Daphne Hessels

DD3PCA3-based molecular urine analysis for the diagnosis of prostate cancer

BACKGROUND: DD3(PCA3) is the most prostate cancer-specific gene described to date. To assess the clinical utility of DD3(PCA3) a time-resolved fluorescence-based, quantitative RT-PCR analysis for DD3(PCA3) was developed. METHODS: The diagnostic potential of DD3(PCA3) was determined by quantitative measurement of DD3(PCA3) transcripts in non-malignant and malignant prostate specimens. Moreover, DD3(PCA3) transcripts were determined quantitatively in urine sediments obtained after prostatic massage. A cohort of 108 men, admitted for prostate biopsies based on a PSA of >3ng/ml, was studied. RESULTS: Prostate tumors showed a 66-fold up-regulation of DD3(PCA3) (median 158.4.10(5) copies/microg tissue RNA) when compared to benign prostate tissue (median 2.4.10(5) copies/microg tissue RNA). This up-regulation was found in more than 95% of prostate cancer specimens studied. These data revealed that specimens with less than 10% of cancer cells could be accurately discriminated from non-cancer tissues. Hence, detection of a small fraction of prostate cancer cells in a background of normal cells seemed feasible. Therefore, this DD3(PCA3)-based RT-PCR assay was used for the identification of prostate cancer in urine sediments obtained after prostatic massage. From 108 men with a serum PSA value >3ng/ml, 24 men were shown to have prostate cancer upon biopsy. Of these 24 men, 16 were shown to be positive for DD3(PCA3), indicating a sensitivity of the assay of 67%. Furthermore, a negative predictive value of 90% was calculated. CONCLUSION: The quantitative RT-PCR assay for DD3(PCA3) described, bears great promise as a tool for molecular urine analysis. It has great potential in reducing the number of unnecessary biopsies. A multi-center study using this DD3(PCA3) assay can provide the basis for the utility of molecular diagnostics in clinical urological practice.

Detection of TMPRSS2-ERG Fusion Transcripts and Prostate Cancer Antigen 3 in Urinary Sediments May Improve Diagnosis of Prostate Cancer

Purpose: Early detection of prostate cancer can increase the curative success rate for prostate cancer. We studied the diagnostic usefulness of TMPRSS2-ERG fusion transcripts as well as the combination of prostate cancer antigen 3 (PCA3) RNA and TMPRSS2-ERG fusion transcripts in urinary sediments after digital rectal examination (DRE). Experimental Design: A total of 78 men with prostate cancer–positive biopsies and 30 men with prostate cancer–negative biopsies were included in this study. After DRE, the first voided urine was collected, and urinary sediments were obtained. We used semiquantitative reverse transcription-PCR (RT-PCR) analysis followed by Southern blot hybridization with a radiolabeled probe for the detection TMPRSS2-ERG fusion transcripts in these urinary sediments. A quantitative RT-PCR assay for PCA3 was used to determine the PCA3 score in the same sediments. Results: TMPRSS2-ERG fusion transcripts can be detected in the urine after DRE with a sensitivity of 37%. In this cohort of patients, the PCA3-based assay had a sensitivity of 62%. When both markers were combined, the sensitivity increased to 73%. Especially in the cohort of men with persistently elevated serum prostate-specific antigen levels and history of negative biopsies, the high positive predictive value of 94% of TMPRSS2-ERG fusion transcripts could give a better indication which patients require repeat biopsies. Conclusion: In this report, we used for the first time the combination of the prostate cancer–specific biomarkers TMPRSS2-ERG and PCA3, which significantly improves the sensitivity for prostate cancer diagnosis.

Prospective Multicentre Evaluation of PCA3 and TMPRSS2-ERG Gene Fusions as Diagnostic and Prognostic Urinary Biomarkers for Prostate Cancer ☆

BACKGROUND Prostate cancer antigen 3 (PCA3) and v-ets erythroblastosis virus E26 oncogene homolog (TMPRSS2-ERG) gene fusions are promising prostate cancer (PCa) specific biomarkers that can be measured in urine. OBJECTIVE To evaluate the diagnostic and prognostic value of Progensa PCA3 and TMPRSS2-ERG gene fusions (as individual biomarkers and as a panel) for PCa in a prospective multicentre setting. DESIGN, SETTING, AND PARTICIPANTS At six centres, post-digital rectal examination first-catch urine specimens prior to prostate biopsies were prospectively collected from 497 men. We assessed the predictive value of Progensa PCA3 and TMPRSS2-ERG (quantitative nucleic acid amplification assay to detect TMPRSS2-ERG messenger RNA [mRNA]) for PCa, Gleason score, clinical tumour stage, and PCa significance (individually and as a marker panel). This was compared with serum prostate-specific antigen and the European Randomised Study of Screening for Prostate Cancer (ERSPC) risk calculator. In a subgroup (n=61) we evaluated biomarker association with prostatectomy outcome. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Univariate and multivariate logistic regression analysis and receiver operating curves were used. RESULTS AND LIMITATIONS Urine samples of 443 men contained sufficient mRNA for marker analysis. PCa was diagnosed in 196 of 443 men. Both PCA3 and TMPRSS2-ERG had significant additional predictive value to the ERSPC risk calculator parameters in multivariate analysis (p<0.001 and resp. p=0.002). The area under the curve (AUC) increased from 0.799 (ERSPC risk calculator), to 0.833 (ERSPC risk calculator plus PCA3), to 0.842 (ERSPC risk calculator plus PCA3 plus TMPRSS2-ERG) to predict PCa. Sensitivity of PCA3 increased from 68% to 76% when combined with TMPRSS2-ERG. TMPRSS2-ERG added significant predictive value to the ERSPC risk calculator to predict biopsy Gleason score (p<0.001) and clinical tumour stage (p=0.023), whereas PCA3 did not. CONCLUSIONS TMPRSS2-ERG had independent additional predictive value to PCA3 and the ERSPC risk calculator parameters for predicting PCa. TMPRSS2-ERG had prognostic value, whereas PCA3 did not. Implementing the novel urinary biomarker panel PCA3 and TMPRSS2-ERG into clinical practice would lead to a considerable reduction of the number of prostate biopsies.

The time-resolved fluorescence-based PCA3 test on urinary sediments after digital rectal examination; a Dutch multicenter validation of the diagnostic performance.

Purpose: To improve the specificity in prostate cancer diagnosis and to prevent unnecessary prostate biopsies, especially in the serum prostate-specific antigen (PSA) “gray zone” between 3 and 15 ng/mL, the implementation of prostate cancer–specific markers is urgently needed. The recently discovered prostate cancer antigen 3 (PCA3) is such a promising prostate cancer marker. In a previous single institution study, the PCA3 urine test clearly proved to be of diagnostic value. Therefore, the diagnostic performance of the PCA3 urine test was validated in a multicenter study. Experimental Design: The first voided urine after digital rectal examination was collected from a total of 583 men with serum PSA levels between 3 and 15 ng/mL who were to undergo prostate biopsies. We determined the PCA3 score in these samples and correlated the results with the results of the prostate biopsies. Results: A total of 534 men (92%) had an informative sample. The area under the receiver-operating characteristic curve, a measure of the diagnostic accuracy of a test, was 0.66 for the PCA3 urine test and 0.57 for serum PSA. The sensitivity for the PCA3 urine test was 65%, the specificity was 66% (versus 47% for serum PSA), and the negative predictive value was 80%. Conclusions: In this multicenter study, we validated the diagnostic performance of the PCA3 urine test in the largest group studied thus far using a PCA3 gene-based test. This study shows that the PCA3 urine test, when used as a reflex test, can improve the specificity in prostate cancer diagnosis and could prevent many unnecessary prostate biopsies.

Predictive value of PCA3 in urinary sediments in determining clinico-pathological characteristics of prostate cancer.

PCA3 urine tests have shown to improve the specificity in prostate cancer (PCa) diagnosis, and have thus the potential to reduce the number of unnecessary prostate biopsies and to predict repeat biopsy outcomes. In this study, PCA3 was correlated with clinical stage, biopsy Gleason score (GS), radical prostatectomy GS, tumor volume, and pathological stage to assess its potential as predictor of PCa aggressiveness.

New targets for therapy in prostate cancer: differential display code 3 (DD3PCA3), a highly prostate cancer–specific gene

Identification of new markers for diagnosis and new targets for therapy would represent a considerable advance in the treatment of prostate cancer. Differential display code 3 (DD3(PCA3)) is a novel gene with characteristics that indicate its potentially valuable role in early identification of malignancy and in the construction of interventions directed specifically toward malignantly transformed cells. DD3(PCA3) has a messenger RNA product that is highly overexpressed in tumors. Compared with other genetic markers that are associated with prostate tissue, DD3(PCA3) is the most specific marker for malignant disease. Indeed, it is not expressed in any other normal human tissue, including breast, bladder, testis, gastrointestinal organ, and musculoskeletal tissue. This specific relation of DD3(PCA3) to prostate tissue has been confirmed by reverse transcription-polymerase chain reaction analysis. Clonal investigation of the DD3(PCA3) transcription unit indicates that the gene has 4 distinct exons, which can give rise to a number of differently sized transcripts. Open reading frame analysis has also confirmed that the DD3(PCA3) exons are populated by an unusual number of stop codons. The dramatic prostate-specific expression and pronounced upregulation of DD3(PCA3) in prostate cancer suggest a unique transcriptional regulation. A quantitative assay for DD3(PCA3) would be a potentially valuable tool for the detection of malignant cells in blood, urine, or other clinical specimens, and it could have important implications for the earlier diagnosis and molecular staging of prostate cancer. Although further studies are needed, gene therapies based on identification to delineate the range of transcription factors that interact with the DD3(PCA3) promoter represent a promising area for preclinical investigation.

Detailed analysis of histopathological parameters in radical prostatectomy specimens and PCA3 urine test results

Due to the drawbacks of serum prostate‐specific antigen, there is an ongoing search for new diagnostic and prognostic prostate cancer (PCa) markers. PCA3 has proven to be of value in the diagnosis of PCa. However, so far few attempts have been made to investigate the prognostic value of PCA3. Our objective was to further investigate the prognostic value of PCA3.

Detection of High-grade Prostate Cancer Using a Urinary Molecular Biomarker-Based Risk Score.

BACKGROUND To reduce overdiagnosis and overtreatment, a test is urgently needed to detect clinically significant prostate cancer (PCa). OBJECTIVE To develop a multimodal model, incorporating previously identified messenger RNA (mRNA) biomarkers and traditional risk factors that could be used to identify patients with high-grade PCa (Gleason score ≥7) on prostate biopsy. DESIGN, SETTING, AND PARTICIPANTS In two prospective multicenter studies, urine was collected for mRNA profiling after digital rectal examination (DRE) and prior to prostate biopsy. The multimodal risk score was developed on a first cohort (n=519) and subsequently validated clinically in an independent cohort (n=386). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The mRNA levels were measured using reverse transcription quantitative polymerase chain reaction. Logistic regression was used to model patient risk and combine risk factors. Models were compared using the area under the curve (AUC) of the receiver operating characteristic, and clinical utility was evaluated with a decision curve analysis (DCA). RESULTS AND LIMITATIONS HOXC6 and DLX1 mRNA levels were shown to be good predictors for the detection of high-grade PCa. The multimodal approach reached an overall AUC of 0.90 (95% confidence interval [CI], 0.85-0.95) in the validation cohort (AUC 0.86 in the training cohort), with the mRNA signature, prostate-specific antigen (PSA) density, and previous cancer-negative prostate biopsies as the strongest, most significant components, in addition to nonsignificant model contributions of PSA, age, and family history. For another model, which included DRE as an additional risk factor, an AUC of 0.86 (95% CI, 0.80-0.92) was obtained (AUC 0.90 in the training cohort). Both models were successfully validated, with no significant change in AUC in the validation cohort, and DCA indicated a strong net benefit and the best reduction in unnecessary biopsies compared with other clinical decision-making tools, such as the Prostate Cancer Prevention Trial risk calculator and the PCA3 assay. CONCLUSIONS The risk score based on the mRNA liquid biopsy assay combined with traditional clinical risk factors identified men at risk of harboring high-grade PCa and resulted in a better patient risk stratification compared with current methods in clinical practice. Therefore, the risk score could reduce the number of unnecessary prostate biopsies. PATIENT SUMMARY This study evaluated a novel urine-based assay that could be used as a noninvasive diagnostic aid for high-grade prostate cancer (PCa). When results of this assay are combined with traditional clinical risk factors, risk stratification for high-grade PCa and biopsy decision making are improved.

Applicability of biomarkers in the early diagnosis of prostate cancer

Early diagnosis of prostate cancer can increase the curative success rate for this disease. Although serum prostate-specific antigen measurement is regarded as the best conventional tumor marker available, there is little doubt that it has great limitations. The threshold above which biopsies are indicated has now decreased to a serum prostate-specific antigen value of 3 ng/ml, which results in a negative biopsy rate of 70–80%. This can readily be explained by the fact that prostate-specific antigen is not specific for prostate cancer. Clinicians need more sensitive tools to help diagnose prostate cancer and monitor progression of the disease. Molecular oncology is playing an increasing role in the fields of diagnosis and therapy for prostate cancer and has already been instrumental in elucidating many of the basic mechanisms underlying the development and progression of this disease. The identification of new prostate cancer-specific genes, such as DD3PCA3, would represent a considerable advance in the improvement of diagnostic tests for prostate cancer. This could subsequently lead to a reduction of the number of unnecessary biopsies.

Identification of a Candidate Gene Panel for the Early Diagnosis of Prostate Cancer

Purpose: Serum PSA (sPSA) testing has led to the identification of patients with indolent prostate cancer, and inevitably overtreatment has become a concern. Progensa PCA3 urine testing was shown to improve the diagnosis of prostate cancer, but its diagnostic value for aggressive prostate cancer is limited. Therefore, urinary biomarkers that can be used for prediction of Gleason score ≥7 prostate cancer in biopsies are urgently needed. Experimental Design: Using gene expression profiling data, 39 prostate cancer biomarkers were identified. After quantitative PCR analysis on tissue specimens and urinary sediments, eight promising biomarkers for the urinary detection of prostate cancer were selected (ONECUT2, HOXC4, HOXC6, DLX1, TDRD1, NKAIN1, MS4A8B, PPFIA2). The hypothesis that biomarker combinations improve the diagnostic value for aggressive prostate cancer was tested on 358 urinary sediments of an intention-to-treat cohort. Results: A urinary three-gene panel (HOXC6, TDRD1, and DLX1) had higher accuracy [area under the curve (AUC), 0.77; 95% confidence interval (CI), 0.71–0.83] to predict Gleason score ≥7 prostate cancer in biopsies compared with Progensa PCA3 (AUC, 0.68; 95% CI, 0.62–0.75) or sPSA (AUC, 0.72; 95% CI, 0.65–0.78). Combining the three-gene panel with sPSA further improved the predictive accuracy (AUC, 0.81; 95% CI, 0.75–0.86). The accuracy of the three-gene predictive model was maintained in subgroups with low sPSA concentrations. Conclusions: The urinary three-gene panel (HOXC6, TDRD1, and DLX1) represents a promising tool to identify patients with aggressive prostate cancer, also in those with low sPSA values. The combination of the urinary three-gene panel with sPSA bears great potential for the early diagnosis of patients with clinically significant prostate cancer. Clin Cancer Res; 21(13); 3061–70. ©2015 AACR.