adjustment, continent diversion patients had 1.4 (95% CI: 1.1-1.7) and 1.7 (95% CI: 1.4-2.1) times the odds of experiencing a postoperative complication or all-cause readmission, respectively. There was no statistically significant difference in mortality (OR 1.2, 95% CI: 0.5-2.9).CONCLUSION:Compared to ileal conduit creation, continent urinary diversion is associated with increased odds of postoperative complications and readmission to the hospital within 30 days of surgery. Bladder cancer patients undergoing cystectomy and seeking continent diversion should be counseled on the increased short-term morbidity associated with this specific type of diversion.