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Tumour-infiltrating lymphocytes (TIL), known to be of prognostic value in various solid tumours, have been in the focus of research in the last years. TIL are often quantified via IMMUNOSCORE ® (IS), a scoring system based on TIL cell densities. Recent studies were able to replicate these findings for muscle-invasive bladder cancer (MIBC), however data regarding non-muscle-invasive bladder cancer (NMIBC) are scarce. This study aimed to evaluate the value of a modified Immunoscore (mIS) as a predictive marker for NMIBC prognosis using tissue-micro-arrays (TMAs). We analysed two TMAs containing 316 samples from 158 patients with NMIBC, stained for CD3, CD8, CD45RO and FOXP3. Stained TIL were captured by digital pathology, cumulated, averaged, and reported as density (stained cells per mm²). The mIS was then constructed based on density of all four immune-cell types. Clinical, pathological and follow-up data were collected retrospectively. Univariable and multivariable cox regression analysis was performed to assess the potential value of mIS as a predictor for progression free survival (PFS) and recurrence-free-survival (RFS). Patients within “European Organisation for Research and Treatment of Cancer” (EORTC) risk groups were further substratified in high mIS and low mIS subgroups. Finally log-rank test was used to compare the different survival curves. The median age in our cohort was 68 years (Interquartile Range (IQR): 60 - 76), and 117 (74%) patients were male. A total of 26 patients (16.5%) were classified as EORTC low risk, 45 (28.5%) as intermediate risk and 87 (55.1%) as high risk. Patients in the EORTC high risk group with low mIS showed a shorter PFS in comparison to high mIS (HR 2.9, CI 0.79 – 11.0, p=0.082). In contrast, no predictive potential regarding PFS was observed in intermediate or low risk groups. Furthermore, mIS was not able to predict RFS in any EORTC risk group. mIS could be utilized to predict prognosis more accurately in high-risk patients with NMIBC by identifying those with higher or lower risk of progression. Therefore, mIS could be used to allocate these highrisk patients to more streamlined follow-up or more aggressive treatment strategies.

To evaluate the prognostic value of a modified Immunoscore (mIS) in a cohort of bladder cancer (BC) patients undergoing radical cystectomy (RC), two tissue microarrays of 159 BC patients were immunohistochemically stained for CD3/CD8/FOXP3 and CD45RO to detect Tumor-Infiltrating Lymphocytes (TIL). To predict progression free survival (PFS) and cancer specific survival (CSS), a predictive model cumulatively incorporating all four components was constructed and labeled as mIS. Patients were stratified into two risk groups; “high mIS/favorable risk” and “low mIS/unfavorable risk”. Kaplan–Meier analysis was used to test mIS within each American Joint Committee on Cancer (AJCC) stage group for BC. In a univariable cox regression analysis all single components used for mIS, showed a significant association with CSS. Patients with high mIS (all components) in the AJCC stage IIIa group additionally showed a significantly longer PFS (Hazard Ratio (HR): 2.7; p = 0.008) and CSS (HR: 3.5; p = 0.006) as compared to patients with low mIS. mIS is of prognostic value in BC patients undergoing RC and was able to stratify patients within AJCC stage IIIa and might thus serve as a prognostic marker to guide risk-adapted treatment or follow-up strategies after RC.

ISSN:0022-3417 ISSN:1096-9896

The bromodomain and extra-terminal (BET) family of proteins, comprised of four members including BRD2, BRD3, BRD4 and the testis-specific isoform BRDT, largely function as transcriptional co-activators 1–3 and play critical roles in various cellular processes, including cell cycle, apoptosis, migration and invasion 4,5. As such, BET proteins enhance the oncogenic functions of major cancer drivers by either elevating their expression such as c-Myc in leukemia 6,7 or by promoting transcriptional activities of oncogenic factors such as AR and ERG in the prostate cancer setting 8. Pathologically, BET proteins are frequently overexpressed and clinically linked to various types of human cancers 5,9,10, therefore pursued as attractive therapeutic targets for selective inhibition in patients. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed 11,12 and shown promising outcomes in early clinical trials. Despite resistance to BET inhibitor has been documented in pre-clinical models 13–15 the molecular mechanisms underlying acquired resistance are largely unknown. Here, we report that Cullin 3SPOP earmarks BET proteins including BRD2, BRD3 and BRD4 for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of BET proteins, leading to their elevated abundance in SPOP-deficient prostate cancer. As a result, prostate cancer cells and prostate cancer patient-derived organoids harboring SPOP mutations are more resistant to BET inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor suppressor role of SPOP in prostate cancer by negatively controlling BET protein stability, and also provide a molecular mechanism for BET inhibitor resistance in prostate cancer patients bearing SPOP mutations.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.