Purpose: Currently, markers are lacking that can identify patients with high risk nonmuscle invasive bladder cancer who will fail bacillus Calmette-Gu erin treatment. Therefore, we evaluated the prognostic value of T1 substaging in patients with primary high risk nonmuscle invasive bladder cancer. Materials and Methods: Patients with primary high risk nonmuscle invasive bladder cancer who received !5 bacillus Calmette-Gu erin induction instillations were included. All tumors were centrally reviewed, which included T1 substaging (microinvasion vs extensive invasion of the lamina propria). T1 patients were stratified into high risk or highest risk subgroups according to major urology guidelines. Primary end point was bacillus Calmette-Gu erin failure, defined as development of a high grade recurrence. Secondary end points were high grade recurrence-free survival, defined as time from primary diagnosis to biopsyproven high grade recurrence and progression-free survival. Time-to-event analyses were used to predict survival. Results: A total of 264 patients with high risk nonmuscle invasive bladder cancer had tumor invasion of the lamina propria, of which 73% were classified as extensive invasion and 27% as microinvasion. Median followup was 68 months (IQR 43e98) and bacillus Calmette-Gu erin failure was more common among patients with extensive vs microinvasive tumors (41% vs 21%, p[0.002). The 3-year high grade recurrence-free survival (defined as bacillus Calmette-Guerin failure) for patients with extensive vs microinvasive tumors was 64% vs 83% (p[0.004). In multivariate analysis, T1 substaging was an independent predictor of high grade recurrence-free survival (HR 3.2, p[0.005) and progression-free survival (HR 3.0, p[0.009). Patients with highest risk/microinvasive disease have an improved progression-free survival as compared to highest risk/T1e disease (p.adj[0.038). Conclusions: T1 substaging provides important prognostic information on patients with primary high risk nonmuscle invasive bladder cancer treated with

SamenvattingDeze casus beschrijft een patiënt met progressieve onderbuikspijn en onvermogen tot mictie op basis van een uitzonderlijk grote mediane intraprostatische cyste. Een dergelijke cyste is een zeldzame, benigne, vaak asymptomatische aandoening en kan onderverdeeld worden in een utriculus cyste en een Müllerian duct cyste. Na drainage van de cyste bleef de patiënt in deze beschreven casus klachtenvrij.

Intravesical instillations with Bacillus Calmette-Guérin (BCG) is the recommended treatment for T1 high-grade (HG) bladder cancer (BC) patients. Unfortunately, risk stratification is insufficient to identify patients at risk of BCG treatment failure. Another challenge is the worldwide BCG-shortage, which emphasizes the need for alternative therapeutic strategies. Thus, we aimed to investigate molecular signal transduction pathway activities using Philips' OncoSignal consumer-ready test in BCG treated T1HG BC in order to identify differentially expressed pathways that may predict clinical outcome or provide a rationale for alternative therapeutic strategies. Primary T1HG BC patients treated with ≥5/6 BCG induction instillations were retrospectively included. RNA-sequencing (50 million paired-end reads, containing ≥80% tumor) was performed on: 1) BCG-naïve tumors from patients with complete response to BCG at study inclusion (BCG-responders) and 2) BCG-naïve tumors with matching muscle-invasive bladder cancer (MIBC) recurrences during BCG therapy (BCG-failures). Philips OncoSignal analysis was performed to quantify functional signal transduction pathway activities. Molecular subtyping was performed using non-muscle invasive and muscle-invasive classifiers. Clinical and molecular subtyping results were combined with Philips OncoSignal to explore differences between BCG responders and BCG failures and between molecular subtypes. OncoSignal was performed in 20 tumors: 14 BCG-naïve (7 BCG-responders vs 7 BCG-failures) and 6 MIBC recurrences. Median follow-up for BCG-responders was 99 months (IQR 74-120); median follow-up for BCG-failures was 49 months (IQR 25-79), with a median time to progression of 14 months (IQR 10-41). Based on pilot data, MAPK and TGFβ pathway activity was significantly higher in tumors from BCG failures vs BCG-responders (both p<0.05). Using TCGAs MIBC classifier, 10/14 pre-BCG samples were luminal-papillary (LumP), 1/14 was luminal-infiltrated (lum-inf) and 3/14 basal-squamous (bas-sq). In post-BCG MIBC, 2/6 tumors were lum-inf, 3/6 bas-sq and 1/6 neuronal. Using Erasmus MCs T1-BCG classifier (unpublished), 7/14 pre-BCG tumors were luminal-like, the other 7/14 were lum-inf/basal-like. All 6 post-BCG MIBCs were lum-inf/basal-like. Importantly, TCGAs bas-sq/lum-inf subtype and Erasmus MCs lum-inf/basal-like subtype had the highest MAPK, TGFβ and JAK-STAT1/2 activity (all p<0.05). Based on our pilot study, results indicate that Philips OncoSignal is able to stratify BCG failures from BCG responders. Furthermore, OncoSignal results discern LumP tumors from lum-inf/basal-like tumors. Philips OncoSignal might be useful to stratify patients for alternative treatments. Further investigation on a large cohort of BC patients is required to determine whether Philips OncoSignal can be of value during clinical-decision making. Citation Format: Florus C. De Jong, Teemu D. Laajala, Robert F. Hoedemaeker, Sébastien Rinaldetti, Jolien T. Mensink, Angelique C. Van Der Made, Deric K. Van der Schoot, Egbert R. Boevé, Ellen C. Zwarthoff, Joost L. Boormans, Dan Theodorescu, James C. Costello, Tahlita C. Zuiverloon. Differential pathway analyses of BCG-treated T1HG bladder cancer using Philips OncoSignal: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 615.

Intravesical instillations with Bacillus Calmette-Guérin (BCG) are the recommended treatment in T1 high-grade (HG) bladder cancer (BC) patients. However, even with BCG treatment, 50% of patients develop a HG recurrence or progression to advanced disease. Current risk stratification is insufficient to identify patients at risk of BCG failure. We aimed to identify molecular predictors of BCG failure in T1HG BC patients with the objective to improve clinical decision-making. Primary T1HG BC patients treated with ≥5/6 BCG induction instillations were retrospectively included. RNA-sequencing (50 million paired-end reads, ≥80% tumor) was performed on centrally reviewed tumors from: 1) BCG-naïve tumors and recurrent tumors from patients that failed BCG therapy (BCG non-responders), matched to an equal number of 2) patients who had an ongoing complete response until study inclusion (BCG responders). Endpoints were HG recurrence-free survival defined as BCG-failure (RFS) and progression-free survival (PFS), assessed by survival analyses. BCG-failure was defined as development of a biopsy-proven HG recurrence or progression to muscle-muscle invasion. Using RNA-seq, we sought to identify genes, pathways and signatures that stratified patients with respect to these endpoints. 132 BCG-naïve T1HG tumors were sequenced and 45 post-BCG tumors. Median follow-up for all patients was 75 months (IQR 48-106), 99 months for n=63 BCG-responders (IQR 74-120) and 49 months for n=69 BCG-non-responders (IQR 25-79). Three molecular subtypes were identified: BCG1 (32%), BCG2 (40%) and BCG3 (28%). BCG3 patients had a significantly worse RFS (HR 2.4 p<0.01) and PFS (HR 2.7; p<0.01) compared to BCG1 or BCG2 patients. BCG3 tumors expressed high EMT and basal markers and had an immune suppressive tumor microenvironment. Immune deconvolution revealed significantly increased immune cell infiltration (i.e. B cells, T regulatory cells and tumor associated macrophages), while CD14 and FOXP3 were identified as significant regulons in BCG3 patients. BCG1 patients showed a favorable PFS and overexpression of genes associated with BCG processing and antigen presentation. BCG2 patients overexpressed luminal BC markers, such as FGFR3, showed MYC pathway activity and were mostly luminal papillary (consensus muscle-invasive BC classifier). Interestingly, post-BCG tumor recurrences were strongly enriched for the BCG3 subtype; 30/45 (67%) of recurring tumors were classified as BCG3. Gene expression profiling of BCG-naïve primary T1HG BC patients identified three molecular subtypes that corresponded to clinical outcome and response to BCG therapy. The EMT-basal/immune suppressive BCG3 patients are not candidates for treatment with BCG given the poor RFS and PFS and might be candidates for early radical cystectomy or immune-modulating therapy. Citation Format: Florus C. De Jong, Teemu D. Laajala, Robert F. Hoedemaeker, Sébastien Rinaldetti, Jolien T. Mensink, Angelique C. Van der Made, Deric K. Van der Schoot, Egbert R. Boevé, Ellen C. Zwarthoff, Joost L. Boormans, Dan Theodorescu, James C. Costello, Tahlita C. Zuiverloon. Transcriptomic analysis of BCG-treated T1HG bladder cancer patients identifies an EMT-basal subgroup with immune suppressive characteristics at high risk of BCG-failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 614.