Greater utility of molecular subtype rather than epithelial‐to‐mesenchymal transition (EMT) markers for prognosis in high‐risk non‐muscle‐invasive (HGT1) bladder cancer

Abstract Approximately 75% of bladder cancers are non‐muscle invasive (NMIBC). Of these, up to 53% of cases progress to life‐threatening muscle‐invasive bladder cancer (MIBC). Patients with high‐grade stage T1 (HGT1) NMIBC frequently undergo radical cystectomy (RC), although this represents overtreatment for many. Identification of progressors versus non‐progressors could spare unnecessary treatment. Recent studies have confirmed that urothelial carcinoma is composed of two main molecular subtypes, basal and luminal, with 12% of basal tumours exhibiting epithelial‐to‐mesenchymal transition (EMT). Levels of immune cell infiltration have been shown to be subtype‐specific. Here, we performed immunohistochemistry (IHC) for 11 antibodies relating to molecular subtypes or EMT in 26 cases of HGT1 urothelial carcinoma cases with 6 matched samples subsequently obtained at cystectomy (n = 6; 1 muscle‐invasive, 5 non‐muscle‐invasive; 3 = pTis, 1 = pT1, 1 = pTa) and at recurrence (n = 2, pT2). RNAScope was also conducted in a subset. Expression patterns in HGT1 specimens versus MIBC (pT2+) were examined, and correlated with disease‐specific survival (DSS). Levels of stromal tumour‐infiltrating lymphocytes (sTILs) were assessed manually to determine whether lymphocyte infiltration was associated with DSS and whether differences existed between HGT1 and MIBC. Molecular subtype markers demonstrated increased prognostic potential compared to the EMT markers assessed. Increased expression of the luminal markers FOXA1 and SCUBE2, were found to be significantly associated with better DFS. No EMT markers were significantly associated with DFS. In areas of non‐invasive papillary urothelial carcinoma, but not invasive carcinoma, sTIL levels were found to be significantly associated with DFS. While differences were observed between HGT1 cases that progressed versus those that did not, a larger cohort study is required for validation of these findings. Taken together, an emphasis on molecular subtype markers, rather than EMT markers, may be preferable when studying biomarkers of HGT1 urothelial carcinoma in the future.


RNAScope in-situ hybridisation (ISH)
Probe choice: Two basal and luminal targets for the ISH probes were chosen based on a study by Ochoa et al. (2016) which clustered molecular subtypes with respect to miRNA targets [34].
SCUBE2 and FGFR3 were chosen as luminal targets and EGFR and ZEB2 as basal targets.
Sample preparation and treatment: Tissue sections (6 µm) baked onto SuperFrost Plus slides (ThermoFisher) were baked again in a dry oven 1 hr prior to commencing the protocol. Sections were de-paraffinised with xylene for 2 x 5 min followed by 100% ethanol for 2 x 1 min and allowed to air-dry for 5 min at room temperature. Hydrogen peroxide (RNAScope, #322335) was added to the sections for 10 min at room temperature and placed in distilled water for 2 x 1 min. Target retrieval was conducted by placing the tissue sections into 1 x Target Retrieval Reagent (RNAScope, #322000) pre-heated to 100°C using a pressure cooker (Biocare Medical, DC2002) for 30 min. Slides were removed and placed in distilled water rinsed for 15 s and placed into 100% ethanol for 3 min. Slides were allowed to dry at room temperature. A hydrophobic barrier was drawn around each tissue section (ImmEdge pen, Vector Laboratories, #H-4000), allowed to dry, and RNAScope Protease Plus solution (RNAScope, #322331) was added for 30 min at 40°C. Slides were then placed in distilled water and washed for 1 x 1 min.
Detection: For detection the RNAScope 2.5 Detection Reagent -Red kit was used (RNAScope #322360). Initially, the hybridisation probes (Supplementary material, Table S2) were added to each tissue section and allowed to incubate for 2 hr at 40 °C. After hybridisation, slides were washed for 2 x 2 min with 1 x RNAScope wash buffer. A series of six hybridisation amplification (AMP 1-6) steps were then conducted, by placing the hybridisation amplification solution onto the tissue at 40°C for 30 min for AMP 1, 3 and 5 and 15 min for AMP 2, 4 and 6. Amplification steps 1-4 were conducted at 40°C and the remaining amplification steps were conducted at room temperature. A wash step was conducted for 2 x 2 min following each amplification incubation. Following amplification, detection substrate (RED working solution, 1:60 ratio of Fast RED-B to Fast RED-A) was added and incubated for 10 min at room temperature. The slides were then rinsed under running tap water. Slides were counterstained with aqueous haematoxylin for 30 sec and placed immediately into tap water. Slides were placed into 0.5% lithium carbonate solution for 10 dips and rinsed with tap water. Sections were dried completely for 15 min in a 60°C oven, dipped into fresh pure xylene and mounted with DPX mountant (Sigma, Cat#06522) and coverslip. Slides were dried overnight before scanning.

IHC and ISH assessment
Antibodies: Antibodies utilised are shown in supplementary material, Table S1. To assess EMT, N/E-cadherin, Axl, vimentin and slug/snail antibodies were used. N/E-cadherin and vimentin collectively represent the hallmarks of EMT [6] and were therefore chosen as core EMT markers. In addition, receptor tyrosine kinases (RTKs) have been shown to promote EMT [6]. Axl is a RTK which has been shown to be regulated by vimentin [5,44] [41] and also to be increased in luminal tumours and so was included as a molecular subtype marker.
Immunohistochemistry assessment: For Cytokeratins 5/6, 14 and 20, CD44, vimentin and Axl, cases were classified as negatively or positively stained based on criteria from the literature (Table 1).
For FOXA1, GATA3, E-cadherin, N-cadherin and slug/snail, staining was assessed using a semi-quantitative method. For each papillary and invasive region, up to 10 high-magnification images were acquired using ImageScope software (Aperio, Leica). Images were assessed for staining intensity on a four-point grading scale (0+ = negative, 1+ = weak, 2+ = moderate and 3+ = strong), and the percentage of cells staining positively were visually estimated. The overall semi-quantitative H-score was defined as the product of staining intensity and percentage of positive staining (intensity x percentage positive), as previously described [21].
Staining intensity scales were pre-determined for each antibody individually (as outlined in supplementary material, Figure S3). For FOXA1 and GATA3, nuclear staining was assessed, and membranous staining was assessed for E-cadherin and N-cadherin. Although the Slug/Snail antibody stains both nuclear and cytoplasmic components, only nuclear staining was assessed as increased nuclear expression of slug/snail indicates activation of EMT signalling