Validación externa de los ARNm: FXYD3 y KRT20 como biomarcadores predictivos de la presencia de micrometástasis en ganglios de pacientes con tumor vesical infiltrante / External validation of FXYD3 and KRT20 as predictive biomarkers for the presence of micrometastasis in muscle invasive bladder cancer lymph nodes

Introducción: Recientes estudios han propuesto que los ARNm FXYD3 y KRT20 cuantificados por qrtPCR en material parafinado podrían ser biomarcadores capaces de detectar los ganglios portadores de micrometástasis que se escapaban al análisis convencional por hematoxilina-eosina (HE). Se decidió hacer un estudio de validación en ganglios de pacientes a los que se les practicó una cistectomía radical. Objetivo: Clasificar el estado adenopático de una muestra de pacientes cistectomizados, según la expresión ganglionar de FXYD3 y KRT20. Como objetivo secundario valorar si existe una evolución oncológica diferencial de los pacientes, según la expresión ganglionar de dichas proteínas. Material y método: Se incluyeron ganglios linfáticos de 64 pacientes cistectomizados por tumor vesical infiltrante. El modelo se desarrolló a expensas de ganglios metastásicos de 15 pacientes y ganglios de 4 pacientes sin tumor conocido. La expresión génica se midió mediante PCR cuantitativa en tiempo real. Se calculó la expresión mediana mediante q-rtPCR de los ARNm de FXYD3 y KRT20 en el tejido ganglionar. Se continuó con un análisis de curvas ROC, según la función y = 0.1400 + 0.250FXYD3-2.532. Se estableció el punto de corte mediante una curva ROC. Dicha fórmula se aplicó al tejido ganglionar restante; en función del punto de corte antes establecido la muestra quedó clasificada en 4 subgrupos: HE- qrtPCR-, HE- qrtPCR+, HE+ qrtPCR+ y HE+ qrtPCR-. Se procedió a un análisis descriptivo, comparativo y a un análisis de supervivencia libre de progresión metastásica, calculando las curvas de Kaplan y Meyer para los 3 subgrupos establecidos. Los test se consideraron estadísticamente significativos cuando p < 0,05. Resultados: Mediante q-rtPCR se comprobó que había diferencias en la expresión mediana de FXYD3 (p = 0,05) y de KRT20 (p = 0,009) entre el tejido ganglionar de los pacientes con HBP y los pacientes con metástasis adenopáticas. Se asignó como punto de corte de 0,377. La muestra se clasificó en: un 37,5% de los pacientes eran pN0 por HE y pN0 por qrtPCR (-HE -qrtPCR), el 39,1% eran pN0 por HE pero eran metastásicos por qrtPCR (-HE +qrtPCR) y 15 pacientes (23,4%) eran metastásicos por ambas técnicas (+HE +qrtPCR). Las curvas de Kaplan y Meyer mostraron una peor supervivencia libre de progresión metastásica para los pacientes (+HE +qrtPCR) que para el resto de los subgrupos, no observando diferencias significativas entre (-HE +qrtPCR) y (-HE -qrtPCR). Conclusiones: Según nuestros resultados un 39,1% de los pacientes con tumor vesical infiltrante sobreexpresarían los biomarcadores FXYD3 y KRT20, siendo N0 por HE. No observamos un comportamiento clínico diferencial de los pacientes cistectomizados según su expresión de FXYD3 y KRT20 cuando son N0 por HE

Introduction: Recent studies have proposed that FXYD3 and KRT20 mRNA quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in paraffin could be biomarkers to detect lymph nodes with micrometastases that avoid detection by conventional analysis with hematoxylin-eosin (HE). A validation study was conducted on the lymph nodes of patients who underwent radical cystectomy. Objective: To classify the adenopathic state of a sample of patients who underwent cystectomy, based on the lymph node expression of FXYD3 and KRT20. The secondary objective was to assess whether there is a differential oncologic evolution for the patients, depending on the lymph node expression of these proteins. Material and method: The study included lymph nodes from 64 patients who underwent cystectomy for infiltrating bladder tumor: The model was developed using metastatic lymph nodes from 15 patients and lymph nodes from 4 patients with no known tumor. Genetic expression was measured using real-time qRT-PCR. We calculated (using qRT-PCR) the median expression of FXYD3 and KRT20 mRNA in the lymph node tissue. We then analyzed the receiver operating characteristic (ROC) curves, according to the function y = 0.1400 + 0.250FXYD3-2.532. The cutoff was established using an ROC curve. The formula was applied to the remaining lymph node tissue, based on the previously established cutoff. The sample was classified into 4 subgroups: HE- qRT-PCR-, HE- qRT-PCR+, HE+ qRT-PCR+ y HE+, qRT-PCR-. A descriptive, comparative analysis was performed, as well as a metastatic progression-free survival analysis, calculating the Kaplan and Meyer curves for the 3 established subgroups. The test results were considered statistically significant at P < .05. Results: Using qRT-PCR, we verified that there were differences in the median expression of FXYD3 (P = .05) and KRT20 (P = .009) between the lymph node tissues of patients with benign prostate hyperplasia and those of patients with lymph node metastasis. A cutoff was assigned to 0.377. The sample was classified as follows: 37.5% of the patients were pN0 by HE and pN0 by qRT-PCR (-HE -qRT-PCR), 39.1% were pN0 by HE but metastatic by qRT-PCR (-HE +qRT-PCR), and 15 patients (23.4%) were metastatic by both techniques (+HE +qRT-PCR). The Kaplan and Meyer curves showed poorer metastatic progression-free survival for the patients who were +HE and +qRT-PCR than for the other subgroups, with no significant differences between -HE +qRT-PCR and -HE -qRT-PCR. Conclusions: According to our results, 39.1% of the patients with infiltrating vesical tumors overexpressed the FXYD3 and KRT20 biomarkers and were N0 by HE. We observed no differential clinical behavior among the patients who underwent cystectomy according to their expression of FXYD3 and KRT20 when they were N0 by HE

[Update on the diagnosis of PCa in urine. The current role of urine markers]. / Actualización en el diagnóstico del CaP en orina. Papel actual de los marcadores urinarios.

Prostate cancer (PCa) is still a main health issue, in fact it is responsible for 10% of cancer deaths across Europe. The morphology of the prostate gland makes urine an ideal sample, non invasive, for determination both diagnostic and prognostic biomarkers. We use urinary PCA3 levels to indicate a prostate biopsy, and it is the only urinary biomarkers in PCa with FDA approval for clinical use. Many other biomarkers based on the expression of specific genes of PCa are being studied and validated, for instance the fusion gene TMPRSS2-ERG with a commercial kit available, while another approach is to test the expression of a panel of genes. An emerging focus of research, which deserves attention, is miRNAs. Other newer approaches such as epigenetics, proteomics and metabolomics also would be very useful in the future for the development and validation of new biomarkers. In this paper we review the state of the art in the field of urinary biomarkers in PCa.

External validation of FXYD3 and KRT20 as predictive biomarkers for the presence of micrometastasis in muscle invasive bladder cancer lymph nodes.

INTRODUCTION: Recent studies have proposed that FXYD3 and KRT20 mRNA quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in paraffin could be biomarkers to detect lymph nodes with micrometastases that avoid detection by conventional analysis with hematoxylin-eosin (HE). A validation study was conducted on the lymph nodes of patients who underwent radical cystectomy. OBJECTIVE: To classify the adenopathic state of a sample of patients who underwent cystectomy, based on the lymph node expression of FXYD3 and KRT20. The secondary objective was to assess whether there is a differential oncologic evolution for the patients, depending on the lymph node expression of these proteins. MATERIAL AND METHOD: The study included lymph nodes from 64 patients who underwent cystectomy for infiltrating bladder tumor: The model was developed using metastatic lymph nodes from 15 patients and lymph nodes from 4 patients with no known tumor. Genetic expression was measured using real-time qRT-PCR. We calculated (using qRT-PCR) the median expression of FXYD3 and KRT20 mRNA in the lymph node tissue. We then analyzed the receiver operating characteristic (ROC) curves, according to the function y=0.1400+0.250FXYD3-2.532. The cutoff was established using an ROC curve. The formula was applied to the remaining lymph node tissue, based on the previously established cutoff. The sample was classified into 4 subgroups: HE- qRT-PCR-, HE- qRT-PCR+, HE+ qRT-PCR+ y HE+, qRT-PCR-. A descriptive, comparative analysis was performed, as well as a metastatic progression-free survival analysis, calculating the Kaplan and Meyer curves for the 3 established subgroups. The test results were considered statistically significant at P<.05. RESULTS: Using qRT-PCR, we verified that there were differences in the median expression of FXYD3 (P=.05) and KRT20 (P=.009) between the lymph node tissues of patients with benign prostate hyperplasia and those of patients with lymph node metastasis. A cutoff was assigned to 0.377. The sample was classified as follows: 37.5% of the patients were pN0 by HE and pN0 by qRT-PCR (-HE -qRT-PCR), 39.1% were pN0 by HE but metastatic by qRT-PCR (-HE +qRT-PCR), and 15 patients (23.4%) were metastatic by both techniques (+HE +qRT-PCR). The Kaplan and Meyer curves showed poorer metastatic progression-free survival for the patients who were +HE and +qRT-PCR than for the other subgroups, with no significant differences between -HE +qRT-PCR and -HE -qRT-PCR. CONCLUSIONS: According to our results, 39.1% of the patients with infiltrating vesical tumors overexpressed the FXYD3 and KRT20 biomarkers and were N0 by HE. We observed no differential clinical behavior among the patients who underwent cystectomy according to their expression of FXYD3 and KRT20 when they were N0 by HE.

Actualización en el diagnóstico del CaP en orina. Papel actual de los marcadores urinarios / Update on the diagnosis of PCa in urine. The current role of urine markers

El cáncer de próstata (CaP) es un problema sanitario de primer orden, de lo que da idea el hecho de que es responsable del 10% de muertes por cáncer en Europa. La naturaleza y anatomía de la glándula prostática hace que la orina sea una muestra ideal y no invasiva para la determinación de biomarcadores tanto diagnósticos como pronósticos. Como punta de lanza de la nueva generación de biomarcadores de CaP tenemos los niveles urinarios de PCA3 usados para la indicación de biopsias prostáticas y el único de los biomarcadores urinarios que cuenta con aprobación de la FDA para su uso clínico. Muchos otros marcadores basados en la expresión de genes específicos del CaP están siendo estudiados y validados tanto en solitario como formando parte de paneles, tal es el caso del gen de fusión TMPRSS2-ERG que cuenta con un kit comercial. También es de resaltar la importancia del estudio de los miARNs como campo emergente. Otros enfoques más novedosos como la epigenética, proteómica y metabolómica también pueden en un futuro cercano ser claves en el descubrimiento, desarrollo y validación de nuevos biomarcadores útiles. En este trabajo se revisa el estado actual del desarrollo de todos estos marcadores biológicos

Prostate cancer (PCa) is still a main health issue, in fact it is responsible for 10% of cancer deaths across Europe. The morphology of the prostate gland makes urine an ideal sample, non invasive, for determination both diagnostic and prognostic biomarkers. We use urinary PCA3 levels to indicate a prostate biopsy, and it is the only urinary biomarkers in PCa with FDA approval for clinical use. Many other biomarkers based on the expression of specific genes of PCa are being studied and validated, for instance the fusion gene TMPRSS2-ERG with a commercial kit available, while another approach is to test the expression of a panel of genes. An emerging focus of research, which deserves attention, is miRNAs. Other newer approaches such as epigenetics, proteomics and metabolomics also would be very useful in the future for the development and validation of new biomarkers. In this paper we review the state of the art in the field of urinary biomarkers in PCa

Optimización de un programa de cribado oportunista de cáncer de próstata; ensayo aleatorizado prospectivo del papel del PSA y del PCA3 en uso secuencial / Optimizing prostate cancer screening; prospective randomized controlled study of the role of PSA and PCA3 testing in a sequential manner in an opportunistic screening program

Objetivos: Reducir el número de biopsias (Bx) innecesarias en un programa de cribado oportunista en cáncer de próstata (CaP). Material y métodos: Estudio prospectivo y aleatorizado evaluando el PCA3 como biomarcador de segunda línea. De septiembre de 2010 a septiembre de 2012 2.366 hombres con edad en rango 40-74 años, y más de 10 años de expectativa de vida, fueron estudiados mediante PSA y tacto rectal (TR), excluyendo los biopsiados previamente o con infección urinaria reciente. Ante un TR sospechoso y/o PSA > 3 ng/ml se les realizó un PCA3. A todos aquellos con PCA3 ≥ 35 se les realizó una Bx inicial (IBx) -12 cilindros-. Con PCA3 < 35 fueron aleatorizados 1:1 a IBx u observación. Los criterios de rebiopsia (16-18 cilindros) durante el seguimiento fueron un incremento de PSA > 0,5 ng/ml a 6 meses o PSAv > 0,75 ng/ml/año. Resultados: Con un seguimiento medio de 10,1 meses se testó el PCA3 en 321/2.366 hombres (13,57%), 289 en la primera visita y 32 durante el seguimiento. Entre los 110 hombres con PCA3+ (34,3%) se identificó CaP en 43 en IBx (39,1%). En el brazo aleatorizado 110 se observaron y 101 se biopsiaron, encontrando 12 CaP (11,9%), mostrando un reducción en la detección de CaP estadísticamente significativa en esta cohorte (p < 0,001). Las tasas de detección global de CaP fueron de 40,9 y 9,5% para las ramas PCA3+ y PCA3- respectivamente (p < 0,001). AUC para PSA y PCA3 fueron 0,601 y 0,74. Este es un protocolo abierto en este momento, limitado por su seguimiento insuficiente. Conclusiones: El PCA3 como biomarcador de segunda línea en un programa de cribado oportunista podría potencialmente evitar un 65,7% de IBx y 50,1% a 10 meses de seguimiento, dejando de diagnosticar 3,2% de CaP de alto grado

Objectives: To reduce unnecessary biopsies (Bx) in an opportunistic screening programme of prostate cancer. Material and methods: We perform a prospective evaluation of PCA3 as a second line biomarker in an opportunistic screening for prostate cancer (PCa). From September-2010 until September-2012, 2,366 men, aged 40-74 years and with > 10 years life expectancy, were initially screened with PSA/digital rectal examination (DRE). Men with previous Bx or with recent urine infections were excluded. Men with abnormal DRE and/or PSA > 3 ng/ml were submitted for PCA3. All men with PCA3 ≥ 35 underwent an initial biopsy (IBx) -12 cores-. Men with PCA3 < 35 were randomized 1:1 to either IBx or observation. Re-biopsy (16-18 cores) criteria were PSA increase > 0.5 ng/ml at 4-6months or PSAv > 0.75 ng/ml/year. Results: With median follow-up (FU) of 10.1 months, PCA3 was performed in 321/2366 men (13.57%), 289 at first visit and 32 during FU. All 110 PCA3+ men (34.3%) were biopsied and PCa was identified in 43 men in IBx (39.1%). In the randomized arm, 110 were observed and 101 underwent biopsy, finding 12 PCa (11.9%), showing a statistically significant reduction of PCa detection rate in this cohort (P < 0.001). Global PCa detection rates were 40.9% and 9.5% for the PCA3+ and PCA3- branches, respectively (P < 0.001). Area under the curve for PSA and PCA3 were 0.601 and 0.74, respectively. This is an ongoing prospective study limited by its short follow-up period and still limited enrolment. Conclusions: PCA3 as a second line biomarker within an opportunistic dual screening protocol, can potentially avoid 65.7% and 50.1% biopsies at first round and at median FU of 10.1 months, respectively, just missing around 3.2% of high grade PCa

Optimizing prostate cancer screening; prospective randomized controlled study of the role of PSA and PCA3 testing in a sequential manner in an opportunistic screening program.

OBJECTIVES: To reduce unnecessary biopsies (Bx) in an opportunistic screening programme of prostate cancer. MATERIAL AND METHODS: We perform a prospective evaluation of PCA3 as a second line biomarker in an opportunistic screening for prostate cancer (PCa). From September-2010 until September-2012, 2,366 men, aged 40-74 years and with >10 years life expectancy, were initially screened with PSA/digital rectal examination (DRE). Men with previous Bx or with recent urine infections were excluded. Men with abnormal DRE and/or PSA >3 ng/ml were submitted for PCA3. All men with PCA3 ≥ 35 underwent an initial biopsy (IBx) -12cores-. Men with PCA3 < 35 were randomized 1:1 to either IBx or observation. Re-biopsy(16-18 cores) criteria were PSA increase >.5 ng/ml at 4-6 months or PSAv > .75 ng/ml/year. RESULTS: With median follow-up (FU) of 10.1 months, PCA3 was performed in 321/2366 men (13.57%), 289 at first visit and 32 during FU. All 110 PCA3+ men (34.3%) were biopsied and PCa was identified in 43 men in IBx (39.1%). In the randomized arm, 110 were observed and 101 underwent biopsy, finding 12 PCa (11.9%), showing a statistically significant reduction of PCa detection rate in this cohort (P<.001). Global PCa detection rates were 40.9% and 9.5% for the PCA3+ and PCA3- branches, respectively (P<.001). Area under the curve for PSA and PCA3 were .601 and .74, respectively. This is an ongoing prospective study limited by its short follow-up period and still limited enrolment. CONCLUSIONS: PCA3 as a second line biomarker within an opportunistic dual screening protocol, can potentially avoid 65.7% and 50.1% biopsies at first round and at median FU of 10.1 months, respectively, just missing around 3.2% of high grade PCa.

Diagnóstico no invasivo del cáncer de próstata: marcadores séricos y en orina / Non-invasive diagnosis of prostate cancer: serum and urine markers

El gran número de biomarcadores que la investigación básica plantea en distintos escenarios clínicos de cáncer de próstata (CaP) exige de la comunidad científica un rigor en su desarrollo molecular y clínico para la selección de aquellos que puedan aportar información diagnóstica o pronóstica a los nomogramas de factores clínico-patológicos establecidos. El CaP necesita por su prevalencia y heterogenicidad un diagnóstico más dirigido, la caracterización de su potencial maligno y la monitorización de sus múltiples tratamientos. En este artículo de revisión pretendemos repasar la reciente incorporación de nuevos biomarcadores séricos y en orina en el manejo clínico de este tumor, haciendo hincapié en aquellos con mayor desarrollo clínico (AU)

The great number of biomarkers basic research is presenting in different clinical scenarios of prostate cancer demands the scientific community rigor in their molecular and clinical development for the selection of those which could supply diagnostic and prognostic information for the established nomograms of clinical-pathological factors. Prostate cancer, due to its prevalence and heterogeneity, needs a more directed diagnosis, characterization of malignant potential and monitoring of its multiple therapies. In this review article we try to go over the recent incorporation of new serum and urine markers in the clinical management of this tumor, emphasizing those with greater clinical development (AU)

miRNAs as biomarkers in prostate cancer

Current prostate cancer (PCa) diagnosis is based in the serum prostate-specific antigen biomarker and digital rectal examination. However, these methods are limited by a low predictive value (24-37 %) and a high risk of mistaken results. During last years, new promising biomarkers such as Prostate Cancer Antigen 3 (PCA-3) and TMPRSS2-ETS fusion genes have been evaluated for their clinical use. However, the search of new biomarkers that could be used for PCa diagnosis and prognosis is still needed. Recent studies have demonstrated that the aberrant expression of microRNAs (miRNAs), small non-coding RNAs that negatively regulate gene expression, is related with the development of several cancers, including PCa. Since miRNAs serve as phenotypic signatures of different cancers, they appear as potential diagnostic, prognostic and therapeutic tools. Here, we review the current knowledge of miRNA expression patterns in PCa and their role in PCa prognosis and therapeutics (AU)