| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Abstracts of Oak Ridge Posters |
1
Matritech, Inc., 330 Nevada St., Newton, MA 02460;
2
The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, Baltimore, MD 21287-0033
aauthor for correspondence: fax 617-928-0821, e-mail jhlavaty{at}matritech.com
Prostate cancer is the second most common malignancy in men, after skin cancer, and the second most common cause of cancer death in men over age 60 years, after lung cancer. This year, 
198 100 new cases of prostate cancer will be diagnosed in the US, and an estimated 31 500 men will die of prostate cancer (1). Five-year survival is close to 100% when the disease is diagnosed and treated with definitive local therapy while it is still organ-confined, but in approximately one-third of men diagnosed with clinically localized disease, the disease has spread beyond the confines of the prostate at the time of surgery (2)(3).
The Food and Drug Administration approved a serum test for prostate-specific antigen (PSA) in the 1980s. With an upper reference limit in serum of 4 µg/L, 67–80% of prostate cancers can be detected, for a positive predictive value of 24% (4)(5). Combining the serum PSA test with a digital rectal examination can improve the positive predictive value (3)(6)(7). Despite the availability of the PSA test and the moderately high compliance with routine testing recommendations, 20–30% of prostate cancers are missed by the current early detection protocols. The identification of more accurate serum markers for prostate cancer could improve the current clinical capabilities for cancer detection and may reduce cancer mortality.
Proteomics, the large-scale comparison of protein expression patterns, can be used to identify proteins that are associated with disease states such as cancer. These studies have been enhanced by the development of powerful and sensitive new methods, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). In this technique, proteins are adsorbed to a solid matrix, desorbed with a pulsed laser beam to produce gas-phase ions that traverse a field-free flight tube, and then separated according to their velocities, which depend on their mass/charge ratio. The sensitivity of this method for protein identification has been improved by the development of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-MS). SELDI is an affinity-based MS method in which proteins are selectively adsorbed to a chemically modified surface, impurities are removed by washing with buffer, an energy-absorbing material is layered on top, and the proteins are identified by laser desorption mass analysis. SELDI protein analysis has been used to detect prostate cancer-associated proteins in cancer cell lysates, seminal plasma, and serum (8)(9). We report here the use of SELDI to identify a putative prostate cancer-specific protein in the preoperative serum of patients with histologically confirmed prostate cancer.
Serum samples were obtained from The Johns Hopkins School of Medicine (Baltimore, MD). For men with prostate cancer, serum samples were obtained before surgery. Serum samples also were obtained from age-matched controls clinically determined to be cancer-free (serum PSA concentration <2 µg/L and an unremarkable digital rectal exam). All samples were collected with informed consent according to protocols approved by the Institutional Review Board. Serum samples were stored at -80 °C before analysis.
The serum samples were partially purified to remove interfering serum components, fractionated by ion-exchange chromatography, and analyzed by SELDI in a process known as "retentate mapping". Briefly, sera were treated with 1,1,2-trichloro-trifluoroethane to remove lipids, passed over a HiTrap Protein G column (Pharmacia Biotech) to remove immunoglobulins, and then passed over a HiTrap Blue column (Pharmacia Biotech) to remove human serum albumin. The samples were fractionated over a Protein-Pak Q 8HR column (Waters) with a 14-step NaCl step gradient in the concentration range of 0–1 mol/L. Throughout sample preparation, 50 mmol/L NaH2PO4, pH 7.0, was used as the buffer.
Cancer-specific serum protein markers were identified by a three-stage screening strategy. In the first stage, a set of putative prostate cancer biomarkers was identified by comparing sera from five patients whose prostate cancer showed capsular penetration with sera from five cancer-free controls. Each of the 14 fractions from each sample was applied to four different ProteinChipsTM (Ciphergen Biosystems): H4, which has a hydrophobic surface for reversed-phase binding; WCX-2, which binds cationic proteins; IMAC-3-Ni2+, which binds proteins with an affinity for nickel; and SAX-2, which binds anionic proteins. Samples were analyzed in a Ciphergen Series PBS-I ProteinChip System (SELDI mass spectrometer). Ciphergen system software was used to produce composite spectra for each fraction assayed on each chip from the prostate cancer and control samples. The software generated difference spectra that identified four novel peaks (59.7, 22.7, 21.4, and 50.8 kDa) that were present in the cancer samples but not in the controls. These peaks were identified with the H4, WCX-2, and IMAC-3 ProteinChips. A novel peak was defined as having an amplitude at least threefold greater than the baseline.
In the second stage of biomarker identification, an additional 15 cancer serum samples and 15 healthy serum controls were analyzed under the specific conditions used to identify the four putative markers, i.e., each marker was analyzed from a single ion-exchange fraction with a single ProteinChip. On the basis of combined analysis of all of the samples, one novel protein peak was found in all 20 cancer samples but not in any of the prostate cancer-free controls. This was a 50.8-kDa protein identified in the 125 mmol/L salt fraction with the WCX-2 ProteinChip. Representative SELDI profiles are illustrated in Fig. 1
.
|
In the third stage of biomarker identification, 16 additional prostate cancer serum samples were assayed for the presence of the 50.8-kDa peak. The 50.8-kDa protein peak was found in all 36 cancer serum samples (5 from the first stage, 15 from the second stage, and 16 from the third stage of biomarker identification). Studies are now underway to purify and identify the 50.8-kDa polypeptide. This will allow us to elucidate its biochemical association with prostate cancer and develop a sensitive and specific assay that is suitable for prostate cancer screening.
All of the controls and all but two of the cancer patients had serum PSA concentrations tested. Eight of the 34 prostate cancer patients who were tested (24%) had preoperative serum PSA concentrations <4 µg/L, the recommended age-adjusted cutoff for men at least 50 years of age who are otherwise healthy, nonsymptomatic, and have no prior risk for prostate cancer. One of these cancers was organ-confined, and the other seven demonstrated capsular penetration on histologic examination of the resected prostate. All eight of these cancer patients were missed by PSA serum testing but were identified by SELDI analysis of the 50.8-kDa serum protein.
Four of the 18 patients (22%) with PSA between 4.1 and 10 µg/L had tumors with capsular penetration, whereas all 5 patients with PSA between 10.1 and 20 µg/L and all 3 patients with PSA >20 µg/L had tumors with capsular penetration. The probability of organ confinement decreases with increasing preoperative serum PSA concentrations, although PSA testing alone is not completely predictive (2). SELDI analysis conducted in the way we describe is only qualitative, so no direct correlation of concentration could be made with either PSA concentration or tumor grade.
PSA is synthesized predominately in the epithelium of the prostate gland and the periurethral glands; therefore, serum PSA should disappear after radical prostatectomy because the tissue source is removed. Serum PSA concentration is used as a surrogate endpoint for postoperative disease management: the failure of serum PSA to disappear after surgery indicates the presence of persistent disease, and the recurrence of serum PSA signals either cancer recurrence or metastasis. Patients with metastatic prostate cancer typically undergo androgen ablation therapy, which can be effective in suppressing recurrence and metastasis. However, because PSA synthesis and secretion require hormonal influence, androgen suppression can reduce PSA production (4). Therefore, even if occult metastatic sites should develop, they might not secrete enough PSA into the serum to exceed the diagnostic threshold set for routine screening. Another serum marker, preferably androgen-independent, would be a great asset for monitoring prostate disease after surgery.
All of the serum samples tested in this study were collected preoperatively. The findings in this work are promising, but preliminary. It would be informative to test postoperative serum samples for the disappearance of the 50.8-kDa protein and to compare this with serum PSA and clinical disease progression.
Acknowledgments
We acknowledge Natalie S. Rudolph for help in preparing the manuscript.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  J. Li, Z. Zhang, J. Rosenzweig, Y. Y. Wang, and D. W. Chan Proteomics and Bioinformatics Approaches for Identification of Serum Biomarkers to Detect Breast Cancer Clin. Chem., August 1, 2002; 48(8): 1296 - 1304. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
