Authorship:
Kenji Nakayama(1, 2), Takahiro Inoue(1), Sadanori Sekiya(3), Yu Miyazaki(1), Naoki Terada(1), Takayuki Goto(1), Koichi Kojima(3), Shinichi Iwamoto(3), Koichi Tanaka(3) and Osamu Ogawa(1)
(1) Department of Urology, Graduate School of Medicine, Kyoto University; (2) Shimadzu Techno-Research, Inc.; and (3) Shimadzu Corporation, Kyoto, JAPAN.
| Short Abstract Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, PSA is not a cancer-specific marker. Therefore, our aim was to discover new biomarkers for the diagnosis of PCa. We focused on urine samples voided following prostate massage and conducted a peptidomic analysis of them using MALDI-TOF/MS. Multivariate analysis of the mass spectra of urinary extracts revealed a 2331 Da peptide as a C-terminal PSA fragment. Their quantitative analyses using MALDI-TOF/MS revealed that the peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects. Those results indicate that the peptide may become a new pathognomonic biomarker for the PCa diagnosis. |
Long Abstract
Introduction: Prostate cancer (PCa) is one of the most common cancers and the leading cause of cancer-related deaths in men [1]. The mechanisms underlying the development of PCa have not yet been determined because of its clinical and histological heterogeneity. The incidence for PCa has markedly increased in Japan recently [2, 3]. The large-scale clinical detection of prostate-specific antigen (PSA) levels in the serum as a PCa biomarker has been carried out since the 1990s [3-7]. Although the overall benefits and risks of population PSA screening for prostate cancer continue to be assessed [8], PSA is known to be an excellent organ-specific, but not a cancer-specific marker [9], which continues to be a clinical problem. This is further compounded by the longer-living, aging population and elevated PSA levels associated with increasing age [3, 10].
Although the sensitivity of PSA in the detection of cancer is high, its specificity is limited, and screening healthy men often causes false cancer alarms (e.g. due to inflammation or benign hyperplasia) and unnecessary prostate biopsies [3, 11]. Several issues have been identified regarding the sub-optimal sensitivity of PSA testing for PCa screening, which lead to unnecessary biopsies, over-diagnoses, and overtreatments [12-17]. A significant amount of effort in research is currently being directed towards improving the accuracy of PCa screening [12]. Moreover, a strong emphasis has been placed on the need to identify novel biomarkers for the diagnosis of PCa.
Proteomic techniques applied to serum, plasma, and urine may provide valuable information regarding biomarkers and marker patterns, which may be used to improve the detection of cancer [18]. In the present study, we focused on urine samples voided following prostate massage (digital rectal examination [DRE]), which were expected to contain many peptides and protein fragments secreted from prostatic microenvironments that could enable the detection of secreted prostate products as potential sources of PCa-specific biomarkers [18,19]. Therefore, we conducted peptidomic and proteomic analyses of urine samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) in order to discover new potential pathognomonic biomarker candidates for the diagnosis of PCa [20].
Materials and Methods: This study was conducted with the approval of the Ethics Committee of the Kyoto University Graduate School of Medicine. Informed consent was obtained from all cases for the examinations and experiments conducted. Clinical materials were used after written informed consent was obtained, according to protocols approved by the Institutional Review Board of Kyoto University Hospital.
We focused on urine samples voided following DRE and conducted a peptidomic analyses of these samples using MALDI-TOF/MS. The individuals from which urine samples were collected following DRE were classified into 2 groups; i.e., PCa patients and non-cancer subjects. The confirmatory diagnosis of PCa was made by a histological diagnosis from prostate biopsy specimens or prostate glands removed following surgery, when performed. All urine samples from the non-cancer group were collected prior to Holmium Laser Enucleation of the prostate (HoLEP). The diagnoses of non-cancer subjects were defined as non-malignant by histological diagnoses of prostate glands removed by HoLEP. All histological diagnoses were confirmed by genitourinary pathologists in Kyoto University Hospital.
Urinary biomaterials were concentrated and desalted using CM-Sepharose prior to the following analyses being performed by MALDI-TOF/MS: 1) differential analyses of mass spectra; 2) determination of amino acid sequences; and 3) quantitative analyses using a stable isotope-labeled internal standard.
Results: Multivariate analysis of the MALDI-TOF/MS mass spectra of urinary extracts revealed a 2331 Da peptide in urine samples following DRE. This peptide was identified as a C-terminal PSA fragment composed of 19 amino acid residues. Moreover, quantitative analysis of the relationship between isotope-labeled synthetic and intact peptides using MALDI-TOF/ MS revealed that this peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects.
Conclusions: The results of the present study indicate that the 2331 Da peptide fragment of PSA may become a new pathognomonic biomarker for the diagnosis of PCa. A further large-scale investigation is currently underway to assess the possibility of using this peptide in the early detection of PCa. In the conference, its several unique chemical characteristics are reported and discussed.
References
1. Siegel R, Naishadham D, Jemal A. (2013) CA Cancer J Clin 63: 11-30.
2. Marugame T, Mizuno S. (2005) Jpn J Clin Oncol 35: 690-691.
3. Ito K. (2009) Int J Urol 16: 458–464.
4. Catalona WJ, Smith DS, Ratliff TL, Dodds KM, Coplen DE, et al. (1991) N Engl J Med 324: 1156-1161.
5. Shibata A, Ma J, Whittemore AS. (1998) J Natl Cancer Inst 90: 1230-1231.
6. Barry MJ. (2001) N Engl J Med 344: 1373-1377.
7. Catalona WJ, Loeb S. (2010) J Natl Compr Canc Netw 8: 265-270.
8. Lin K, Lipsitz R, Miller T, Janakiraman S. (2008) Ann Intern Med 149: 192–199.
9. Roobol MJ, Zappa M, Maattanen L, Ciatto S. (2007) Prostate 67: 439–446.
10. Oesterling JE, Jacobsen SJ, Chute CG, Guess HA, Girman CJ, et al. (1993) JAMA 270: 860-864.
11. Stephan C, Jung K, Lein M, Diamandis EP. (2007) Eur J Cancer 43: 1918–1926.
12. Loeb S, Catalona WJ. (2010) Nat Rev Urol 7: 184-185.
13. Mistry K, Cable G. (2003) J Am Board Fam Pract 16: 95–101.
14. Pashayan N, Powles J, Brown C, Duffy SW. (2006) Br J Cancer 95: 401–405.
15. Etzioni R, Penson DF, Legler JM, di Tommaso D, Boer R, et al. (2002) J Natl Cancer Inst 94: 981–990.
16. Ilic D, Neuberger MM, Djulbegovic M, Dahm P. (2013) Cochrane Database Syst Rev 1: CD004720. doi: 10.1002/14651858. CD004720.pub3.
17. Andriole GL, Grubb RL 3rd, Buys SS, Chia D, Church TR, et al. (2009) N Engl J Med 360: 1310–1319.
18. Schiffer E. (2007) World J Urol 25: 557-562.
19. Drake RR, White KY, Fuller TW, Igwe E, Clements MA, et al. (2009) J Proteomics 72: 907-917.
20. Nakayama K, Inoue T, Sekiya S, Terada N, Miyazaki Y, et al. (2014) PLoS One 9(9): e107234. doi: 10.1371/journal.pone.0107234.