A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers

Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation—the area under the curve was 0.84 with a p value below 10⁻⁶. Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair.     

Prostate cancer: risk assessment and diagnostic approaches

The successful treatment of prostate cancer relies on detection of the disease at its earliest stages. Although prostate-specific antigen (PSA)-based screening has been a significant advance in the early diagnosis of prostate cancer, identifying specific genetic alterations in a given family or patient will allow more appropriate screening for early disease. Mapping and identification of specific prostate cancer susceptibility genes is slowly becoming a reality. Other prostate cancer risks include a family history, race, and possibly serum markers such as insulin-like growth factor-I (IGF-I). Once a high-risk man is identified, transrectal ultrasound (TRUS)-guided biopsies are the standard to diagnose prostate cancer. Although TRUS is an advance over traditional digitally directed biopsies, it represents a random sampling of the prostate since most lesions cannot be visualized. Newer modalities such as ultrasound contrast agents, pattern recognition, and artificial neural networks (ANNs), applied to TRUS images, may improve diagnostic accuracy. If a man at risk for prostate cancer has undergone a negative TRUS biopsy, the decision for the need for additional biopsies is problematic. Use of PSA derivatives such as free and total PSA and the initial biopsy abnormalities such as atypia or high-grade prostatic intraepithelial neoplasia may define those patients in need of follow-up biopsy.     

p63 in prostate biology and pathology

The identification of stem cells and differentiation programs regulating the development and maintenance of the normal prostate epithelium is essential for the identification of the cell type(s) and molecular alterations involved in the development and propagation of prostate cancer (CaP). The p53-homologue p63 is highly expressed in normal prostate basal cells and is a clinically useful biomarker for the diagnosis of CaP. Importantly, p63 has been shown to play a critical role in prostate development. Recent experimental evidence also suggests that this gene is essential for normal stem cell function in the prostate as well as other epithelial organs. Future studies aimed at better defining the role of p63 in the renewal of the adult prostate epithelium are likely to shed new light on the mechanisms involved in prostate carcinogenesis.     

BMI1, Stem Cell Factor Acting as Novel Serum-biomarker for Caucasian and African-American Prostate Cancer

Background

Lack of reliable predictive biomarkers is a stumbling block in the management of prostate cancer (CaP). Prostate-specific antigen (PSA) widely used in clinics has several caveats as a CaP biomarker. African-American CaP patients have poor prognosis than Caucasians, and notably the serum-PSA does not perform well in this group. Further, some men with low serum-PSA remain unnoticed for CaP until they develop disease. Thus, there is a need to identify a reliable diagnostic and predictive biomarker of CaP. Here, we show that BMI1 stem-cell protein is secretory and could be explored for biomarker use in CaP patients.

Methodology/Principal Findings

Semi-quantitative analysis of BMI1 was performed in prostatic tissues of TRAMP (autochthonous transgenic mouse model), human CaP patients, and in cell-based models representing normal and different CaP phenotypes in African-American and Caucasian men, by employing immunohistochemistry, immunoblotting and Slot-blotting. Quantitative analysis of BMI1 and PSA were performed in blood and culture-media of siRNA-transfected and non-transfected cells by employing ELISA. BMI1 protein is (i) secreted by CaP cells, (ii) increased in the apical region of epithelial cells and stromal region in prostatic tumors, and (iii) detected in human blood. BMI1 is detectable in blood of CaP patients in an order of increasing tumor stage, exhibit a positive correlation with serum-PSA and importantly is detectable in patients which exhibit low serum-PSA. The clinical significance of BMI1 as a biomarker could be ascertained from observation that CaP cells secrete this protein in higher levels than cells representative of benign prostatic hyperplasia (BPH).

Conclusions/Significance

BMI1 could be developed as a dual bio-marker (serum and biopsy) for the diagnosis and prognosis of CaP in Caucasian and African-American men. Though compelling these data warrant further investigation in a cohort of African-American patients.     

Are plasma insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) useful markers of prostate cancer?

Increased concentrations of insulin-like growth factor I (IGF-I) and decreased insulin-like growth factor binding protein 3 (IGFBP-3) in serum have been proposed as markers of prostate cancer (CaP). The evidence for this, however, is contradictory. We assayed serum for IGF-I, IGFBP-3 and prostate-specific antigen (PSA) in patients with CaP and benign prostatic hyperplasia (BPH) and in healthy controls (HC). The mean +/- SD concentration of IGF-I in CaP (98.3 +/- 39.3 ng/mL; n = 15) was lower than in BPH (119 +/- 31.1 ng/mL; n=24) and HC (119 +/- 36.1 ng/mL; n=46), but the differences between the three groups were not statistically significant (p > 0.05). The mean IGFBP-3 concentrations in CaP (2691 +/- 1105 ng/mL; n = 16; p = 0.029) and BPH (2618 +/- 816 ng/mL; n = 26; p = 0.006) patients were significantly lower than that of the HC (3119 +/- 618 ng/mL; n=59), but the difference between the two groups of patients was not significant (p > 0.05). PSA concentrations in CaP (median = 80.8 ng/mL; n = 25) were significantly higher than those in BPH (median = 8.6 ng/mL; n = 39) (p < 0.001). Ninety-six percent of CaP and 72% of BPH patients had PSA concentrations >4.0 ng/mL; the proportions of patients with concentrations exceeding 20 ng/mL were 76% and 10%, respectively. We conclude that IGF-I and IGFBP-3 are inferior to PSA for CaP detection.     

Identification of prostate-specific antigen (PSA) isoforms in complex biological samples utilizing complementary platforms

Measurements of the prostate-specific antigen (PSA) levels in blood are widely used as diagnostic, predictive and prognostic marker of prostate disease. The selective detection of molecular forms of PSA can contribute clinically to meaningful enhancements of the conventional PSA-test. As it is plausible that an in-depth search for structural variants of PSA gene products may increase our ability to discriminate distinct patho-biological basis and stages of prostate diseases, we have developed a multi-step protocol comprising gel-based methods followed by mass spectrometric identification.Our current aim was to provide a comprehensive identification of PSA variants occurring in seminal fluid. We provide a proof-of-principle for this multiple step analytical approach to identify multiple PSA variants from complex biological samples that revealed distinct molecular characteristics. In addition, sequence-annotated protein bands in SDS–PAGE gels were compared to those detected by Western blots, and by monitoring the enzymatic activity in zymogram gels, using gelatin as a substrate. The high accuracy annotations were obtained by fast turnaround MALDI-Orbitrap analysis from excised and digested gel bands. Multiple PSA forms were identified utilizing a combination of MASCOT and SEQUEST search engines.     

Benign prostatic hyperplasia: does prostate size matter?

Recent studies and analyses have confirmed that baseline prostate volume is related to progression of benign prostatic hyperplasia (BPH) as well as to negative outcomes related to BPH, such as acute urinary retention (AUR) and need for surgery, and can also predict response to therapy. Other investigations have determined that prostate-specific antigen (PSA) level has good predictive value for assessing prostate volume. Strong evidence exists that baseline serum PSA level, like baseline prostate volume, predicts future prostate growth. Randomized placebo-controlled finasteride trials have shown that men with larger prostate volumes and higher PSA levels experience a clinically significant response to therapy compared with those with smaller prostate volumes and lower PSA levels. It has also been demonstrated that men with larger prostate glands and higher PSA levels are at increased risk for AUR and BPH-related surgery but that finasteride reduces these risks. Moreover, doxazosin and finasteride, alone and in combination, have been shown to significantly reduce BPH clinical progression.     

Imaging in the diagnosis and management of prostate cancer

The current diagnosis and management of prostate cancer is largely based on the use of serum prostate-specific antigen (PSA) and pathologic risk factors such as Gleason score and clinical stage. The use of serum PSA in clinical practice has resulted in significant stage migration and, as such, imaging modalities historically utilized to stage prostate cancer are no longer able to reliably identify the small amounts of prostate cancer most often found at presentation. Molecular imaging techniques have focused on improving sensitivity and specificity for cancer detection through knowledge of specific attributes of disease biology. The evolution of imaging techniques has created a new role for imaging in the management of prostate cancer.     

Value of percent free prostate-specific antigen for the prediction of pathological stage in men with clinically localized prostate cancer

PURPOSE: To analyze if the percentage of free prostate-specific antigen (PSA) can provide additional information to the combination of local clinical stage, serum PSA and Gleason score in the prediction of final stage and pathological features of prostate cancer. MATERIALS AND METHODS: A group of 480 men with clinically localized prostate cancer underwent lymphadenectomy and radical prostatectomy. Total and free PSA were measured in preoperative serum. Clinical stage was T1 in 70.4% of patients and T2 in 29.6%. The biopsy Gleason score ranged between 2 and 4 in 5.6%, between 5 and 7 in 78.4%, and was higher than 7 in 16%. Total serum PSA was below 4.1 ng/mL in 4.3%, between 4.1 and 10 ng/mL in 66.4%, between 10.1 and 20 ng/mL in 22.5%, and higher than 20 in 6.7% of patients. The tumor was organ-confined in 49.8% and specimen-confined in 64.2%, and its pathological features were favorable in 35%. RESULTS: Multiple logistic regression analysis demonstrated that percent free PSA has independent predictive value for pathological stage only in the subset of patients with cT1 tumors and serum PSA between 4.1 and 10 ng/mL. In this group the probability of organ-confined cancer was 68.3% if the percent free PSA was above 15 and 56.3% if it was lower (p<0.001). The probability of specimen-confined disease was 86.6% and 71.3%, respectively (p<0.007), and the probability of favorable pathology was 59.8% and 39.6%, respectively (p<0.002). We also found higher rates of organ- and specimen-confined tumors and favorable pathology for every Gleason score when the percent free PSA was higher than 15. CONCLUSIONS: Percent free PSA seems to provide additional information to the combination of clinical stage and Gleason score for the prediction of pathological features only in patients with clinical stage T1c and serum PSA between 4.1 and 10 ng/mL.     

Hospital parking garage

Prostate cancer is a serious health care problem in the United States. Whether or not to screen for it has become a timely issue. Although a large number of men have clinically important, asymptomatic, undetected prostate cancer, an even larger number have clinically unimportant cancer. To justify screening programs, not only must we avoid detecting biologically unimportant cancers, we must also detect and effectively treat that subset of tumors that, if undiagnosed, would progress, produce symptoms, and reduce life expectancy. Serum prostate-specific antigen (PSA) assay, or its variations such as PSA density, PSA velocity, and age-specific reference ranges, and the digital rectal examination are the best tests for detecting clinically important, asymptomatic, curable tumors. Recent data suggest that using serum PSA levels does not result in an overdetection of unimportant tumors. Highly effective, curative treatment of localized prostate cancer is available. These factors promote optimism that screening for prostate cancer will ultimately prove beneficial. Nonetheless, men should be informed regarding the benefits and possible risks before being screened for prostate cancer.     

Measurement of aberrant glycosylation of prostate specific antigen can improve specificity in early detection of prostate cancer

Introduction

We previously identified prostate cancer (PCa)-associated aberrant glycosylation of PSA, where α2,3-linked sialylation is an additional terminal N-glycan on free PSA (S2,3PSA). We then developed a new assay system measuring S2,3PSA using a magnetic microbead-based immunoassay. We compared the diagnostic accuracy of conventional PSA and percent-free PSA (%fPSA) tests.

Methods

We used MagPlex beads to measure serum S2,3PSA levels using anti-human fPSA monoclonal antibody (8A6) for capture and anti-α2,3-linked sialic acid monoclonal antibody (HYB4) for detection. We determined the cutoff values in a training test and measured serum S2,3PSA levels in 314 patients who underwent biopsy, including 138 PCa and 176 non-PCa patients with PSA of <10.0 ng/ml. Serum S2,3PSA levels were presented as mean fluorescence intensity (MFI). Receiver operating characteristic curves were used to evaluate the diagnostic accuracy of total PSA, %fPSA, and S2,3PSA.

Results

We determined an MFI cutoff value of 1130 with a sensitivity of 95.0% and specificity of 72.0% for the diagnosis of PCa in the training test. In the validation study, the area under the curve for the detection of PCa with S2,3PSA was 0.84, which was significantly higher than that with PSA or %fPSA.

Conclusions

Although the present study is small and preliminary, these results suggest that the measurement of serum S2,3PSA using a magnetic microbead-based immunoassay may improve the accuracy of early detection of PCa and reduce unnecessary prostate biopsy.     

The possible role of chromogranin A as a prognostic factor in organ-confined prostate cancer

The clinical significance of neuroendocrine differentiation in patients who have undergone surgery for localized prostate cancer is still unclear. The aims of this study were to assess the relationship between serum neuroendocrine markers and well-known prognostic factors in prostate cancer (pathological staging, definitive Gleason score and serum PSA) and to search for correlations between serum chromogranin A (CgA) levels and pathological findings. Forty-one consecutive patients who had undergone radical retropubic prostatectomy for clinically localized prostate cancer were evaluated. Serum PSA, CgA and neuron-specific enolase were measured immediately before surgery. Twenty-six surgical specimens were phenotypically and immunohistochemically evaluated using an antibody against CgA. Significant correlations were found between serum CgA, pathological staging and Gleason score (p=0.049 and p=0.038, respectively). Serum CgA did not correlate with PSA, patient age, or immunohistochemical findings. There was a significant correlation between positive immunohistochemical CgA staining and Gleason score (p=0.014). An increase in serum CgA levels, independent of PSA values, might be the expression of pathologically more advanced tumor stage and higher Gleason score; this could help to identify a high-risk patient group eligible for adjuvant therapy.     

Induction of Tc2 cells with specificity for prostate-specific antigen from patients with hormone-refractory prostate cancer

Prostate-specific antigen (PSA) is a potentially useful antigen for targeted T-cell immunotherapy of prostate cancer (CaP). Our laboratory has identified a synthetic nonamer peptide (PSA 146-154) homologue of PSA, which binds to the prevalent human leukocyte antigen, HLA-A2, and elicits specific cytotoxic T-lymphocyte (CTL) responses from normal individuals of the HLA-A2 phenotype. In the present study, we report on the induction of CTL from peripheral blood mononuclear cells (PBMC) of patients with hormone-refractory CaP, which exhibit the same specificity. T-cell lines were established from two patients by stimulation of PBMC with PSA 146-154 peptide in vitro. The T-cell lines exhibited specific cytolytic activity against T2 cells pulsed with PSA 146-154 peptide, but not a control HLA-A2 binding peptide (HIV-RT 476-484) via chromium release assay (CRA). The T-cell lines also showed PSA 146-154 peptide-specific IL-4 responses, but no detectable interferon-gamma (IFN-gamma) responses via enzyme-linked immuno-spot assays. Magnetic immuno-selection studies of one of the T-cell lines demonstrated that both cytolytic and interleukin-4 (IL-4) responses were mediated by CD8(+), but not by CD4(+) T cells. This Tc2 line was further characterized for the ability to recognize endogenously processed PSA epitopes. The line specifically secreted IL-4 in response to HLA-A2(+) target cells transfected to express PSA and specifically lysed the PSA(+) target cells, but not control transfected cells. The results indicate that the PSA 146-154 peptide emulates a naturally processed and presented peptide epitope of PSA that is within the T-cell repertoire of HLA-A2(+)patients with CaP.     

Evolution of the clinical presentation and outcomes after radical prostatectomy for patients with clinically localized prostate cancer--changing trends over a ten year period

We demonstrate the evolution of the clinical presentation and outcomes for patients with clinically localized prostate cancer (PC) treated with radical retropubic prostatectomy (RRP) at our department, emphasizing epidemiologic significance of changes during the 10-year period. We assessed the annual trends for changes in patients age, preoperative prostate specific antigen (PSA), preoperative versus postoperative stages and Gleason grades, organ confined status and surgical margin status. A total of 488 RRPs were performed from January 1996 to December 2005 with the annual frequency increased from 8 to 129 (1512.5%). Mean patient age increased from 61.5 to 66.12 years in 2005, with the percentage of men aged more than 70 years increased from 12.5 to 26.5%, respectively. The detection of PC based solely on pathological PSA levels (as indication for prostate biopsy) rose impressively from 25.5 to 70% and the rates of postoperative organ-confined disease also increased significantly from 25 to 74.7%. Mean preoperative PSA decreased from 16.7 to 9.89 ng/mL. On the contrary, there was an increase in percentage of patients with preoperative PSA values ranging from 4 to 10 ng/mL (from 20 to 65.4%). Positive surgical margin rate decreased from 49.4 to 25% and percent of patients receiving neoadjuvant therapy decreased from 78.5 to 5.4%. Proportion of patients who were undergraded decreased from 75.1 to 31.7%. The rates of understaging have remained relatively stable over the years. During the study period, PC was increasingly detected by prostate biopsy on the basis of a pathological PSA level only and shifted significantly to more organ-confined stages with more favourable outcomes for pathological variables due to a more accurate assessment of clinical stage prior to surgery, reduced use of neoadjuvant therapy and improved surgical technique. Our data also argue strongly that routine PSA testing should be expanded and not restricted.