Lower Urinary Tract Symptoms

The BCG treatment protocol has been previously reported.^{22 (link)} In brief, patients received 1 vial of BCG TICE strain (Organon Teknika Corporation) with or without 50 million U of interferon α-2b (IFN-α-2b) instilled simultaneously into the bladder for approximately 90 to 120 minutes. The starting BCG dose of the induction cycle was altered to one-third of the dose during times when BCG availability was limited at our institution or when a patient experienced substantial lower urinary tract symptoms before treatment. The BCG maintenance regimen consisted of a reduced (one-third to one-tenth) dose administered over three 3-week courses at 3 months, 9 months, and 15 months after the end of the last induction treatment, as previously described.^{22 (link)}

After institutional review board approval, patient medical records were retrospectively reviewed, and data were stored in a Research Electronic Data Capture (REDCap) database supported by the University of Iowa (via funding from the National Institutes of Health/Clinical and Translational Science Awards program). Patients’ clinicopathological features, treatment history, tolerance to therapy, and oncological outcomes were analyzed. The primary outcome was high-grade recurrence-free survival (RFS). Secondary outcomes included RFS, progression-free survival (PFS), cystectomy-free survival (CFS), cancer-specific survival (CSS), and overall survival (OS). Adverse events were classified using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 5. When symptoms had a unifying cause (eg, lower urinary tract symptoms during urinary tract infection), only the latter unifying CTCAE term was reported. Intolerance was defined as an inability to continue treatment due to the development of AEs.
We used χ² tests to compare categorical variables and t tests to compare continuous variables (baseline characteristics and AEs) between treatment groups. Survival probabilities were estimated and plotted using the Kaplan-Meier method. Estimates along with pointwise 95% CIs were reported.
Recurrence-free survival was defined as time from the start of therapy induction to recurrence, and high-grade RFS was defined as time from the start of therapy induction to high-grade recurrence, respectively. Recurrence was defined as tumor relapse in the bladder or prostatic urethra (for male patients). Progression-free survival was defined as time from the start of therapy induction to progression, with progression defined as the development of a T2 or greater disease, lymph node or metastatic disease, or receipt of any cystectomy. Otherwise, patients were censored at the last urological follow-up visit. Cancer-specific survival was defined as time from the start of therapy induction to death due to bladder cancer. Patients were censored at the date of death due to other causes or at the date they were last known to be alive. Overall survival was defined as time from the start of therapy induction to death due to any cause. Duration of response was defined as time from initial surveillance to recurrence (high or low grade) among those who hadn’t experienced recurrence at the initial surveillance visit. Patients still alive were censored at the last known follow-up visit.
Cox regression models were used to evaluate the associations of patient, disease, and treatment characteristics with RFS and high-grade RFS. All statistical testing was 2-sided, with P = .05 set as the significance threshold. Data were analyzed using SAS software, version 9.4 (SAS Institute Inc).

The raw sequencing reads of a urinary metagenome sample was retrieved from GenBank (SRA Accession No. SRR19149281). This data represents DNA sequencing of a urine sample (sample ID 6162). In other words, both bacterial and viral constituents of the urinary microbiota are expected to be present. Details regarding the sample collection, DNA extraction, and sequencing was previously reported [51 ]. Briefly, a urine sample was obtained via transurethral catheterization of a female without lower urinary tract symptoms as part of a prior IRB-approved study (Loyola University Chicago, Maywood, IL, USA, IRB # 207102). A total of 10% by volume of AssayAssure^® was added to 2 mL of the collected urine and stored at −80 °C. DNA was extracted from the thawed urine sample using the Norgen Urine DNA according to the manufacturer’s protocol with one exception: a starting volume of 500 uL was used and the binding solution was adjusted accordingly. The DNA sequenced at MIGS (Pittsburgh, PA, USA) using the Illumina DNA Prep kit and IDT 10 bp UDI indices on an Illumina NextSeq 2000 (paired-end reads, 2 × 151 bp). This raw data set includes human reads; the urine was not processed prior to DNA extraction to remove human cells (if present). We thus removed human reads from further analysis using Bowtie 2 v.2.4.5 and the application’s publicly available human genome index (GRCh38) [52 (link)]. Only reads that did not map to the human genome were considered further. This included 1,403,038 read pairs, 56.90% of the raw data. These filtered sequencing reads were assembled using metaSPAdes v.3.15.4 [53 (link)] with default parameters. Assembled contigs were then evaluated by VirSorter2 to identify putative viral sequences [31 (link)], the results of which were queried against the NCBI nr/nt nucleotide database via megaBLAST (using default parameters) to identify the most similar sequence record. The filtered reads and predicted viral sequences were next analyzed using PIE. Viral sequences exceeding the 99% threshold in PIE were queried against the NCBI nr/nt nucleotide database via BLAST in an effort to identify their putative taxonomy and annotated via the Bacteriophage “annotation recipe” on BV-BRC v.3.28.9 [54 (link)].