Carme Mallofré

Is histone H4 acetylation (H4ac) altered in the seminiferous tubules of patients affected by testicular tumours? A considerable dysregulation of H4ac was detected in the cells of the seminiferous tubules adjacent to testicular tumours of different aetiology and prior to any treatment, while no comparable alterations were observed in patients with disrupted spermatogenesis. Altered H4ac levels have been associated with a variety of testicular pathological conditions. However, no information has been available regarding potential alterations in the spermatogenic cells adjacent to the neoplasia in testicular tumour patients. A retrospective analysis using testicular sections from 33 men aged between 21 and 74 years old was performed. Three study groups were defined and subjected to double-blind evaluation: a control group with normal spermatogenesis (n = 6), patients with testicular tumours (n = 18) and patients with spermatogenic impairments (n = 8). One additional sample with normal spermatogenesis was used as a technical internal control in all evaluations. Immunohistochemistry against H4ac and, when needed, Placental-like alkaline phosphatase and CD117, was performed on testicular sections. The H4ac H-score, based on the percentage of detection and signal intensity, was used as the scoring method for statistical analyses. Protein expression data from the Human Protein Atlas were used to compare the expression levels of predicted secreted proteins from testicular tumours with those present in the normal tissue. We revealed, for the first time, a dramatic disruption of the spermatogenic H4ac pattern in unaffected seminiferous tubule cells from different testicular tumour patients prior to any antineoplastic treatment, as compared to controls (P < 0.05). Since no similar alterations were associated with spermatogenic impairments and the in silico analysis revealed proteins potentially secreted by the tumour to the testicular stroma, we propose a potential paracrine effect of the neoplasia as a mechanistic hypothesis for this dysregulation. Statistical analyses were not performed on the hypospermatogenesis and Leydig cell tumour groups due to limited availability of samples. To the best of our knowledge, this is the first report showing an epigenetic alteration in cells from active seminiferous tubules adjacent to tumour cells in testicular tumour patients. Our results suggest that, despite presenting spermatogenic activity, the global epigenetic dysregulation found in the testicular tumour patients could lead to molecular alterations of the male germ cells. Since testicular tumours are normally diagnosed in men at reproductive age, H4ac alterations might have an impact when these testicular tumour patients express a desire for fatherhood. This work was supported by the European Union Marie Curie European Training Network actions and by grants to R.O. from the ‘Ministerio de Economía y Competividad (Spain)’ (fondos FEDER ‘una manera de hacer Europa’, PI13/00699, PI16/00346 and PI20/00936) and from EU-FP7-PEOPLE-2011-ITN289880. J.C. was supported by the Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109. J.C. is a Serra Húnter fellow (Universitat de Barcelona, Generalitat de Catalunya). F.B. has received grants from the Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario (Spain) (FPU15/02306). A.d.l.I. is supported by a fellowship of the Ministerio de Economía, Industria y Competitividad (Spain) (PFIS, FI17/00224). M.J. is supported by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337). The authors have no conflicts of interest to declare. N/A.

Prognostic microRNAs in upper tract urothelial carcinoma: multicenter and international validation study Prognostic microRNAs in Upper Tract Urothelial Carcinoma: Multicenter and International Validation Study

The aim of this study was to assess the accuracy of Ultrasound (US) and contrast-enhanced ultrasound (CEUS) in the characterization of renal nodules indeterminate on CT by identifying benign cystic lesions not requiring further examination. 72 patients with 83 indeterminate renal nodules on CT underwent baseline US and CEUS that classified lesions as benign (Bosniak I, II or IIF cysts) or potentially malignant (Bosniak III or IV cysts, solid nodules). The accuracy of US and CEUS in the differentiation between benign cysts and potentially malignant nodules was analyzed and compared with the final diagnosis obtained by histology or follow-up of at least 23 months with CEUS ± a conclusive CT/MR study. Final diagnoses comprised 50 benign complex cysts, 1 focal nephritis, 1 multilocular cystic nephroma, 3 oncocytomas, 1 transitional cell carcinoma and 27 renal cell carcinomas. Unenhanced US correctly classified 18/50 (36%) benign cysts and 17/33 (51.5%) of the potentially malignant lesions obtaining a sensitivity of 36%, specificity of 51.5%, and overall accuracy of 42.2%. The addition of CEUS allowed a correct diagnosis of 48 /50 (96%) benign cysts and of 31/33 (93.9%) nodules as potentially malignant, with a sensitivity of 96%, specificity of 93.9%, and overall accuracy of 95.2%. CEUS is very useful in the differentiation between benign complex cysts and other lesions that require further investigation in non-conclusive renal nodules detected on CT, improving the accuracy of baseline US from 42.2 to 95.2%.

533 Background: Sunitinib is a standard first line treatment of metastatic renal cell carcinoma (ccRCC). Approximately 20% of patients experience primary resistance to therapy and no predictive biomarkers are available. The aim of our study is to discover novel biomarkers to predict response to sunitinib and to generate hypothesis about mechanisms of intrinsic resistance. Methods: Gene expression analysis was performed in formalin-fixed paraffin embedded (FFPE) samples from 44 patients with metastatic ccRCC treated with sunitinib in our institution. Affymetrix Human Gene 2.0 ST array was performed in primary tumors from 6 extremely sensitive (progression free survival (PFS) > 24 months) and 8 refractory (progression disease as best response) ccRCC. Technical validation with qPCR (Fluidigm Dynamic 96.96 Array) was performed in the whole cohort. The ΔΔCt method was used to quantify the relative amount of mRNA. Clinical and pathological data were correlated with gene expression. Results: 330 genes were differentially expressed between refractory and sensitive patients (p≤0.05). Network analysis showed 16 significant networks represented. Gene expression of 96 selected markers was tested in 47 primary tumors and 24 metastases. We found IL8, VEGF and NOTCH pathways upregulated on refractory patients. Survival analysis showed that the overexpression of IL8 correlated with a worse PFS (HR: 2.42, 95%CI1.27 – 4.63, p=0.0075) and overall survival (OS) (HR: 3.42, 95%CI 1.50 – 7.79, p=0.0034). Overexpression of VEGFBcorrelated with a prolonged PFS (HR 0.52, 95% CI 0.28 – 0.98, p=0.0415). Conclusions: Our results confirm the predictive value of IL8 expression in a cohort of ccRCC patients treated with sunitinib and suggests novel biomarkers of response to sunitinib. These data will be further validated in an independent cohort of patients.

Objective: To examine the microRNA ( miRNA ) expression pattern in tumour samples from patients with progressing and non‐progressing upper tract urothelial carcinoma ( UTUC ) in order to identify putative miRNAs that may be used as prognostic markers. Patients and Methods: We conducted a multicentre, retrospective study of formalin‐fixed paraffin‐embedded tissue samples from 150 patients with UTUC who had undergone radical nephroureterectomy. Global mi RNA expression patterns were analysed in 18 selected samples from patients with UTUC using T aq M an arrays. The differential expression of five key miRNAs was validated by quantitative polymerase chain reaction in an independent cohort of 132 samples from patients with UTUC . Models to predict tumour progression and cancer‐specific survival that included miRNA expression patterns were developed by C ox regression analysis. Results: Twenty‐six miRNAs were found to be aberrantly expressed between samples from patients with progressing and non‐progressing UTUC and five of these were selected for subsequent studies. The regression analysis identified tumour stage and miR ‐31 and miR ‐149 expression as independently associated with tumour progression and tumour stage and miR ‐149 expression as independently associated with cancer‐specific survival. The risk scores derived from these miRNA models were able to discriminate two groups with a highly significantly different probability of tumour progression (hazard ratio [ HR ] 4.78; P < 0.001) and death ( HR 276; P = 0.004). Conclusions: There is a differential m iRNA expression pattern between patients with progressing and non‐progressing UTUC . The identification of new miRNAs associated with a high probability of tumour recurrence and cancer‐specific survival in patients with UTUC and their combination in a robust, easy‐to‐use and reliable algorithm may help tailor treatment and surveillance strategies in these patients.

OBJECTIVES: To evaluate α, β and γ‐catenin expression in upper urinary tract urothelial tumours (UUTC) and determine their value as prognostic factors; to investigate the correlation between the catenin complex and the AKT pathway. PATIENTS AND METHODS: We retrospectively analysed 114 consecutive patients treated at our institution from 1990 to 2004; the mean follow‐up was 54 months. Tumour samples were available from 70 patients, and included in tissue microarrays for immunohistochemical analysis. The antibodies used were anti‐α, ‐β and γ‐catenin, and antiphospho‐AKT. The prognostic value of the expression of these molecules was analysed using tumour progression and cancer‐specific survival as end‐points. RESULTS: Of the 114 patients, 27% developed tumour progression; the cancer‐specific and overall survival were 77% and 60.6%, respectively. Abnormal α, β and γ‐catenin expression was found in 44 (63%), 22 (31%) and 28 (41%) patients, respectively; the abnormal catenin expression patterns correlated with each other. Positive cytoplasm phospho‐AKT expression was found in 27 (39%) patients. Three of them were found to have cytoplasmic β‐catenin accumulation and none of them nuclear expression. β‐catenin expression was the only one that was an independent marker of tumour progression, with a hazard ratio (95% confidence interval) of 3.1(1.2–8.6), together with grade (7.1, 1.2–55.8) and stage (4.6, 2.1–10). In the cancer‐specific survival analysis, again β‐catenin was an independent prognostic factor (3.4, 1–11.5) together with stage (4.6, 2.2–9.8). CONCLUSIONS: The loss of the normal membrane β‐catenin expression constitutes an independent factor of tumour progression and cancer‐specific survival. Our data suggest that the AKT/GSK3β/β‐catenin signalling pathway is not activated in the UUTC carcinogenesis.