GoKawiilPatient recruitment and sample collection
Specimens were obtained from 20 patients, including histopathologically normal tissue (n = 20, flash-frozen tissue), primary tumour samples (n = 24; 16 FFPE and 8 flash-frozen tissue) and 1–7 metastases per patient (n = 80, flash-frozen tissue). Details of tissue source, location, histology, among other information, for each sample are provided in Supplementary Table 1. Autopsy samples (n = 108), which included all normal, all metastatic and 8 primary tumour samples, were obtained within 11.5 h (median 3.7 h) of death as part of the rapid autopsy programme (Supplementary Table 1). For the logistical framework of the rapid autopsy programme, see Fig. 1a. A representative selection of the metastases observed at autopsy (excluding bone metastases) was chosen for sequencing, in addition to autopsy-derived primary tumour samples when available. Archival FFPE primary tumour samples (n = 16) included diagnostic (pre-treatment) TURBT (n = 5) and surgical specimens (n = 11) (Supplementary Table 1). All primary tumour and metastatic specimens selected for sequencing had more than 80% tumour cellularity based on genitourinary pathologist review (F.V.-L.). All normal tissues were confirmed to be normal by histology.
Study approval
All samples were obtained from patients with signed informed consent documents under the aegis of the Genitourinary Cancer Biorepository at the University of Washington (University of Washington IRB 2341). All 20 patients signed written informed consent for the rapid autopsy programme. Metastases and the primary tumour (if present) were identified and collected. In accordance with study protocols approved by the institutional review board, no metastatic biopsy samples collected from living patients were obtainable for use in this study.
Sectioning, H&E staining and pathologist assessment of tumour sections
All visceral metastases and matched primary tumour were embedded in Optimal Cutting Temperature compound (OCT; Tissue-Tek, Sakura Finetek) or FFPE. Haematoxylin and eosin (H&E) staining was completed as previously described18,61. H&E-stained sections (5 µm) of the OCT-embedded and FFPE tissues were reviewed by an independent, dedicated genitourinary pathologist (F.V.-L.). The percentage of tumour, necrosis and histological subtypes were assessed and recorded for each section. Between one and ten sections per tumour or normal sample were assessed, depending on the size of the specimen available. The areas of >80% tumour were marked for macrodissection for DNA and RNA extraction purposes.
Histology was defined both at the patient level and the tumour (sample) level. All classifications were based on pathology assessment of FFPE H&E-stained specimens by pathologist (F.V.-L.) review based on the WHO classifications of ‘Urinary and Male Genital Tumours’ (5th edition)4. NE histology was further verified by synaptophysin positivity. PUC, UC-Sarc and UCSD tumours are UC with plasmacytoid, sarcomatoid or squamous differentiation, respectively. Patients and tumours were designated as non-UC if there was any evidence of variant histology. For patient-level histology, the histology was defined as a variant if there was evidence of subtype histology in any tumour in that patient (for example, if a patient had any tumour exhibiting any squamous differentiation, they were classified as UCSD). Patients 16-070, 17-030, 18-101 and 19-022 were designated UCSD because they had a mix of UC and UCSD tumours, whereas all tumours in patients 16-097 and 17-026 exhibited UCSD histology. Sample-level histology was defined for each tumour individually (for example, the primary tumour for patient 16-070 is UCSD, whereas their metastases are UC at the sample level). Further details on the percentage of histological subtypes in each tumour are given in Supplementary Table 1 (first sheet).
HER2 immunohistochemistry
FFPE sections (5 μm) were deparaffinized and rehydrated in sequential xylene and graded ethanol series17. HER2 protein expression was evaluated using a PATHWAY anti-HER2/neu (clone 4B5) kit on a Ventana BenchMark ULTRA platform (Ventana/Roche Tissue Diagnostics) following the manufacturer’s standardized protocols. Staining intensity (scored 0 to 3+) and the percentage of positive tumour cells were assessed by two pathologists (M.C.H. and E.S.).
Radiological assessment of CT scans
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