Basal/squamous and Mixed subtype bladder cancers present poor outcomes after neoadjuvant chemotherapy in the VESPER trial.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard treatment for muscle-invasive bladder cancer (MIBC), yet 40% of patients progress, emphasizing the need for biomarkers predictive for response or chemoresistance. Gene expression-based subtypes may serve as biomarkers, though which subtypes will respond, notably when it comes to the basal subtype, remains contentious. PATIENTS AND METHODS: This post-hoc study analyzed 300 NAC-treated patients enrolled in the GETUG/AFU VESPER trial, with transurethral diagnostic FFPE which underwent pathological review prior to being sequenced. "Mixed" subtype was defined for tumors displaying at least two different Consensus molecular subtypes in separate regions. We evaluated the association between molecular subtypes and outcome after NAC. Tumours with remaining tissue at cystectomy (n=83) were compared with pre-treatment tumours. RESULTS: Cases were classified Basal/squamous (Ba/Sq) (n=84), Luminal Unstable (n=57), Stroma-rich (n=53), Mixed (n=48), Luminal Papillary (n=39), Luminal Non-Specific (n=18) and Neuroendocrine-like (n=1), with 30/48 Mixed cases including a Ba/Sq component. Compared with other molecular subtypes in a multivariate Cox model, Ba/Sq (Pure or Mixed) patients had an increased hazard ratio of progression free survival (HR:2.0, 95% CI 1.36-3.0). Mixed tumors were associated with decreased metabolic activity that could account for chemoresistance. Ba/Sq and Mixed non-responders mostly maintained their subtype at cystectomy and have fewer myeloid dendritic cells after NAC. Tumors classified luminal papillary at TURBT exhibited an increase of T CD4+ and macrophage signatures after NAC. Other subtypes did not show significant immune changes after NAC. Our study design relied on detailed pathological review, which precluded evaluating the Mixed subtype in published datasets. Furthermore, the sample size for post-NAC analyses constrained the statistical power of these findings. CONCLUSIONS: Our findings underscore the importance of recognizing intra-tumor heterogeneity in MIBC, its role in chemoresistance associated with Ba/Sq subtype and provide valuable insights that could help future treatment development and improve patient outcomes.

'Desperate house genes': the dramatic example of hypoxia.

BACKGROUND: Microenvironmental conditions in normal or tumour tissues and cell lines may interfere on further biological analysis. To evaluate transcript variations carefully, it is common to use stable housekeeping genes (HKG) to normalise quantitative microarrays or real-time polymerase chain reaction results. However, recent studies argue that HKG fluctuate according to tissues and treatments. So, as an example of HKG variation under an array of conditions that are common in the cancer field, we evaluate whether hypoxia could have an impact on HKG expression. METHODS: Expression of 10 commonly used HKG was measured on four cell lines treated with four oxygen concentrations (from 1 to 20%). RESULTS: Large variations of HKG transcripts were observed in hypoxic conditions and differ along with the cell line and the oxygen concentration. To elect the most stable HKG, we compared the three statistical means based either on PCR cycle threshold coefficient of variation calculation or two specifically dedicated software. Nevertheless, the best HKG dramatically differs according to the statistical method used. Moreover, using, as a reference, absolute quantification of a target gene (here the proteinase activating receptor gene 1 (PAR1) gene), we show that the conclusions raised about PAR1 variation in hypoxia can totally diverge according to the selected HKG used for normalisation. CONCLUSION: The choice of a valid HKG will determine the relevance of the results that will be further interpreted, and so it should be seriously considered. The results of our study confirm unambiguously that HKG variations must be precisely and systematically determined before any experiment for each situation, to obtain reliable normalised results in the experimental setting that has been designed. Indeed, such assay design, functional for all in vitro systems, should be carefully evaluated before any extension to other experimental models including in vivo ones.

Comparative expression of Hedgehog ligands at different stages of prostate carcinoma progression.

Recent studies have revealed the potential involvement of Hedgehog (Hh) signalling in proliferation and invasive behaviour of prostate carcinoma (PCa). The aim of this study was to specify the role of Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh) in the natural history of PCa. Hh ligands expression was compared in primary hormone-naive PCa (HNPC), hormone-treated PCa (HTPC) and hormone-refractory PCa (HRPC), using immunohistochemistry. Shh and Dhh were expressed by both epithelial and stromal cells of prostate tissues. Ihh was only expressed by stromal cells. For the three ligands, mRNA and immunostaining were not correlated. In HNPC, Shh epithelial expression was significantly associated with high Gleason scores (p = 0.03), metastatic lymph nodes (p = 0.004) and Dhh epithelial staining was associated with high pT stages (p = 0.003), seminal vesicle invasion (p = 0.03) and bladder neck invasion (p = 0.0008). Negative Shh staining in stromal cells was associated with high Gleason scores (p = 0.015), high pT stages (p = 0.01) and bladder neck invasion (p = 0.04). Concomitant absence of Shh and Dhh expression in stromal cells was an independent prognostic parameter for biological recurrence on multivariate analysis (p = 0.01). Epithelial expression of Shh and Dhh was increased in HTPC compared to HNPC (p = 0.02 and p = 0.04). Interestingly, in vitro, transcript analysis also showed increased expression of these 2 Hh ligands when androgen-sensitive LNCaP cells were maintained in androgen-free medium mimicking hormonal therapy. Epithelial expression of Dhh was increased (p < 0.0001) in HRPC compared to HNPC, while stromal expression of Shh and Dhh was decreased (p < 0.0001). In conclusion, the Hh signalling pathway is associated with pejorative pathological parameters in HNPC and is up-regulated in epithelial cells of HTPC and HRPC. Moreover, the lack of Hh molecules in stromal cells seems to be associated with invasive and hormone-refractory behaviours and suggests specific changes in stromal-epithelial crosstalks during PCa progression.