Helmut Klocker

Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy

We report the development of a previously undescribed gold nanoparticle bio-barcode assay probe for the detection of prostate specific antigen (PSA) at 330 fg/mL, automation of the assay, and the results of a clinical pilot study designed to assess the ability of the assay to detect PSA in the serum of 18 men who have undergone radical prostatectomy for prostate cancer. Due to a lack of sensitivity, available PSA immunoassays are often not capable of detecting PSA in the serum of men after radical prostatectomy. This new bio-barcode PSA assay is ≈300 times more sensitive than commercial immunoassays. Significantly, with the barcode assay, every patient in this cohort had a measurable serum PSA level after radical prostatectomy. Patients were separated into categories based on PSA levels as a function of time. One group of patients showed low levels of PSA with no significant increase with time and did not recur. Others showed, at some point postprostatectomy, rising PSA levels. The majority recurred. Therefore, this new ultrasensitive assay points to significant possible outcomes: (i) The ability to tell patients, who have undetectable PSA levels with conventional assays, but detectable and nonrising levels with the barcode assay, that their cancer will not recur. (ii) The ability to assign recurrence earlier because of the ability to measure increasing levels of PSA before conventional tools can make such assignments. (iii) The ability to use PSA levels that are not detectable with conventional assays to follow the response of patients to adjuvant or salvage therapies.

Circulating Tumor-Specific DNA: A Marker for Monitoring Efficacy of Adjuvant Therapy in Cancer Patients

Adjuvant systemic therapy (a strategy that targets potential disseminated tumor cells after complete removal of the tumor) has clearly improved survival of patients with cancer. To date, no tool is available to monitor efficacy of these therapies, unless distant metastases arise, a situation that unavoidably leads to death. We analyzed RASSF1A DNA methylation in pretherapeutic sera and serum samples collected 1 year after surgery from 148 patients with breast cancer who were receiving adjuvant tamoxifen; 19.6% and 22.3% of patients with breast cancer showed RASSF1A DNA methylation in their pretherapeutic and 1-year-after serum samples, respectively. RASSF1A methylation 1 year after primary surgery (and during adjuvant tamoxifen therapy) was an independent predictor of poor outcome, with a relative risk (95% confidence interval) for relapse of 5.1 (1.3-19.8) and for death of 6.9 (1.9-25.9). Measurement of serum DNA methylation allows adjuvant systemic treatment to be monitored for efficacy: disappearance of RASSF1A DNA methylation in serum throughout treatment with tamoxifen indicates a response, whereas persistence or new appearance means resistance to adjuvant tamoxifen treatment. It remains to be seen whether modifications made in adjuvant therapeutic strategies based on detection of circulating nucleic acids will improve survival as well as quality of life.

Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides.

Currently available methods for treatment of human prostatic carcinoma aim to inactivate the androgen receptor (AR) by androgen deprivation or blockade with anti-androgens. Failure of endocrine therapy and tumor progression is characterized by androgen-independent growth despite high levels of AR expression in metastatic disease. We inhibited AR expression in LNCaP prostate tumor cells by using antisense AR oligodeoxynucleotides (ODNs) and explored whether antisense AR treatment would be conceivable as a therapy for advanced prostate cancer. Among the various AR antisense ODNs tested, a 15-base ODN targeting the CAG repeats encoding the poly-glutamine region of the AR (as750/15) was found to be most effective. Treatment of LNCaP cells with as750/15 reduced AR expression to ∼2% within 24 hours compared with mock-treated controls. AR down-regulation resulted in significant cell growth inhibition, strongly reduced secretion of the androgen-regulated prostate-specific antigen, reduction of epidermal growth factor receptor expression, and an increase in apoptotic cells. Mis-sense and mismatched control ODNs had no or only slight effects. Antisense inhibition was also very efficient in LNCaP-abl cells, a subline established after long-term androgen ablation of LNCaP cells, resulting in inhibition of AR expression and cell proliferation that was similar to that seen for parental LNCaP cells. This study shows that inhibition of AR expression by antisense AR ODNs may be a promising new approach for treatment of advanced human prostate cancer.

Prostate-Specific Antigen (PSA) Isoform p2PSA in Combination with Total PSA and Free PSA Improves Diagnostic Accuracy in Prostate Cancer Detection

BACKGROUND Novel markers for prostate cancer (PCa) detection are needed. Total prostate-specific antigen (tPSA) and percent free prostate-specific antigen (%fPSA=tPSA/fPSA) lack diagnostic specificity. OBJECTIVE To evaluate the use of prostate-specific antigen (PSA) isoforms p2PSA and benign prostatic hyperplasia-associated PSA (BPHA). DESIGN, SETTING, AND PARTICIPANTS Our study included 405 serum samples from the Rotterdam arm of the European Randomised Study of Screening for Prostate Cancer and 351 samples from the Urology Department of Innsbruck Medical University. MEASUREMENTS BPHA, tPSA, fPSA, and p2PSA levels were measured by Beckman-Coulter Access Immunoassay. In addition, the Beckman Coulter Prostate Health Index was calculated: phi=(p2PSA/fPSA)×√(tPSA). RESULTS AND LIMITATIONS The p2PSA and phi levels differed significantly between men with and without PCa. No difference in BPHA levels was observed. The highest PCa predictive value in both cohorts was achieved by phi with areas under the curve (AUCs) of 0.750 and 0.709, a significant increase compared to tPSA (AUC: 0.585 and 0.534) and %fPSA (AUC: 0.675 and 0.576). Also, %p2PSA (p2PSA/fPSA) showed significantly higher AUCs compared to tPSA and %fPSA (AUC: 0.716 and 0.695, respectively). At 95% and 90% sensitivity, the specificities of phi were 23% and 31% compared to 10% and 8% for tPSA, respectively. In both cohorts, multivariate analysis showed a significant increase in PCa predictive value after addition of p2PSA to a model consisting of tPSA and fPSA (increase in AUC from 0.675 to 0.755 and from 0.581 to 0.697, respectively). Additionally, the specificity at 95% sensitivity increased from 8% to 24% and 7% to 23%, respectively. Furthermore, %p2PSA, phi, and the model consisting of tPSA and fPSA with or without the addition of p2PSA missed the least of the tumours with a biopsy or pathologic Gleason score ≥7 at 95% and 90% sensitivity. CONCLUSIONS This study shows significant increases in PCa predictive value and specificity of phi and %p2PSA compared to tPSA and %fPSA. p2PSA has limited additional value in identifying aggressive PCa (Gleason score ≥7).

Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells.

We investigated the effects of androgen receptor (AR) down regulation with a small interference RNA molecule (siRNA_AR(start)) on androgen sensitive LNCaP and androgen independent LNCaPabl prostate cancer cells, the latter representing an in vitro model for the development of therapy resistance in prostate cancer. Although LNCaPabl cells express increased levels of AR in comparison with androgen sensitive LNCaP cells, the protein was significantly down regulated in response to siRNA_AR(start) treatment. This AR down regulation resulted in a marked cell growth inhibition in both cell lines. By contrast, DU-145 prostate cancer cells, which lack AR expression, were not inhibited by the siRNA_AR(start). In consequence to AR down regulation, both cell lines, LNCaP and LNCaPabl, shared a highly similar gene expression profile in terms of major changes in cell cycle regulatory genes. The cell cycle inhibitor p21(Waf1/Cip1) as well as cyclin D1 were significantly up regulated by siRNA_AR(start) treatment, considering a switch in cyclin expression towards cell cycle retardation. Control molecules had moderate effects on cell proliferation and gene expression, respectively. In summary, we found that AR inhibition with siRNA induces cell growth retardation in androgen sensitive as well as in androgen independent prostate cancer cells and thus may represent an interesting approach to combat hormone-refractory prostate cancer.

Epithelial-to-Mesenchymal Transition Leads to Docetaxel Resistance in Prostate Cancer and Is Mediated by Reduced Expression of miR-200c and miR-205

Docetaxel is a standard chemotherapy for patients with metastatic prostate cancer. However, the response is rather limited and not all of the patients benefit from this treatment. To uncover key mechanisms of docetaxel insensitivity in prostate cancer, we have established docetaxel-resistant sublines. In this study, we report that docetaxel-resistant cells underwent an epithelial-to-mesenchymal transition during the selection process, leading to diminished E-cadherin levels and up-regulation of mesenchymal markers. Screening for key regulators of an epithelial phenotype revealed a significantly reduced expression of microRNA (miR)-200c and miR-205 in docetaxel-resistant cells. Transfection of either microRNA (miRNA) resulted in re-expression of E-cadherin. Functional assays confirmed reduced adhesive and increased invasive and migratory abilities. Furthermore, we detected an increased subpopulation with stem cell-like properties in resistant cells. Tissue microarray analysis revealed a reduced E-cadherin expression in tumors after neoadjuvant chemotherapy. Low E-cadherin levels could be linked to tumor relapse. The present study uncovers epithelial-to-mesenchymal transition as a hallmark of docetaxel resistance. Therefore, we suggest that this mechanism is at least in part responsible for chemotherapy failure, with implications for the development of novel therapeutics.

Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity

BackgroundMassively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings.MethodsUsing identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity.ResultsWe show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance.Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations.ConclusionsThe application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.

Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor

We have shown recently that a 15-mer phosphorothioate oligodeoxynucleotide (ODNas750/15) that hybridizes to the (CAG)n polyglutamine region of mRNA encoding human androgen receptor (AR) inhibits the expression of AR in LNCaP prostate cancer cells in vitro. This AR downregulation was accompanied by significant cell growth inhibition and reduced PSA secretion. In the present study we investigated the effects of this antisense AR ODN on prostate tumor growth in vivo using a mouse xenograft model. Via subcutaneously implanted diffusion pumps, either ODNas750/15 or a scrambled control sequence ODNsr750/15 was continuously administered into LNCaP tumor–bearing male nude mice for 7 weeks. Compared with untreated control animals, treatment with ODNas750/15 resulted in significant tumor growth inhibition. Retardation of tumor growth was also significant in castrated mice, whereas the scrambled control ODN did not exert any effects. No side effects such as loss of body weight were observed at any time of treatment. ODN treatment was well tolerated and, in contrast to castration, did not induce shrinkage of mouse prostates. Both AR expression in the tumor and PSA levels in mouse serum correlated with tumor size. However, we failed to demonstrate a correlation between tumor retardation and Ki-67 antigen expression and the number of apoptotic cells, respectively. Testing of antisense-treated LNCaP cells revealed that expression levels of other proteins that contain shorter polyglutamine sequence stretches such as HDAC2, TFIID, and c-jun were not affected. The present study demonstrates that downregulation of AR with antisense ODNas750/15 causes prostate tumor growth inhibition. These results further point out the important role of the AR in prostate tumors and support further testing of AR downregulation for treatment of prostate cancer.

Type IV collagenase (matrix metalloproteinase-2 and -9) in prostate cancer

Background: The type IV collagenases/gelatinases matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) play an important role in cancer invasion and metastasis. In the present study, we measured the expression of mRNAs and enzymatic activities of MMP-9 and -2 in prostate tissues and serum samples from men with or without prostate cancer.Methods: A total of 44 tissue samples (three from healthy volunteers, 21 from patients with benign prostate hyperplasia, 10 from patients with localized prostate cancer and 10 from patients with metastatic disease) and 71 serum samples were collected (20 from healthy volunteers, 26 from patients with benign prostatic hyperplasia, 10 from patients with localized cancer, 15 from patients with metastatic cancer). The level of mRNA for MMP-2 and -9 was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The enzymatic activity of MMPs was determined by zymography.Results: Expression of MMP-9 mRNA was significantly higher in malignant than in nonmalignant prostate tissues (P<0.001), while no significant difference of MMP-2 expression was detected in different prostate tissues. Results of zymography showed that there was significant difference in the enzymatic activity of MMP-9, but not MMP-2, among normal prostate, BPH, localized and metastatic prostate cancer tissues, serum samples (P<0.05). The active form of MMP-2, with a molecular mass of 62 kDa, was detected in normal prostate, BPH and prostate cancer tissues, but not in the serum samples. Moreover, there was a significant difference in the ratio of the active form (62 kDa) and proform (72 kDa) of MMP-2 among normal, BPH and prostate cancer tissues. This ratio was further increased in metastatic prostate cancer tissues.Conclusion: The activity of MMP-9 and the ratio of active form/proform of MMP-2 are associated with the progression and metastasis of prostate cancer.

Expression and prognostic relevance of annexin A3 in prostate cancer.

OBJECTIVES By differential quantitative protein expression, it has previously been shown that annexin A3 (ANXA3) expression is associated with prostate cancer. However little is known about the role and biology of ANXA3 in the human prostate. The aim of this study was to thoroughly analyze ANXA3 expression patterns and its potential as a prognostic marker in a large set of benign, preneoplastic, and neoplastic prostate tissue samples. METHODS Immunohistochemistry-based ANXA3 protein expression was analyzed for 1589 prostate cancers as well as smaller subsets of benign epithelium and high-grade prostatic intraepithelial neoplasia (PIN) in a tissue microarray format. RESULTS All samples of benign prostatic epithelium and PIN showed ANXA3 protein expression, with PIN lesions showing a decreased staining intensity compared with benign epithelium (p<0.0001). In cancer, ANXA3 protein expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT stage and Gleason score (p<0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor and enabled substratification of the large group of intermediate-risk patients (n=969) into high- and low-risk subgroups. CONCLUSIONS ANXA3 represents a promising candidate tissue marker, and when combined with the standard prognostic parameters, is suggested to provide a more precise prediction of prognosis in the individual patient, therefore harboring the potential to contribute to future patient management.