PCA3 as a second-line biomarker in a prospective controlled randomized opportunistic prostate cancer screening programme. / PCA3 como biomarcador de segunda línea en un programa de screening oportunista prospectivo, aleatorizado y controlado.

OBJECTIVES: PCA3 performance as a single second line biomarker is compared to the European Randomised Study of Screening for Prostate Cancer risk calculator model 3 (ERSPC RC-3) in an opportunistic screening in prostate cancer (PCa). MATERIAL AND METHODS: 5,199 men, aged 40-75y, underwent prostate-specific antigen (PSA) screening and digital rectal examination (DRE). Men with a normal DRE and PSA ≥3ng/ml had a PCA3 test done. 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. We compared them to those obtained with ERSPC RC-3. RESULTS: PCA3 test was performed on 838 men (16.1%). In PCA3(+) and PCA3(-) groups, global PCa detection rates were 40.9% and 14.7% with a median follow-up (FU) of 21.7 months (P<.001). In the PCA3(+) arm (n=301, 35.9%), PCa was identified in 115 men at IBx (38.2%). In the randomized arm, 256 underwent IBx and PCa was found in 46 (18.0%) (P<.001). The biopsy-sparing potential would have been 64.1% as opposed to 76.6% if we had used ERSPC RC-3. However, the estimated false negative cases for HGPCa would have been reduced by 37.1% (89 to 56 patients). Moreover, if we had applied PCA3-35 to avoid IBx, 14.7% PCa and 9.1% of clinical significant PCa patients would not have been diagnosed during this FU. CONCLUSIONS: When PCA3-35 is used as a second-line biomarker when PSA ≥3ng/ml and DRE is normal, IBx could be avoided in 12.5% less than if ERSPC RC-3 is used and would reduce the false negative cases by 36.2%. At a FU of 21.7 months, this dual protocol would miss 9.1% of clinically significant PCa, so strict FU is mandatory with established biopsy criteria based on PSA and DRE in cases with PCA3 <35.

[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.

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.

Molecular characterization and clinical impact of TMPRSS2-ERG rearrangement on prostate cancer: comparison between FISH and RT-PCR.

Prostate cancer (PCa) is a very heterogeneous disease, and there are constraints in its current diagnosis. Serum PSA levels, digital rectal examination (DRE), and histopathologic analysis often drive to overdiagnosis and overtreatment. Since 2005, the presence of the genetic rearrangement between transmembrane-serine protease gene (TMPRSS2) and the erythroblast transformation-specific (ETS) member ERG (v-ets erythroblastosis virus E26 oncogene homolog avian) has been demonstrated in almost half of PCa cases. Both FISH and RT-PCR are useful tools for detecting these rearrangements, but very few comparatives between both techniques have been published. In this study, we included FFPE tumors from 294 PCa patients treated with radical prostatectomy with more than 5 years of followup. We constructed a total of 20 tissue microarrays in order to perform break-apart and tricolor probe FISH approaches that were compared with RT-PCR, showing a concordance of 80.6% (P < 0.001). The presence of TMPRSS2-ERG rearrangement was observed in 56.6% of cases. No association between TMPRSS2-ERG status and clinicopathological parameters nor biochemical progression and clinical progression free survival was found. In conclusion, this study demonstrates that both FISH and RT-PCR are useful tools in the assessment of the TMPRSS2-ERG fusion gene status in PCa patients and that this genetic feature per se lacks prognostic value.

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

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.

[Outcomes of expanded use of PCA3 testing in a Spanish population with clinical suspicion of prostate cancer]. / Resultados del uso expandido del PCA3 score en una población española con sospecha de cáncer de próstata.

OBJECTIVES: DD3(PCA3) (PCA3) gene expression is prostate cancer-specific. Routine use of this biomarker has resulted in a 35-67% reduction in the number of required biopsies. The aim of this study is to evaluate our outcomes in its routine use and to establish in which group of patients this is the most efficient, depending on the number of previous PCA3 biopsies. MATERIAL AND METHODS: A total of 474 consecutive patients who had previously undergone a biopsy (group A, n=337) or not (group B, n=134) for whom a PCA3 was requested were analyzed. We subdivided group A into A(1) (a previous biopsy, n=182) and A(2) (<1 previous biopsy, n=155). The recommendation of whether to perform a biopsy or not was made independently by each of the 11 clinicians and guided by prostatic specific antigen (PSA) levels and digital rectal examination. RESULTS: Median age was 65 years (range 38 to 84). PCA3 score had an informative ratio of 99.6%, with a median of 29 (range 1-3245). The percentage of biopsy sparing was 49% of the cases. ROC analysis demonstrated an AUC for PSA and PCA3 of 0.532 (P=.417) and 0.672 (P<.0001), respectively. Sensitivities of PSA≥ 4 and PCA3≥ 35 were 87% vs. 85%, with specificities of 12% vs. 33%, PPV 34% vs. 39% and NPV 63% vs. 81%, respectively. The PCA3 score showed direct correlation with the percentage of positive biopsies (P<.0001). CONCLUSIONS: Routine use of PCA3, due to its high NPV, results in a significant reduction in the number of biopsies. PCA3 appears to be more efficient in biopsy-naive patients. Among patients already biopsied, the results are superior in those biopsied only once.

[Prostate cancer: the revolution of the fusion genes]. / Cáncer de próstata: la revolución de los genes de fusión.

BACKGROUND: TMPRSS2-ETS fusion gene rearrangements constitute a very common and specific alteration in prostate cancer cells. These genetic alterations lead the overexpression of ETS genes which encode the E26 family of transcription factors involved in cell proliferation. Of this family, the ERG oncogene is overexpressed in almost 50% of prostate cancer cases. EVIDENCE SYNTHESIS: TMPRSS2-ERG overexpresses ERG through an androgen-mediated response. Structurally, the rearrangement is due to interstitial deletion and to a lesser extent to reciprocal translocation and plays a key role in cellular metabolism. Almost all fusion gene transcripts produce a truncated ERG protein and the presence of a specific isoform of this gene suggests the clonality of the tumor; hence, metastasis shares the fusion gene status of their primary lesion. Although the prognostic implications of TMPRSS2-ERG have not been fully elucidated, they constitutes a field of great diagnostic potential and, therefore, the development of techniques to identify and to analyze the presence and characteristics of this gene in a non-invasive fashion deserves great interest in this area. Currently, there is evidence supporting the hypothesis that the presence of fusion gene differentiates two molecular groups within prostate cancer with a differential behaviour making the fusion gene a potential therapeutic target. In this regard, the use of anti-HDAC (trichostatin), antagonists of estrogen receptor alpha and abiraterone acetate have shown promising results. CONCLUSIONS: This review describes the great potential offered by the investigation of fusion genes in PC and the need for further studies.

[Immunohistochemical expression of microvascular density and carbonic anhidrase IX in renal carcinoma. Relation to histological type and tumoral progression]. / Expresión inmunohistoquímica de la densidad microvascular y de la anhidrasa carbónica IX en carcinoma renal. Relación con el tipo histológico y con la progresión tumoral.

PURPOSE: to correlate the immunohistochemical expression of microvascular density (MVD) and the carbonic anhydrase IX (CAIX) with the different histological subtypes of renal carcinoma and its progression. MATERIAL AND METHODS: we studied 93 patients with renal cell carcinoma operated between 1990 and 2008. Antibodies employed for immunohistochemistry (IHC); CD31 (1: 40, Dako) and CD34 (1: 50, Dako) for MVD and CAIX (1: 100, Santa Cruz). CAIX was validated semiquantitatively as: strongly positive (>85%); weakly positive (10% -85%); and negative (< 10%), independently of the intensity of the stain. MVD was validated with both anti-CD31 and anti-CD34 by means of a whole section, to select the microscopic field (x100) with highest density of stained vessels, counting the number of vessels in a photographic field of 0.53 mm(2). Results are expressed as the maximal number of vessels by mm(2) of tumour tissue. RESULTS: median follow up was 40 months (1-160). We found no differences of expression with any of the 3 IHC markers between tumours that progressed (49) and tumours that did not progress (44). The IHC expression of CAIX was strongly related to MVD, measured for both CD31 and CD34 (p<0.0001). MVD with both antibodies was inversely related to tumour size and Fuhrman grade and was also stronger in clear cell carcinomas compared to the rest of histological subtypes, measured by CD31 (p = 0.001) and CD34 (p = 0.003). CONCLUSIONS: neither MVD nor CAIX expressions were related to tumour progression, but were related to histological subtypes. This fact, added to their co-expression, could prompt the use of the CAIX expression, which is far more reproducible, as a quick and easy approximation to MVD. More research should be done to use it as marker for targeted therapy.