Prognostic value of circulating microRNAs in upper tract urinary carcinoma

The identification of upper tract urinary carcinoma (UTUC) prognostic biomarkers is urgently needed to predict tumour progression. This study aimed to identify serum microRNAs (miRNAs) that may be useful as minimally invasive predictive biomarkers of tumour progression and survival in UTUC patients. To this end, 33 UTUC patients who underwent radical nephroureterectomy at the Hospital Clinic of Barcelona were prospectively included. Expression of 800 miRNAs was evaluated in serum samples from these patients using nCounter® miRNA Expression Assays. The study was divided into an initial discovery phase (n=12) and a validation phase (n=21). Cox regression analysis was used for survival analysis. The median follow-up (range) of the series was 42 months (9-100 months). In the discovery phase, 38 differentially expressed miRNAs were identified between progressing and non-progressing UTUC patients (p<0.05). Validation of these 38 miRNAs in an independent set of UTUC patients confirmed the differential expression in 18 of them (p<0.05). Cox Regression analysis showed miR-151b and pathological stage as significant prognostic factors for tumour progression (HR=0.33, p<0.001 and HR=2.62, p=0.006, respectively) and cancer specific survival (HR=0.25, p<0.001 and HR=3.98, p=0.003, respectively). Survival curves revealed that miR-151b is able to discriminate between two groups of UTUC patients with a highly significant different probability of tumour progression (p=0.006) and cancer specific survival (p=0.034). Although the data needs to be externally validated, miRNA analysis in serum appears to be a valuable prognostic tool in UTUC patients. Particularly, differential expression of miR-151b in serum may serve as a minimally invasive prognostic tool in UTUC

Ability of a urine gene expression classifier to reduce the number of follow up cystoscopies in bladder cancer patients

This study aimed to improve our previous urine gene expression classifiers focusing on the detection of non-high-risk non-muscle-invasive bladder cancer (NMIBC), and develop a new classifier able to decrease the frequency of cystoscopies during bladder cancer (BC) patients' surveillance. A total of 597 urines from BC patients, controls and patients in follow-up for BC (PFBC) were included. The study has 3 phases. In the urinary biomarker discovery phase, 84 urines from BC and control patients were retrospectively included and analyzed by Ribonucleic Acid (RNA) sequencing. In the classifier development phase, a total of 132 selected genes from previous phase were evaluated by nCounter in 214 prospectively collected urines from PFBC (98 with tumor). A diagnostic classifier was generated by logistic regression. Finally, in the classifier validation phase, a multicentric and international cohort of 248 urines (134 BC and 114 nonrecurrent PFBC) was used to validate classifier performance. A total of 521 genes were found differentially expressed between non-high-risk NMIBC samples and all other groups (P < 0.05). An 8-gene diagnostic classifier with an area under curve (AUC) of 0.893 was developed. Validation of this classifier in a cohort of PFBC achieved an overall sensitivity (SN) and a negative predictive value (NPV) of 96% and 97%, respectively (AUC = 0.823). Notably, this accuracy was maintained in non-high-risk NMIBC group (SN = 94%; NPV = 98%). In conclusion, this 8-gene expression classifier has high SN and NPV in a real clinical scenario. The use of this classifier can reduce the number of follow-up cystoscopies in PFBC, although assessing its final place in clinical setting is necessary

Prognostic microRNAs in upper tract urothelial carcinoma: multicenter and international validation study

Objective: To validate previously discovered miRNAs (miR-31-5p and miR-149-5p) as prognostic factors for UTUC in an independent cohort of UTUC patients.Patients and Methods: Multicenter, international and retrospective study of formalin-fixed paraffin-embedded tissue samples from 103 UTUC patients (45 progressing and 58 non-progressing) who underwent radical nephroureterectomy. Total RNA was isolated and reverse transcribed. The expression of target miRNAs (miR-31-5p and miR-149-5p) and the endogenous control miR-218-5p was evaluated in all samples by reverse transcription quantitative PCR. Normalized miRNA expression values were evaluated by multivariate forward stepwise Cox regression analysis. Kaplan Meier curves were used to discriminate between two groups of patients with a different probability of tumour progression.Results: The mean age (range) of the series was 67 (33-94) years. Overall, 45 patients (43.7%) developed tumour progression and 32 patients (31.2%) died, 20 of these (62.5%) due to their UTUC, after a median follow-up of 36 months. The mean time for tumour progression and cancer-specific survival were 15 and 20 months, respectively. Five year tumour progression free survival and cancer-specific survival were 58% for <= pT2, 36% for pT3 and 0% for pT4 and 67.8% for <= pT2, 50.6% for pT3 and 0% for pT4, respectively. In the multivariate analysis, expression of miR-31-5p was found to be an independent prognostic factor of tumour progression (HR 1.1; 95% CI 1.039-1.273; p=0.02). Kaplan Meier curve shows that miR-31-5p expression values are able to discriminate between two groups of UTUC patients with a different probability of tumour progression (p=0.007).Conclusions: We have been able to validate our previous results in an independent multicentre international cohort of UTUC patients, suggesting that miRNA-31-5p could be a useful prognostic marker of UTUC progression. The application of miRNA expression values to clinical practice could refine the currently used clinicopathological-based approach for predicting UTUC patients' outcome

Androgen receptor and its splicing variant 7 expression in peripheral blood mononuclear cells and in circulating tumor cells in metastatic castration-resistant prostate cancer

Androgen receptor (AR) signaling remains crucial in castration-resistant prostate cancer (CRPC). Since it is also essential in immune cells, we studied whether the expression of AR full-length (ARFL) and its splicing variant ARV7 in peripheral blood mononuclear cells (PBMC) predicts systemic treatment response in mCRPC in comparison with circulating-tumor cells (CTC). We measured ARFL and ARV7 mRNA in PBMC and CTC from patients prior to receiving abiraterone (AA), enzalutamide (E), or taxanes by a pre-amplification plus quantitative reverse-transcription PCR. They were also tested in LNCaP-ARV7-transfected and in 22RV1 docetaxel-resistant (22RV1DR) cells. We studied 171 PBMC from 136 patients and from 24 non-cancer controls, and 47 CTC from 22 patients. High PBMC ARV7 levels correlated with worse AA/E and better taxane response. In taxane-treated patients high PBMC ARFL also correlated with longer progression-free survival (PFS). High ARV7 and ARFL expression were independently associated with better biochemical-PFS. Conversely, high CTC ARV7 and ARFL correlated with shorter radiological-PFS and overall survival, respectively. High ARV7 in 22RV1DR and LNCaP-ARV7 cells correlated with taxane resistance. In conclusion, ARFL and ARV7 at PBMC or CTC have a different predictive role in the taxane response, suggesting a potential influence of the AR pathway from PBMC in such response modulation

Estudi del carcinoma urotelial. Identificació de biomarcadors amb valor diagnòstic i pronòstic

Aquesta tesi és una contribució a l’estudi del carcinoma urotelial de vies urinàries superiors i de bufeta. Es centra en la recerca de biomarcadors diagnòstics i pronòstics amb potencial utilitat a la pràctica clínica diària en aquests tipus de tumors. El carcinoma urotelial (CU), també anomenat carcinoma de cèl·lules transicionals, és una malaltia neoplàsica que s’origina a l’uroteli. La localització més freqüent és la bufeta (90%), seguida pel tracte urinari superior (5-10%) i finalment per la uretra (1%). És el novè tumor més freqüent a nivell global, i a la Unió Europea, es posiciona com a cinquè en incidència. El carcinoma urotelial de tracte urinari superior és un tumor de mal pronòstic. En aquests pacients, la supervivència als 5 anys no arriba al 50% per aquells tumors que al diagnòstic són pT2/pT3 i és inferior al 10% quan es tracta de pT4. Actualment, l’estadi patològic i el grau histològic són els factors pronòstics més habitualment utilitzats a la pràctica clínica, tot i que resulten insuficients per a predir l’evolució de la malaltia de manera individualitzada, donat que pacients amb els mateix fenotip tumoral presenten evolucions clíniques diferents. Aproximadament el 75% dels tumors de bufeta es diagnostiquen inicialment com no múscul-invasius (NMIBC). Desafortunadament aquests tumors estan caracteritzats per uns índexs de recidiva que oscil·len entre el 60 i el 85% (un cop que el tumor primari ha estat tractat). A més, entre el 15 i el 30% dels NMIBC progressaran a múscul-invasius (MIBC), podent arribar a produir la mort del pacient. El gold estàndard de diagnòstic i seguiment del CB és la cistoscòpia combinada en alguns casos amb la citologia urinària. La cistoscòpia és un mètode invasiu, incòmode i dolorós pels pacients. D’altra banda, el mètode no invasiu, la citologia urinària, presenta una baixa sensibilitat, sobretot en els tumors de baix grau. Tot i els nombrosos esforços descrits a la literatura per identificar biomarcadors no invasius pel diagnòstic i seguiment del CU de bufeta, els biomarcadors estudiats fins al moment no superen la precisió diagnòstica d’aquesta combinació (aproximadament SN=80%, SP=90%). La identificació de biomarcadors en CU és de gran importància per millorar diferents aspectes del seu diagnòstic , pronòstic o per predir la resposta a tractaments. Cap dels biomarcadors descrits als últims temps ha estat implementat a la pràctica clínica diària. En la tesi per una banda s’identifiquen i validen biomarcadors pronòstics tissulars i sèrics en el carcinoma urotelial de vies urinàries superiors, i per l’ altra, es presenta una nova firma d’expressió gènica amb una alta precisió pel diagnòstic i seguiment del CU de bufeta. Finalment, també s’investiga si és pertinent realitzar anàlisis addicionals en pacients amb citologia urinària sospitosa per CU.This thesis is a contribution to the study of urothelial carcinoma (UC) in the upper urinary tract and in the bladder. It focuses on the search for diagnostic and prognostic biomarkers in these tumors with potential utility in daily clinical practice. Urothelial carcinoma is a neoplastic disease that originates in the urothelium. The most frequent location is the bladder (90%), followed by the upper urinary tract (5-10%) and finally the urethra (1%). It is the ninth most frequent tumor on a global level, and in the European Union, it is ranked fifth in incidence. Urothelial carcinoma of upper urinary tract is a tumor of poor prognosis. In these patients, survival at 5 years does not reach 50% for those with pT2 and/or pT3 and less than 10% for pT4. Nowadays, pathological stage and histological grade are the most commonly used prognostic factors in clinical practice, although they are insufficient to predict the evolution of the disease in an individualized way, since patients with the same tumor phenotype present different follow up. The gold standard for diagnosis and monitoring of bladder cancer is cystoscopy combined in some cases with urinary cytology. Cystoscopy is an invasive, uncomfortable and painful method for patients. The non-invasive method, cytology, has a low sensitivity, especially in low-grade tumors. Consequently, the identification of biomarkers in UC is of great importance to improve different aspects of their diagnosis, prognosis or prediction of the response to treatments. None of the biomarkers described in recent years has been implemented in daily clinical practice yet. In this thesis, on the one hand, prognostic biomarkers for Upper Tact Urothelial Carcinoma are identified and validated in tissue and in serum and on the other hand, a new gene expression signature with high accuracy for the diagnosis and surveillance of bladder cancer is also described. Finally, the utility of an additional analysis in the follow up of patients with cytology suspicious for UC is investigated

Estudi del carcinoma urotelial. Identificació de biomarcadors amb valor diagnòstic i pronòstic

[cat] Aquesta tesi és una contribució a l’estudi del carcinoma urotelial de vies urinàries superiors i de bufeta. Es centra en la recerca de biomarcadors diagnòstics i pronòstics amb potencial utilitat a la pràctica clínica diària en aquests tipus de tumors. El carcinoma urotelial (CU), també anomenat carcinoma de cèl·lules transicionals, és una malaltia neoplàsica que s’origina a l’uroteli. La localització més freqüent és la bufeta (90%), seguida pel tracte urinari superior (5-10%) i finalment per la uretra (1%). És el novè tumor més freqüent a nivell global, i a la Unió Europea, es posiciona com a cinquè en incidència. El carcinoma urotelial de tracte urinari superior és un tumor de mal pronòstic. En aquests pacients, la supervivència als 5 anys no arriba al 50% per aquells tumors que al diagnòstic són pT2/pT3 i és inferior al 10% quan es tracta de pT4. Actualment, l’estadi patològic i el grau histològic són els factors pronòstics més habitualment utilitzats a la pràctica clínica, tot i que resulten insuficients per a predir l’evolució de la malaltia de manera individualitzada, donat que pacients amb els mateix fenotip tumoral presenten evolucions clíniques diferents. Aproximadament el 75% dels tumors de bufeta es diagnostiquen inicialment com no múscul-invasius (NMIBC). Desafortunadament aquests tumors estan caracteritzats per uns índexs de recidiva que oscil·len entre el 60 i el 85% (un cop que el tumor primari ha estat tractat). A més, entre el 15 i el 30% dels NMIBC progressaran a múscul-invasius (MIBC), podent arribar a produir la mort del pacient. El gold estàndard de diagnòstic i seguiment del CB és la cistoscòpia combinada en alguns casos amb la citologia urinària. La cistoscòpia és un mètode invasiu, incòmode i dolorós pels pacients. D’altra banda, el mètode no invasiu, la citologia urinària, presenta una baixa sensibilitat, sobretot en els tumors de baix grau. Tot i els nombrosos esforços descrits a la literatura per identificar biomarcadors no invasius pel diagnòstic i seguiment del CU de bufeta, els biomarcadors estudiats fins al moment no superen la precisió diagnòstica d’aquesta combinació (aproximadament SN=80%, SP=90%). La identificació de biomarcadors en CU és de gran importància per millorar diferents aspectes del seu diagnòstic , pronòstic o per predir la resposta a tractaments. Cap dels biomarcadors descrits als últims temps ha estat implementat a la pràctica clínica diària. En la tesi per una banda s’identifiquen i validen biomarcadors pronòstics tissulars i sèrics en el carcinoma urotelial de vies urinàries superiors, i per l’ altra, es presenta una nova firma d’expressió gènica amb una alta precisió pel diagnòstic i seguiment del CU de bufeta. Finalment, també s’investiga si és pertinent realitzar anàlisis addicionals en pacients amb citologia urinària sospitosa per CU.[eng] This thesis is a contribution to the study of urothelial carcinoma (UC) in the upper urinary tract and in the bladder. It focuses on the search for diagnostic and prognostic biomarkers in these tumors with potential utility in daily clinical practice. Urothelial carcinoma is a neoplastic disease that originates in the urothelium. The most frequent location is the bladder (90%), followed by the upper urinary tract (5-10%) and finally the urethra (1%). It is the ninth most frequent tumor on a global level, and in the European Union, it is ranked fifth in incidence. Urothelial carcinoma of upper urinary tract is a tumor of poor prognosis. In these patients, survival at 5 years does not reach 50% for those with pT2 and/or pT3 and less than 10% for pT4. Nowadays, pathological stage and histological grade are the most commonly used prognostic factors in clinical practice, although they are insufficient to predict the evolution of the disease in an individualized way, since patients with the same tumor phenotype present different follow up. The gold standard for diagnosis and monitoring of bladder cancer is cystoscopy combined in some cases with urinary cytology. Cystoscopy is an invasive, uncomfortable and painful method for patients. The non-invasive method, cytology, has a low sensitivity, especially in low-grade tumors. Consequently, the identification of biomarkers in UC is of great importance to improve different aspects of their diagnosis, prognosis or prediction of the response to treatments. None of the biomarkers described in recent years has been implemented in daily clinical practice yet. In this thesis, on the one hand, prognostic biomarkers for Upper Tact Urothelial Carcinoma are identified and validated in tissue and in serum and on the other hand, a new gene expression signature with high accuracy for the diagnosis and surveillance of bladder cancer is also described. Finally, the utility of an additional analysis in the follow up of patients with cytology suspicious for UC is investigated

Cell Plasticity-Related Phenotypes and Taxanes Resistance in Castration-Resistant Prostate Cancer

Càncer de pròstata resistent a la castració; Plasticitat cel·lular; Resistència als taxansCáncer de próstata resistente a la castración; Plasticidad celular; Resistencia a los taxanosCastration-resistant prostate cancer; Cell plasticity; Taxanes resistanceThe prostatic tumor cells plasticity is involved in resistance to hormone-therapy, allowing these cells to survive despite androgen receptor inhibition. However, its role in taxanes resistance has not been fully established. Gene expression of plasticity-related phenotypes such as epithelial-mesenchymal transition (EMT), stem cell-like and neuroendocrine (NE) phenotypes was studied in vitro, in silico, in circulating tumor cells (CTCs) (N=22) and in tumor samples (N=117) from taxanes-treated metastatic castration-resistant prostate cancer (mCRPC) patients. Docetaxel (D)-resistant cells presented a more pronounced EMT phenotype than cabazitaxel (CZ)-resistant cells. In silico analysis revealed ESRP1 down-regulation in taxane-exposed mCRPC samples. Cell plasticity-related changes occurred in CTCs after taxanes treatment. Tumor EMT phenotype was associated with lower PSA progression-free survival (PFS) to D (P<0.001), and better to CZ (P=0.002). High ESRP1 expression was independently associated with longer PSA-PFS (P<0.001) and radiologic-PFS (P=0.001) in D and shorter PSA-PFS in the CZ cohort (P=0.041). High SYP expression was independently associated with lower PSA-PFS in D (P=0.003) and overall survival (OS) in CZ (P=0.002), and high EZH2 expression was associated with adverse OS in D-treated patients (P=0.013). In conclusion, EMT profile in primary tumor is differentially associated with D or CZ benefit and NE dedifferentiation correlates with adverse taxanes clinical outcome.This work was supported by grants from Instituto de Salud Carlos III-Subdirección General de Evaluación y Fomento de la Investigación [PI12/01226, PI15/676 and PI18/714] and co-funded by the European Regional Development Fund (ERDF). Institutional funding from CERCA Programme/Generalitat de Catalunya is gratefully acknowledged. This work was developed at the Centro Esther Koplowitz, Barcelona, Spain

Prognostic microRNAs in upper tract urothelial carcinoma: multicenter and international validation study

Objective: To validate previously discovered miRNAs (miR-31-5p and miR-149-5p) as prognostic factors for UTUC in an independent cohort of UTUC patients.Patients and Methods: Multicenter, international and retrospective study of formalin-fixed paraffin-embedded tissue samples from 103 UTUC patients (45 progressing and 58 non-progressing) who underwent radical nephroureterectomy. Total RNA was isolated and reverse transcribed. The expression of target miRNAs (miR-31-5p and miR-149-5p) and the endogenous control miR-218-5p was evaluated in all samples by reverse transcription quantitative PCR. Normalized miRNA expression values were evaluated by multivariate forward stepwise Cox regression analysis. Kaplan Meier curves were used to discriminate between two groups of patients with a different probability of tumour progression.Results: The mean age (range) of the series was 67 (33-94) years. Overall, 45 patients (43.7%) developed tumour progression and 32 patients (31.2%) died, 20 of these (62.5%) due to their UTUC, after a median follow-up of 36 months. The mean time for tumour progression and cancer-specific survival were 15 and 20 months, respectively. Five year tumour progression free survival and cancer-specific survival were 58% for <= pT2, 36% for pT3 and 0% for pT4 and 67.8% for <= pT2, 50.6% for pT3 and 0% for pT4, respectively. In the multivariate analysis, expression of miR-31-5p was found to be an independent prognostic factor of tumour progression (HR 1.1; 95% CI 1.039-1.273; p=0.02). Kaplan Meier curve shows that miR-31-5p expression values are able to discriminate between two groups of UTUC patients with a different probability of tumour progression (p=0.007).Conclusions: We have been able to validate our previous results in an independent multicentre international cohort of UTUC patients, suggesting that miRNA-31-5p could be a useful prognostic marker of UTUC progression. The application of miRNA expression values to clinical practice could refine the currently used clinicopathological-based approach for predicting UTUC patients' outcome

Prognostic microRNAs in upper tract urothelial carcinoma: multicenter and international validation study

Objective: To validate previously discovered miRNAs (miR-31-5p and miR-149-5p) as prognostic factors for UTUC in an independent cohort of UTUC patients.Patients and Methods: Multicenter, international and retrospective study of formalin-fixed paraffin-embedded tissue samples from 103 UTUC patients (45 progressing and 58 non-progressing) who underwent radical nephroureterectomy. Total RNA was isolated and reverse transcribed. The expression of target miRNAs (miR-31-5p and miR-149-5p) and the endogenous control miR-218-5p was evaluated in all samples by reverse transcription quantitative PCR. Normalized miRNA expression values were evaluated by multivariate forward stepwise Cox regression analysis. Kaplan Meier curves were used to discriminate between two groups of patients with a different probability of tumour progression.Results: The mean age (range) of the series was 67 (33-94) years. Overall, 45 patients (43.7%) developed tumour progression and 32 patients (31.2%) died, 20 of these (62.5%) due to their UTUC, after a median follow-up of 36 months. The mean time for tumour progression and cancer-specific survival were 15 and 20 months, respectively. Five year tumour progression free survival and cancer-specific survival were 58% for <= pT2, 36% for pT3 and 0% for pT4 and 67.8% for <= pT2, 50.6% for pT3 and 0% for pT4, respectively. In the multivariate analysis, expression of miR-31-5p was found to be an independent prognostic factor of tumour progression (HR 1.1; 95% CI 1.039-1.273; p=0.02). Kaplan Meier curve shows that miR-31-5p expression values are able to discriminate between two groups of UTUC patients with a different probability of tumour progression (p=0.007).Conclusions: We have been able to validate our previous results in an independent multicentre international cohort of UTUC patients, suggesting that miRNA-31-5p could be a useful prognostic marker of UTUC progression. The application of miRNA expression values to clinical practice could refine the currently used clinicopathological-based approach for predicting UTUC patients' outcome

Ability of a urine gene expression classifier to reduce the number of follow up cystoscopies in bladder cancer patients

This study aimed to improve our previous urine gene expression classifiers focusing on the detection of non-high-risk non-muscle-invasive bladder cancer (NMIBC), and develop a new classifier able to decrease the frequency of cystoscopies during bladder cancer (BC) patients' surveillance. A total of 597 urines from BC patients, controls and patients in follow-up for BC (PFBC) were included. The study has 3 phases. In the urinary biomarker discovery phase, 84 urines from BC and control patients were retrospectively included and analyzed by Ribonucleic Acid (RNA) sequencing. In the classifier development phase, a total of 132 selected genes from previous phase were evaluated by nCounter in 214 prospectively collected urines from PFBC (98 with tumor). A diagnostic classifier was generated by logistic regression. Finally, in the classifier validation phase, a multicentric and international cohort of 248 urines (134 BC and 114 nonrecurrent PFBC) was used to validate classifier performance. A total of 521 genes were found differentially expressed between non-high-risk NMIBC samples and all other groups (P < 0.05). An 8-gene diagnostic classifier with an area under curve (AUC) of 0.893 was developed. Validation of this classifier in a cohort of PFBC achieved an overall sensitivity (SN) and a negative predictive value (NPV) of 96% and 97%, respectively (AUC = 0.823). Notably, this accuracy was maintained in non-high-risk NMIBC group (SN = 94%; NPV = 98%). In conclusion, this 8-gene expression classifier has high SN and NPV in a real clinical scenario. The use of this classifier can reduce the number of follow-up cystoscopies in PFBC, although assessing its final place in clinical setting is necessary