Eduardo Reyes

Differential Expression of Matrix Metalloproteinase-2 Expression in Disseminated Tumor Cells and Micrometastasis in Bone Marrow of Patients with Nonmetastatic and Metastatic Prostate Cancer: Theoretical Considerations and Clinical Implications—An Immunocytochemical Study

Matrix metalloproteinase-2 (MMP-2) is important in the dissemination and invasion of tumor cells and activates angiogenesis. We present an immunocytochemical study of MMP-2 expression in circulating prostate cells (CPCs), disseminated tumor cells (DTCs), and micrometastasis (mM) in bone marrow of men with prostate cancer. Methods and Patients. Tumor cells were identified with anti-PSA immunocytochemistry. Positive samples underwent processing with anti-MMP-2, its expression was compared with Gleason score, concordance of expression, and metastatic and nonmetastatic disease. Results. 215 men participated, CPCs were detected in 62.7%, DTCs in 62.2%, and mM in 71.4% in nonmetastatic cancer; in metastatic cancer all had CPCs, DTCs, and mM detected. All CPCs and DTCs expressed MMP-2; in mM MMP-2 expression was positively associated with increasing Gleason score. MMP-2 expression in CPCs and DTCs showed concordance. In low grade tumors, mM and surrounding stromal cells were MMP-2 negative, with variable expression in high grade tumors; in metastatic disease, both mM and stromal cells were MMP-2 positive. Conclusions. CPCs and DTCs are different from mM, with inhibition of MMP-2 expression in mM of low grade tumors. With disease progression, MMP-2 expression increases in both mM and surrounding stromal cells, with implications for the use of bisphosphonates or MMP-2 inhibitors.

Secondary Circulating Prostate Cells Predict Biochemical Failure in Prostate Cancer Patients after Radical Prostatectomy and without Evidence of Disease

Introduction. Although 90% of prostate cancer is considered to be localized, 20%–30% of patients will experience biochemical failure (BF), defined as serum PSA >0.2 ng/mL, after radical prostatectomy (RP). The presence of circulating prostate cells (CPCs) in men without evidence of BF may be useful to predict patients at risk for BF. We describe the frequency of CPCs detected after RP, relation with clinicopathological parameters, and association with biochemical failure. Methods and Patients. Serial blood samples were taken during followup after RP, mononuclear cells were obtained by differential gel centrifugation, and CPCs identified using standard immunocytochemistry using anti-PSA monoclonal antibodies. Age, pathological stage (organ confined, nonorgan confined), pathological grade, margin status (positive, negative), extracapsular extension, perineural, vascular, and lymphatic infiltration (positive, negative) were compared with the presence/absence of CPCs and with and without biochemical failure. Kaplan Meier methods were used to compare the unadjusted biochemical failure free survival of patients with and without CPCs. Results. 114 men participated, and secondary CPCs were detected more frequently in patients with positive margins, extracapsular extension, and vascular and lymphatic infiltration and were associated with biochemical failure independent of these clinicopathological variables, and with a shorter time to BF. Conclusions. Secondary CPCs are an independent risk factor associated with increased BF in men with a PSA <0.2 ng/mL after radical prostatectomy, but do not determine if the recurrence is due to local or systemic disease. These results warrant larger studies to confirm the findings.

Circulating Prostate Cells Found in Men with Benign Prostate Disease Are P504S Negative: Clinical Implications

Introduction. Developments in immunological and quantitative real-time PCR-based analysis have enabled the detection, enumeration, and characterization of circulating tumor cells (CTCs). It is assumed that the detection of CTCs is associated with cancer, based on the finding that CTCs can be detected in all major cancer and not in healthy subjects or those with benign disease. Methods and Patients. Consecutive men, with suspicion of prostate cancer, had blood samples taken before prostate biopsy; mononuclear cells were obtained using differential gel centrifugation and CPCs detecting using anti-PSA immunocytochemistry. Positive samples underwent further classification with anti-P504S. Results. 329 men underwent prostate biopsy; of these men 83 underwent a second biopsy and 44 a third one. Of those with a biopsy negative for cancer, 19/226 (8.4%) had CPCs PSA (+) P504S (−) detected at first biopsy, 6/74 (8.1%) at second biopsy, and 5/33 (15.2%) at third biopsy. Men with cancer-positive biopsies did not have PSA (+) P504S (−) CPCs detected. These benign cells were associated with chronic prostatitis. Conclusions. Patients with chronic prostatitis may have circulating prostate cells detected in blood, which do not express the enzyme P504S and should be thought of as benign in nature.

Primary circulating prostate cells are not detected in men with low grade small volume prostate cancer.

Objective. To determine if primary circulating prostate cells (CPCs) are found in all men with prostate cancer. Methods and Patients. A prospective study, to analyze all men with an elevated PSA between 4.0 and 10.0 ng/mL undergoing initial biopsy. Primary CPCs were obtained by differential gel centrifugation and detected using standard immunocytochemistry using anti-PSA; positive samples underwent a second process with anti-P504S. A malignant primary CPC was defined as PSA (+) P504S (+) and a test positive if 1 cell/4 mL was detected. Biopsy results were registered as cancer/no-cancer, number of cores positive, and percent infiltration of the cores. Results. 328/1123 (29.2%) of the study population had prostate cancer diagnosed on initial biopsy, and 42/328 (12.8%) were negative for primary CPCs. CPC negative men were significantly older, and had lower PSA levels, lower Gleason scores, and fewer positive cores and with infiltration by the cancer. 38/42 (91%) of CPC negative men complied with the criteria for active surveillance in comparison with 34/286 (12%) of CPC positive men. Conclusions. Using primary CPC detection as a sequential test to select men with an elevated PSA for biopsy, the risk of missing clinically significant prostate cancer is minimal when the patient is primary CPC negative; less than 0.5% of all primary CPC negative men had a clinically significant prostate cancer.

Prediction model for early biochemical recurrence after radical prostatectomy based on the Cancer of the Prostate Risk Assessment score and the presence of secondary circulating prostate cells.

To establish a prediction model for early biochemical recurrence based on the Cancer of the Prostate Risk Assessment (CAPRA) score and the presence of secondary circulating prostate cells (CPCs).

A Comparative Performance Analysis of Total PSA, Percentage Free PSA, PSA Velocity, and PSA Density versus the Detection of Primary Circulating Prostate Cells in Predicting Initial Prostate Biopsy Findings in Chilean Men

Introduction. PSA parameters have been used in an attempt to improve the diagnostic yield of prostate screening tests; the detection of primary malignant circulating prostate cells (CPCs) may improve the diagnostic yield of screening and therefore avoid unnecessary biopsies. Patients and Methods. Prospective study of all men undergoing initial prostate biopsy due to an elevated total serum PSA. Free percent PSA, PSA velocity, and PSA density were determined. Primary CPCs were detected using standard immunocytochemistry. A positive test for CPCs was defined as one cell PSA (+) P504S (+) in an 8 ml blood sample. Positive predictive and negative predictive values, specificity, and sensitivity were calculated for each test as well as the number of biopsies avoided and cancers missed. Results. 303 men participated in the study of whom 113/303 (37.3%) men had prostate cancer. Of the three PSA based parameters, free percent PSA was superior, sensitivity 70.8%, and specificity 67.4%. Primary CPCs detection had a sensitivity of 88.5% and a specificity of 88.4% avoiding 181 (59.7%) biopsies, detecting 93/95 (98%) of clinically significant cancers, and missing 13 (11.5%) low grade, small volume tumors. Conclusions. The use of primary CPCs as a sequential test could decrease the number of initial prostate biopsies missing those cancers which are treated by active observation.

Head-to-head comparison of the Montreal nomogram with the detection of primary malignant circulating prostate cells to predict prostate cancer at initial biopsy in Chilean men with suspicion of prostate cancer ☆

INTRODUCTION The limitations of total serum prostate-specific antigen (PSA) level values remain problematic. Nomograms may improve the predictive value of a positive prostate biopsy (PB) finding. We compare in a prospective study of Chilean men suspicious of having prostate cancer (PC), owing to an elevated total serum PSA or abnormal digital rectal examination finding or both, the use of the online Montreal nomogram for the detection of primary malignant circulating prostate cells (mCPCs) to predict a positive PB finding. METHODS AND PATIENTS Consecutive men suspicious of PC underwent 12-core transrectal ultrasound PB; their age, total serum PSA levels and percent free PSA values, and Montreal nomogram scores were registered. Immediately before the PB, an 8-ml blood sample was taken to detect primary mCPCs. Mononuclear cells were obtained by differential gel centrifugation and identified using double immunomarcations with anti-PSA and anti-P504S. Biopsies were classified according to presence of cancer/no cancer. The test results for the detection of mCPC were stated as negative/positive, and the total number of cells/8 ml of blood was registered. Areas under the curve for total serum PSA level, percent free PSA value, Montreal score, and detection of mCPCs were calculated and compared. Diagnostic yields, the number of possible biopsies that could be avoided, and the number of clinically significant cancers that would be missed were calculated. RESULTS Overall, 607 men underwent biopsy, where 197 (32.5%) had cancer. These men were significantly older, had higher total serum PSA level and Montreal score, and lower percent free PSA value. The values for area under the curve were 0.56 for total PSA level, 0.78 for percent free PSA, 0.78 for Montreal score, and 0.84 for mCPC detection; mCPC detection had a significantly superior prediction value (P = 0.018). Using cutoff values of percent free PSA < 10%, Montreal score > 50%, and ≥ 1 mCPC detected, mCPC detection had a higher diagnostic yield. Of the 197 cancers, 41 complied with the criteria for active surveillance; percent free PSA and the Montreal score missed a higher number of significant cancers when compared with mCPC detection. CONCLUSIONS Primary mCPC detection outperformed the use of percent free PSA and the Montreal nomogram in predicting clinically significant PC at initial PB.

Extended use of P504S positive primary circulating prostate cell detection to determine the need for initial prostate biopsy in a prostate cancer screening program in Chile.

BACKGROUND To determine the frequency of primary circulating prostate cells (CPC) detection according to age and serum PSA levels in a cohort of men undergoing screening for prostate cancer and to determine the diagnostic yield in those men complying with the criteria for prostate biopsy. MATERIALS AND METHODS A prospective study was carried out to analyze all men evaluated in a hospital prostate cancer screening program. Primary CPCs were obtained by differential gel centrifugation and detected using standard immunocytochemistry using anti-PSA, positive samples undergoing a second process with anti-P504S. A malignant primary CPC was defined as PSA+ P504S+, and a test positive if 1 cell/4ml was detected. The frequency of primary CPC detection was compared with age and serum PSA levels. Men with a PSA >4.0ng/ml and/or abnormal rectal examination underwent 12 core prostate biopsy, and the results were registered as cancer/no-cancer and compared with the presence/absence of primary CPCs to calculate the diagnostic yield. RESULTS A total of 1,117 men participated; there was an association of primary CPC detection with increasing age and increasing serum PSA. Some 559 men underwent initial prostate biopsy of whom 207/559 (37.0%) were positive for primary CPCs and 183/559 (32.0%) had prostate cancer detected. The diagnostic yield of primary CPCs had a sensitivity of 88.5%, a specificity of 88.0%, and positive and negative predictive values of 78.3% and 94.9%, respectively. CONCLUSIONS The use of primary CPCs for testing is recommended, since its high negative predictive value could be used to avoid prostate biopsy in men with an elevated PSA and/or abnormal DRE. Men positive for primary CPCs should undergo prostate biopsy. It is a test that could be implemented in the routine immunocytochemical laboratory.

Possible Role of HER-2 in the Progression of Prostate Cancer from Primary Tumor to Androgen Independence.

BACKGROUND The expression of HER-2 in prostate cancer has been linked to disease progression. We analysed the presence of HER-2 expression in primary tumors in men undergoing radical prostatectomy, its association with clinical and pathological findings, and its expression in secondary circulating prostate cells (CPCs) during follow up, as well as links with biochemical failure and the effects of androgen blockade. MATERIALS AND METHODS Consecutive men undergoing radical prostatectomy for histologically confirmed prostate cancer were analyzed. HER-2 expression in the primary tumor was assessed using the HercepTest®, CPCs were identified from blood samples using standard immunocytochemistry with anti-PSA and positive samples with the HercepTest® to determine HER-2 expression. The influence of HER-2 expression on the frequency of biochemical failure and effects of androgen blockade was determined. RESULTS 144 men with a mean age of 64.8±10.3 years participated, with a median follow up of 8.2 years. HER-2 was expressed in 20.8% of primary tumors; it was associated with vascular infiltration and older age, but not with other clinical pathological findings. Some 40.3% of men had secondary CPCs detected, of which 38% expressed HER-2. Men CPC (+) had a higher frequency of biochemical failure, but there was no difference in HER-2 expression of CPCs with the frequency of biochemical failure. After androgen blockade, men with HER-2 (+) positive secondary CPCs had a higher frequency of disease progression to castrate resistant disease. CONCLUSIONS HER-2 plays a dual role in the progression of prostate cancer; firstly it may increase the potential of tumor cells to disseminate from the primary tumor via the blood by increasing vascular infiltration. In the presence of androgens, there is no survival advantage of expressing HER-2, but once biochemical failure has occurred and androgen blockade started, HER-2 positive cells are resistant to treatment, survive and grow leading to castration resistant disease.

Effect of Androgen Blockade on HER-2 and Matrix Metalloproteinase-2 Expression on Bone Marrow Micrometastasis and Stromal Cells in Men with Prostate Cancer

Introduction. HER-2 has been associated with castrate resistant prostate cancer and matrix metalloproteinase-2 (MMP-2) in the dissemination and invasion of tumor cells as well as activating angiogenesis. We present an immunocytochemical study of the effect of androgen blockade on the expression of HER-2 and MMP-2 in bone marrow micrometastasis and the surrounding stromal cells in men with prostate cancer. Methods and Patients. A cross-sectional study of men with prostate cancer. Touch preps were obtained from bone marrow biopsies of men with prostate cancer, before and after radical prostatectomy and during androgen blockade. Micrometastasis detected with anti-PSA immunocytochemistry underwent processing with anti-HER-2 and anti-MMP-2 immunocytochemistry. Patients were defined as HER-2 positive or negative, MMP-2 negative or an MMP-2 pattern described as border or central and stromal MMP-2 defined as positive or negative. The expression of the biomarkers was compared before and after primary treatment and during androgen blockade in relation to the serum PSA at the time of sampling and duration of androgen blockade. Results. 191 men participated, 35 men before surgery and 43 after surgery; there were no significant differences in HER-2 expression between groups, there was no MMP-2 expression centrally or stromal expression of MMP-2. In men with androgen blockade, HER-2 expression was significantly higher; there was a trend for increasing HER-2 expression up to 5 years; central MMP-2 expression significantly increased after 3 years, while stromal MMP-2 significantly increased after 6 years. MMP-2 expression both in micrometastasis and stroma was significantly associated with HER-2 expression. Expression of MMP-2 at the border of the micrometastasis was not associated with HER-2 expression and occurred in the absence of androgen blockade. Conclusions. Androgen blockade decreases serum PSA by eliminating HER-2 negative prostate cancer cells. However, there is early selection of HER-2 positive cancer cells which leads to androgen independence and to increased expression of MMP-2 activity in the micrometastasis. The increased MMP-2 activity in the micrometastasis increases the expression of MMP-2 in the surrounding stromal cells and thus could promote angiogenesis and tumor growth resulting in macrometastatic androgen independent disease.