Radical Cystectomy

Formalin‐fixed paraffin‐embedded preoperative TUR‐B specimens reviewed by a uropathologist were collected from consecutive patients who underwent radical cystectomy in four hospitals in southern Sweden from 2006 to 2011. None of the included tumours received any treatment prior to sample‐taking (Trans uretral resection of the bladder (TUR‐B)). The histological variants at pathological review are shown in supplementary material, Table S1. The pathological stage and grade based on TUR‐B specimens are given in supplementary material, Table S2. Sufficient tissue for embedding of dual cores (1.0 mm) in tissue microarrays (TMAs) and for extraction of RNA was obtained from 307 TUR‐B specimens. For three cases, single cores were embedded. Positioning of the cores within the TUR‐B specimen was performed by selecting areas with >80% tumour cells morphologically and histologically representative of the total tumour. RNA extraction was performed on 4–10 10‐µm sections from macrodissected tissue areas located as close as possible to the positions of the TMA cores. Sampling and RNA extraction are described in detail Supplementary materials and methods. The included samples had an average total spectrometric RNA yield of 2.5 µg (range 0.4–9.3 µg). Informed consent was obtained from all patients, and the study was approved by the Local Ethical Committee of Lund University, in accordance with the Helsinki Declaration.

This study was approved by Orlando Health Inc. (Orlando, FL) Institutional Review Board with a waiver of consent. Orlando Health Inc. is a large healthcare system (> 1,000 beds) comprised of eight facilities in central Florida, affiliated with the University of Central Florida College of Medicine and Florida State University School of Medicine. Complications identified within the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) within the Department of Urology in Orlando Health Inc. were queried retrospectively to gather information regarding patient outcomes to urologic surgeries performed from January 1, 2011 to December 31, 2011. Based on the number of procedures performed in 2011, the location of the procedures (in-patient and outpatient) and the difficulty of the procedures, CPT codes associated with 11 diverse procedures covering a wide range of urologic procedures [e.g., insertion or replacement of inflatable penile prosthesis, nephroureterectomy, partial nephrectomy, percutaneous nephrostomy tube placement, radical cystectomy, radical prostatectomy, renal/ureteral/bladder extracorporeal shockwave lithotripsy (ESWL), transurethral destruction of bladder lesion, transurethral prostatectomy, transurethral removal of ureteral obstruction, and ureteral catheterization] were queried and included for analysis. All patients identified in each of the 11 procedures were evaluated (i.e., no patient was excluded from analysis). To establish the true PMI of a procedure in an institution one would expect that >25 patients per group would be needed although the study has not yet been done to determine the exact number.
Individual medical records of the patients who underwent the above procedures were reviewed to determine the incidence of post-operative complications as defined by American College of Surgery National Surgical Quality Improvement Program (ACS NSQIP) within 30 days (any NSQIP 30-day morbidity). The ACS NSQIP complications that were noted included bleeding, superficial wound infection, deep wound infection, organ space infection, wound dehiscence, acute renal failure, progressive renal insufficiency, urinary tract infection, prolonged ileus, pneumonia, failure to wean from ventilator, unplanned intubation, pneumothorax, pulmonary embolus, cardiac arrest, exacerbation of heart failure, deep venous thrombosis, cerebrovascular accident, transient ischemic attack, sepsis, septic shock, and death (all-cause 30-day mortality). The severity of each complication was graded independently by two clinicians (JAB and RS) according to the recently validated Accordion Severity Grading System (Table 1). A third investigator (CJR) reviewed discrepancies and rendered a final score. In cases with multiple ACS NSQIP complications, the case was assigned a grade corresponding to the highest graded complication.
Next, a weighted postoperative morbidity index (PMI) was calculated as previously described [8 (link)] (i.e., to calculate the PMI for each operative procedure, the weights of all the complications for all patients who underwent the corresponding procedure were summed and divided by the total number of patients undergoing that procedure). A PMI of 0 would indicate that no patient having the procedure had any postoperative complications, while on the other hand, and a PMI of 1.000 would indicate that every patient having the procedure suffered postoperative death. In order to analyze complication severity, the sum of severity weights for all patients having any complication after a procedure were divided by the total number of patients with complications in the group (i.e., the denominator was the number of patients having a complication after the procedure, rather than the total number of patients having the procedure). Descriptive statistics were performed in Excel 2007 (Microsoft Corp).

Forty-nine BLCA samples (35 NMIBC and 14 MIBC) were obtained from patients in Shanghai Tenth People’s Hospital who underwent either transurethral resection of bladder tumor or radical cystectomy between November 2019 and April 2020. Informed consent was prior obtained. Total RNA was extracted from the formalin-fixed paraffin-embedded specimens with the RNeasy FFPE Kit (Qiagen, Hilden, Germany) after deparaffinization with Xylen. Paired-end libraries were synthesized from 100 ng/ml of total RNA using ABclonal Whole RNA-seq Lib Prep kit. Sequence data were obtained using the Illumina NovaSeq 6000 platform.
The microarray data sets GSE32894, GSE13507, GSE48277 and GSE31684 with their corresponding clinicopathological features were obtained from the Gene Expression Omnibus (GEO) database of the NCBI database (https://www.ncbi.nlm.nih.gov/). The mRNA (RNA-sequence) Fragments Per Kilobase of transcript per Million Fragments standardized expression data set and corresponding clinicopathological features were downloaded for 403 BLCA patients with prognostic information from The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/). The microarray data of IMvigor210 trial were obtained from the website http://research‐pub.gene.com/IMvigor210CoreBiologies. IMvigor210 trial included patients with metastatic urothelial cancer treated with atezolizumab (PD-L1 inhibitor) (19 (link)). Patients without clinical response in the IMvigor210 trial were excluded. The main clinicopathological characteristics of these data sets were listed in Table 1. A comprehensive immune-related gene list, identified to actively participate in immunological processes, was extracted from the Immunology Database and Analysis Portal database (https://immport.niaid.nih.gov) (20 (link)). Because GSE32894 had the maximum number of patients in NMIBC and MIBC subtypes (including 213 NMIBC patients and 93 MIBC patients), GSE32894 was used to verify the different tumor-infiltrating immune cells (TIICs) and differently expressed immune genes between NMIBC and MIBC.

Clinicopathological data of patients with resected MIBC receiving chemotherapy were obtained from the SEER database utilizing SEER*Stat 8.3.9 software. The inclusion criteria were as follows: (1) diagnosed with MIBC from 2004 to 2015 as the first only malignancy; (2) histological type: Transitional cell carcinoma; (3) patients treated radical cystectomy and chemotherapy (Whether adjuvant chemotherapy or neoadjuvant chemotherapy). Exclusion criteria: (1) M stage: M1 or Mx; (2) patients receiving radiotherapy; (3) the information of N stage, grade, tumor size, race, marital status and regional nodes examined unknown. Our study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University.

The study period for the original cohort was between 2002 and 2018; we chose a 2-year wash-out period (2002 to 2003) and a 2-year follow-up period (2017 to 2018). All the patients had UC (International Classification of Diseases [ICD] diagnostic codes C65 [ureter], C66 [pelvis], and C67 [bladder]) between 2004 and 2016. All cases were confirmed by pathology or cytology with metastatic radiology. Inclusion criteria was the usage of GC or MVAC chemotherapy with UC diagnostic codes. Patients who were diagnosed as having other cancers within 2 years, received adjuvant or neoadjuvant chemotherapy, underwent only surgery, received other chemotherapy, and had inaccurate data were excluded. Neoadjuvant chemotherapy was defined as chemotherapy administered during 3 months before nephroureterectomy (NUx) or radical cystectomy (RCx), and adjuvant chemotherapy was defined as chemotherapy administered during 3 months after NUx or RCx. A flow chart of the study design is displayed in Fig. 1.Figure 1

Flow chart of the study design. UTUC, upper tract urothelial carcinoma; NHIS, National Health Insurance Service; AC, adjuvant chemotherapy; NAC, neoadjuvant chemotherapy; NUx, nephroureterectomy.