Prediction of ovarian cancer prognosis and response to chemotherapy by a serum-based multiparametric biomarker panel.

Currently, there are no effective biomarkers for ovarian cancer prognosis or prediction of therapeutic response. The objective of this study was to examine a panel of 10 serum biochemical parameters for their ability to predict response to chemotherapy, progression and survival of ovarian cancer patients. Sera from ovarian cancer patients were collected prior and during chemotherapy and were analysed by enzyme-linked immunosorbent assay for CA125, kallikreins 5, 6, 7, 8, 10 and 11, B7-H4, regenerating protein IV and Spondin-2. The odds ratio and hazard ratio and their 95% confidence interval (95% CI) were calculated. Time-dependent receiver-operating characteristic (ROC) curves were utilised to evaluate the prognostic performance of the biomarkers. The levels of several markers at baseline (c(0)), or after the first chemotherapy cycle (rc(1)), predicted chemotherapy response and overall or progression-free survival in univariate analysis. A multiparametric model (c(0) of CA125, KLK5, KLK7 and rc(1) of CA125) provided predictive accuracy with area under the ROC curve (AUC) of 0.82 (0.62 after correction for overfitting). Another marker combination (c(0) of KLK7, KLK10, B7-H4, Spondin-2) was useful in predicting short-term (1-year) survival with an AUC of 0.89 (0.74 after correction for overfitting). All markers examined, except KLK7 and regenerating protein IV, were powerful predictors of time to progression (TTP) among chemotherapy responders. Individual and panels of biomarkers from the kallikrein family (and other families) can predict response to chemotherapy, overall survival, short-term (1-year) survival, progression-free survival and TTP of ovarian cancer patients treated with chemotherapy.

"BPSA," a specific molecular form of free prostate-specific antigen, is found predominantly in the transition zone of patients with nodular benign prostatic hyperplasia.

OBJECTIVES: The biologic mechanism for the increased proportion of noncomplexed ("free") prostate-specific antigen (PSA) found in the serum of patients with benign prostate disease is unknown. We recently reported that most of the PSA found in benign, hyperplastic, and cancerous prostatic tissue is in the free form. To determine whether specific molecular forms of free PSA are associated differentially with normal, hyperplastic, or cancerous prostatic tissue, we have further characterized the free PSA in each type of prostatic tissue. METHODS: PSA was purified by immunoaffinity chromatography from matched prostatic tissue samples of peripheral zone cancer (PZ-C), PZ noncancer (PZ-N), and transition zone (TZ) tissue from 10 large-volume (greater than 50 g) and 8 small-volume (less than 25 g) radical prostatectomy specimens. Eight TZ specimens obtained during transurethral resection of the prostate for benign prostatic hyperplasia (BPH) were also analyzed. The different molecular forms of PSA were further resolved by high-performance hydrophobic interaction chromatography. Clipped forms of PSA were identified by N-terminal amino acid sequencing. RESULTS: More than 99% of the PSA in prostatic tissues was in the free, noncomplexed form. Specimens from the prostate TZ were found to contain elevated levels of an altered form of PSA, which we designated BPSA. Purified BPSA contained a distinctive cleavage at lysine 182. The median percent BPSA (%BPSA) was 11.4 in the TZ of specimens with nodular BPH compared with a %BPSA of 4.1 in the TZ of specimens without nodular BPH (P <0.0014). The median %BPSA levels of the PZ-N and PZ-C tissues ranged from 3.2 to 4.9 and were not significantly different from one another or from the %BPSA level of TZ tissues without nodular BPH. CONCLUSIONS: We have identified a specific molecular form of clipped free PSA, called BPSA, that is increased within the prostatic TZ of patients exhibiting nodular BPH. Higher levels of percent free PSA in serum have been found to correlate strongly with prostate volume, which in turn is closely associated with the progressive enlargement of nodular BPH tissue within the TZ of the prostate. Thus, it is possible that a proportion of the serum percent free PSA found in patients with BPH may be composed of BPSA released into the serum.

Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis.

OBJECTIVES: Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha2-macroglobulin and alpha1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. METHODS: Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritech's Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). RESULTS: The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. CONCLUSIONS: In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.

Identification of a novel complex between human kallikrein 2 and protease inhibitor-6 in prostate cancer tissue.

Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.

Use of percentage of free prostate-specific antigen to identify men at high risk of prostate cancer when PSA levels are 2.51 to 4 ng/mL and digital rectal examination is not suspicious for prostate cancer: an alternative model.

OBJECTIVES: Currently, many clinicians do not recommend prostate biopsy for men with digital rectal examination (DRE) results that are not suspicious for cancer and prostate-specific antigen (PSA) values between 2.51 and 4 ng/mL. We propose a new model for the detection of prostate cancer using the percentage of free PSA (%FPSA) in the limited range of PSA values between 2.51 and 4 ng/mL that maximizes clinical specificity (ie, minimizes false-positive results). This model identifies higher risk patients in this relatively low-risk population. METHODS: Three hundred sixty-eight archived serum samples from men evaluated and treated at two academic institutions were reviewed. All men had a histologic diagnosis, findings not suspicious for cancer on DRE, and PSA levels between 2.51 and 4 ng/mL. Samples were tested in Hybritech's Tandem-R PSA and Tandem-R free PSA (FPSA) assays in the same laboratory at each institution. RESULTS: Various models for cancer detection using %FPSA when PSA is 2.51 to 4 ng/mL and DRE is not suspicious for cancer are proposed. These models recommend biopsy for only 10% to 36% of the men in this population and would identify as many as 30% to 54% of the detectable cancers. There is evidence that the cancers that would be detected are the most aggressive cancers in this population. CONCLUSIONS: Our models identified men with a higher risk of prostate cancer in a relatively low-risk population that currently does not routinely undergo biopsy. This may allow for a more cost-effective way to increase cancer detection when PSA values are between 2.51 and 4 ng/mL and DRE is not suspicious for cancer. This model has the potential to detect a greater number of clinically important and potentially curable cancers than would be detected with current practice.

Highly sensitive automated chemiluminometric assay for measuring free human glandular kallikrein-2.

BACKGROUND: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to alpha1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. METHODS: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS(R) analyzer. RESULTS: The detection limit of the assay was 1.5 ng/L, the "functional sensitivity" (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations. CONCLUSION: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.

Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases.

OBJECTIVES: To describe the expression of a potential new tumor marker, human glandular kallikrein 2 (hK2), in primary adenocarcinoma and lymph node metastases that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer. METHODS: We evaluated 151 radical prostatectomy specimens removed at Mayo Clinic with node-positive adenocarcinoma to compare cytoplasmic expression of hK2, pro-hK2, and PSA in benign tissue, prostate adenocarcinoma, and lymph node metastases. Monoclonal antibodies for mature hK2 (hK2-G586), pro-hK2 (pro-hK2-G464), and PSA (PSA-773) were used. A polyclonal antibody for PSA was also used. Immunoreactivity in each case was tested to determine whether cancer recurrence could be predicted. RESULTS: Intense epithelial cytoplasmic immunoreactivity was observed in every case for hK2-G586, pro-hK2-G464, PSA-773, and polyclonal PSA (100% of cases, respectively). The intensity and extent of hK2 expression was greater in lymph node metastases than in primary cancer; furthermore, the expression in primary cancer was greater than in benign epithelium. Pro-hK2 was expressed in a greater percentage of cells in primary cancer than in benign tissue; furthermore, pro-hK2 was expressed to a greater extent in primary cancer than in lymph node metastases. In marked contrast to mature hK2, monoclonal PSA immunoreactivity was expressed to a higher extent in primary cancer than in lymph node metastases. Polyclonal PSA showed an incremental increase in expression from benign tissue to primary cancer and a further increase in expression in lymph node metastases. CONCLUSIONS: hK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to primary cancer and lymph node metastases. Pro-hK2 was expressed to the greatest extent in primary cancer. Monoclonal PSA displayed inverse immunoreactivity compared with hK2. Polyclonal PSA showed incremental increases, suggesting that both hK2 and PSA were being detected. Tissue expression of hK2 appears to be regulated independently of PSA in benign epithelium, adenocarcinoma, and lymph node metastases.

Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay.

The prostate-specific antigen (PSA) immunoassay is an important tool for the detection and monitoring of prostate cancer. PSA exists in serum mainly as complexes with serine protease inhibitors including alpha1 antichymotrypsin (ACT) and alpha2 macroglobulin (MG). PSA-MG complex is not detected by the existing PSA immunoassays since MG (720 kDa) sequesters PSA and masks the antibody binding sites. Existing immunoassays for quantitation of total serum PSA measure PSA-ACT (CPSA) and free PSA (FPSA), which comprise the major and minor components of total PSA, respectively. Monoclonal antibodies (MAb) specific for CPSA alone were generated using a unique immunogen prepared by blocking the major antigenic determinants on FPSA and ACT. The blocked immunogen greatly enhanced the frequency of hybridomas reactive against the CPSA complex. CPSA prototype immunoassays were established using anti-CPSA (PX1G359) or anti-ACT (AC1A212) MAb as tracer MAb and anti-PSA (PSA399) MAb as capture MAb. The complex-selective MAbs demonstrated minimal cross-reactivity with Cathepsin-G (CG) ACT (CG-ACT), ACT or FPSA. CG-ACT complex interfered with the accuracy of initial prototype assays specific for CPSA measurement and caused over-recovery (1 to 3 ng/mL, with 40 to 180 ng/mL range of CG-ACT in serum) of apparent CPSA values. Addition of 0.4% NP-40 and 0.1% 0.088 micron microparticles in clinical specimens eliminated this interference. Specimens from 39 prostate cancer (PCa) patients and 44 benign prostatic hyperplasia (BPH) patients were analyzed with the PX1G359 CPSA assay. In this study, the area under the curve (AUC) values for ROC analysis of total PSA (CPSA+FPSA), FPSA to total PSA ratio (f/t), and FPSA to CPSA ratio (f/c) were 0.357, 0.634, and 0.624, respectively. In a second study using AC1A212 CPSA assay, where specimens from 16 PCa patients and 48 BPH patients were tested, the AUC values for total PSA, f/t and f/c ratios were 0.62, 0.785, and 0.732, respectively. Using the CPSA assay with minimal interference our studies are consistent with previous CPSA data showing that the f/t PSA ratio remains superior to the f/c PSA ratio in differentiating PCa and BPH diseases. Complex PSA by itself or as ratio with free or total PSA does not provide any advantage over the established method of FPSA to total PSA ratio.

Western blotting analysis of antibodies to prostate-specific antigen: cross-reactivity with human kallikrein-2.

Immunoassays for prostate-specific antigen (PSA) using monoclonal or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA shares 80% sequence homology with human glandular kallikrein (hK2). In the present study, we have used SDS-PAGE and Western blotting of a recombinant form of hK2 to determine the degree of cross-reactivity in a panel of 83 antibodies submitted to the ISOBM TD-3 PSA Workshop. From this panel, 24 of the 83 antibodies showed cross-reactivity with hK2. The majority of these antibodies showed binding to conformationally independent or linear epitopes. Fourteen of these antibodies appear to map to the same epitope group, indicating the existence of a dominant and linear immunogenic domain shared by PSA and hK2.

Western blotting analysis of antibodies to prostate-specific antigen: specificities for prostate-specific antigen and prostate-specific antigen fragments.

Approximately 40% of the prostate-specific antigen (PSA) purified from seminal fluid comprises cleaved or fragmented forms of PSA. These fragments are observed by reduced SDS-PAGE and have been identified in preparations of purified seminal plasma. The comparative analysis of 53 antibodies, submitted to an international PSA Workshop, with different PSA variants was studied using SDS-PAGE and Western blotting. Six different patterns of reactivity were observed which may reflect different epitopes recognized by this panel of antibodies. Additional antibody studies to nonreduced intact PSA suggest the epitopes are conformation-dependent.

Identification, purification, and subcellular localization of prostate-specific membrane antigen PSM' protein in the LNCaP prostatic carcinoma cell line.

An alternatively spliced variant of prostate-specific membrane antigen (PSMA) designated PSM' was originally described following identification of its mRNA in normal prostate. We have purified the PSM' protein from LNCaP cells using two immunoaffinity columns in tandem. The first column contained a monoclonal antibody (7E11) that was reactive with the NH2 terminus of PSMA, which specifically depleted the LNCaP lysate of full-length PSMA. The nonbinding fraction was then passed over a second column composed of a monoclonal antibody (PEQ226.5), the epitope of which was located within the 134-437 domain of PSMA and shared with PSM'. The protein eluted from the second immunoaffinity column produced a Mr 95,000 band on SDS-PAGE, which was slightly lower than the full-length PSMA at Mr 100,000. The band was NH2-terminally sequenced through 15 residues, and the assigned sequence coincided with the predicted sequence for PSM' protein minus the first two NH2 terminus amino acids. The PSM' protein, therefore, began with residue 60 of PSMA (alanine). LNCaP cells were fractionated, and PSM' was localized to the cytoplasm.

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia.

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.

Detection of human glandular kallikrein, hK2, as its precursor form and in complex with protease inhibitors in prostate carcinoma serum.

Forms of human glandular kallikrein (kK2) in prostate carcinoma serum were identified using monoclonal antibodies specific for hK2 and prohK2. Recombinant mammalian hK2, prohK2, and prostate = specific antigen (PSA) were utilized to confirm the specificity of monoclonal antibodies for hK2 and the lack of reactivity with PSA. In prostate cancer patient sera containing high levels of hK2 (>100 ng/ml), hK2 exists as a complex with alpha1-antichymotrypsin with a molecular weight of 90 kDa. The kallikrein also exists as a 32-kDa free form, which includes the precursor pro form of hK2. The relative amount of complex and free hK2 varied, but in most sera examined the 32-kDa form predominated. Recombinant hK2 readily formed complexes with alpha2-macroglobulin when the two proteins were incubated together as well as when hK2 was spiked into female serum.

Measurement of serum prostate-specific membrane antigen, a new prognostic marker for prostate cancer.

OBJECTIVES: To describe current results with Western blot assay for prostate specific membrane antigen (PSMA) using 7E11.C5 antibody and the development of an additional antibody measurement for PSMA by a new sandwich immunoassay. METHODS: A population of patients from a screening group, from a difficult diagnostic group, from a pre- and postoperative radical prostatectomy group, and from a group with metastatic disease followed for a serial period, provided the serum values for a prospective assessment of PSMA by Western blot assay. A new monoclonal antibody was sought, reacting to the C-terminal region of PSMA in order to develop a sandwich radioimmunoassay. RESULTS: PSMA values in screened patients correlate with the more advanced stage of the cancers determined. In postprostatectomy patients, the PSMA value corresponds more with preoperative values and with the values of those with a poor clinical course. In difficult diagnostic cases, the PSMA value is increased, specifically in hormone-refractory cases and particularly in those cases judged by other criteria, such as the National Prostatic Cancer Project, to be in clinical progression compared with those judged to be in clinical remission. The level of PSMA value appears to be independent of homogeneous tumor volume and to be more related to that of prior hormone treatment, or to where prostate cancer cells can be documented to be outside the prostate. A new monoclonal antibody, 3F5.4G6, reacts with the extracellular domain of PSMA near the C-terminal region. This is in contrast to the previously measured antibody 7E11.C5, which reacts with an N-terminal epitope. 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5. The epitopes are essentially at opposite ends of the molecule. The 3F5.4G6 antibody reacts with the LNCaP line but not with DU145, or PC3. These two antibodies to PSMA are well suited for use in a new sandwich immunoassay. CONCLUSIONS: PSMA provides a prostatic cancer serum test by using Western blot, which suggests a clinical prognostic value not seen with other markers. New antibodies, such as 3F5.4G6, reacting with the extracellular domain of PSMA combined with 7E11.C5, appear to offer an opportunity for a new sandwich immunoassay.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES: Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS: We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS: The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS: Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

A precursor form of PSA (pPSA) is a component of the free PSA in prostate cancer serum.

OBJECTIVES: Prostate-specific antigen (PSA) is a widely used serum marker for human prostate cancer (PCa). The majority of PSA in serum is present as a complex with alpha-1-antichymotrypsin (ACT). In recent years, the ratio of free (uncomplexed) to total PSA has shown improved discrimination of PCa from benign prostatic hyperplasia. This study examines the nature of the free PSA from detected in PCa serum and shows that some of the uncomplexed PSA is an inactive precursor of PSA (pPSA). METHODS: Western blot analysis was used to detect clipped, fragment forms of PSA in sera and seminal fluid. Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) was used to identify forms of PSA present in the free PSA population. Pooled sera was passed over a PSA immunoaffinity column, and the eluted PSA components were further resolved by HIC-HPLC. RESULTS: Western blot analysis of whole sera showed complexed PSA and the intact, approximately 34 kilodalton free PSA. Only negligible levels of clipped or degraded forms of PSA, as found in seminal fluid, were detected. Column fractions measured for uncomplexed PSA using the Tandem-MP free PSA assay showed that about 25% of the free PSA eluted as pPSA beginning at the [-4]amino acid. Studies with purified recombinant [-4]pPSA showed that this proenzyme form is inactive and does not complex with ACT. CONCLUSIONS: These results suggest that the uncomplexed PSA in PCa serum is primarily unclipped PSA that contains a significant fraction of pPSA.

Early detection of recurrent prostate cancer with an ultrasensitive chemiluminescent prostate-specific antigen assay.

OBJECTIVES: Treatment failure after radical prostatectomy is most commonly heralded by an increase in serum prostate-specific antigen (PSA) to detectable levels. We evaluated the clinical utility of an ultrasensitive chemiluminescent PSA assay. METHODS: We evaluated the assay in banked sera obtained from 170 men after radical prostatectomy. Controls consisted of 142 females, 29 men who had undergone cystoprostatectomy without evidence of prostate cancer, and 25 men without evidence of recurrent disease at least 5 years after prostatectomy for organ-confined disease. Lead time to diagnosis of recurrence was based on comparisons with the IMx or Tandem E assays using a cutoff of 0.1 ng/mL (100 pg/mL). RESULTS: The biologic level of detection of this assay is 8 pg/mL. Serum PSA levels were undetectable in 82.4% of females, 86.2% of the cystoprostatectomy patients, and 96% of the radical prostatectomy controls. After radical prostatectomy, PSA levels were undetectable at last check in 104 of 168 (61.9%) men. In the 24 men with prostate cancer recurrence, the enhanced sensitivity of 8 pg/mL provided a mean lead time based on conservative calculations of 12.7 to 22.5 months over conventional assays. Thirty-four of the 41 men with detectable PSA levels and no evidence of disease recurrence had PSA levels of 30 pg/mL or less. CONCLUSIONS: PSA levels are undetectable in most men who do not have recurrence of disease after radical prostatectomy. Low but detectable serum PSA levels less than or equal to 30 pg/mL can be produced by nonmalignant sources of PSA. PSA assays with enhanced sensitivity can detect recurrent prostate cancer with significant lead time over conventional assays.

Evaluation of PSA, free PSA, PSMA, and total and bone alkaline phosphatase levels compared to bone scans in the management of patients with metastatic prostate cancer.

BACKGROUND: Metastatic prostate cancer clinical evaluation is difficult. A revaluation of new prostate markers with regard to bone scans was performed. METHODS: Serial markers, including bone alkaline phosphatase (BAP), total alkaline phosphatase (TAP), prostate-specific antigen, total (PSA) and free (fPSA), and prostate-specific membrane antigen (PSMA), were obtained in patients under evaluation and treatment for possible or known metastatic prostate cancer. These were correlated with bone scan results (BSR). RESULTS: Seventy patients were observed from mid-October 1996-January 1997, during which time 171 serum samples were obtained and correlated with semiquantitative bone scan status. PSA and fPSA provided some correlation with BAP and BSR, but only at high levels (> 16-50 ng/ml). Receiver-operating curve (ROC) analysis demonstrated that BAP and TAP had a significant discriminating ability for positive and negative bone scans (> .78), compared to PSMA, PSA, and fPSA. However, percent BAP and TAP only correlated with BSR at a level above six lesions. As the lesions detected by BSR increased, the correlation increased. CONCLUSIONS: BAP is a valuable marker for clinical response evaluations to use in the serial follow-up of patients with metastatic prostate cancer, and correlates well with the bone scan as the number of lesions increase to > 6. PSA or fPSA show comparable results, but only at high levels (> 16-50 ng/ml).

Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: a novel prostate cancer marker.

OBJECTIVES: We describe the expression of a potentially new tumor marker, human glandular kallikrein 2 (hK2), that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer. METHODS: We evaluated 257 radical prostatectomy specimens removed at the Mayo Clinic with pathologic Stage 12 adenocarcinoma to compare the cytoplasmic expression of hK2, PSA, and prostatic acid phosphatase (PAP) in benign tissue, high-grade prostatic intraepithelial neoplasia (PIN), and adenocarcinoma. Two monoclonal antibodies, hK2-A523 and hK2-G586, specific for hK2 were used, as well as antibodies against PSA (PSM-773) and PAP (polyclonal). RESULTS: Intense epithelial cytoplasmic immunoreactivity was observed in every case for hK2-A523, hK2-G586, PSA, and PAP (100% of cases, respectively). The intensity and extent of hK2 expression for both antibodies were greater in cancer than high-grade PIN; furthermore, high-grade PIN was greater than benign epithelium. Cases of Gleason primary grade 4 and 5 cancer showed hK2 staining in almost every cell, whereas there was greater heterogeneity of staining in lower grades of cancer. In marked contrast to hK2, PSA and PAP immunoreactivity was most intense in benign epithelium and stained to a lesser extent in PIN and carcinoma. The number of immunoreactive cells for hK2 and PSA was not predictive of cancer recurrence. CONCLUSIONS: hK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to high-grade PIN and adenocarcinoma. PSA and PAP displayed inverse immunoreactivity compared with hK2. The expression of hK2 and PSA was not predictive of cancer recurrence in patients with Stage T2 carcinoma. Expression of hK2 indicates that this kallikrein antigen is both prostate localized and tumor associated. Tissue expression of hK2 appears to be regulated independently of PSA and PAP. Further studies are needed to determine whether tissue immunoreactivity of hK2 will prove clinically useful in the diagnosis and monitoring of prostate cancer.