1. The tNASP protein, a novel diagnostic biomarker for prostate cancer?
Blake Taylor1, Laura Barba1, Zachary Vaskalis1, Oleg Alekseev1,2
1Campbell University School of Osteopathic Medicine, Buies Creek, North Carolina, USA
2University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
INTRODUCTION
MATERIALS and METHODS (continued)
tNASP localization in the low grade (well-differentiated) androgen dependent (AD) prostate adenocarcinoma. Large epithelial cells with prominent nucleoli are tNASP positive. Small epithelial cells are negative.
Nuclear auto-antigenic sperm protein (NASP) is a histone chaperone and a facilitator of chromatin assembly (Richardson R.T., e.a., 2000). NASP is expressed as two splice variants: tNASP, specific to the testis and embryonal tissues, and sNASP, expressed in all somatic cells.
Tissue microarrays and histological sections were immunohistochemically probed with an anti-NASP antibody (specific to both sNASP and tNASP), affinity purified anti-tNASP antibody, and hematoxylin-eosin. Sera from prostate cancer patients and healthy prostate patients (negative control) were both tested for the presence of antibodies against tNASP using ELISA with a recombinant tNASP fragment as bait. Spearman rank correlation was used to analyze if levels of anti-tNASP antibody co-vary with the levels of Prostate Specific Antigen (PSA).
Interestingly, we and others have shown that tNASP acts as a tumor-associated antigen, present in transformed cell lines and cancer cells in addition to its typical expression in testicular tissue (Ali-Fehmi, R., e.a., 2010; Alekseev, O. M., e.a., 2011). Moreover, tNASP is known to be an extremely auto-immunogenic protein.
tNASP localization in the high grade (poorly-differentiated) AD prostate adenocarcinoma. Almost all epithelial cells are tNASP positive. Stromal cells are negative.
SETTING
Surgical specimens of prostate tissues and sera were received from Roswell Park Cancer Institute, Fox Chase Cancer Institute, and University of North Carolina at Chapel Hill School of Medicine. Total 10 samples from normal prostate (negative control), 24 samples from Benign Prostate Hyperplasia, 34 samples from androgen dependent prostate adenocarcinoma, and 16 castration recurrent prostate adenocarcinoma were obtained.
Expression of tNASP in human testis
HYPOTHESIS
tNASP localization in the Benign Prostatic Hyperplasia (BPH) sample. All epithelial cells are tNASP negative. Some stromal cells are positive.
We suggest that while in cancer-free patients tNASP is sequestered in an immunologically-privileged compartment behind the blood-testis barrier, aberrant expression of tNASP protein in cancer tissues may induce a robust humoral immune response.
We hypothesized that cancer-specific expression of tNASP may serve as a tissue-based marker of PC, and that auto-antibodies produced against cancer-expressed tNASP may be detected as a serum-based marker of the disease.
RESULTS and DISCUSSION
ELISA measurements demonstrated significant increase of anti-tNASP antibody concentration in the sera of prostate cancer patients (763±131 ng/ml) compared to sera of control group (526±171 ng/ml). T-test value was
Expression of tNASP was detected in 76% of prostate specimens from patients with androgen-dependent prostate cancer (AD PC), 43.8% of castration recurrent prostate cancer (CR PC), 29.2% of benign prostatic hypertrophy (BPH), and 5.3% of normal prostates. Combined detection of sNASP and tNASP was 88.2% for AD PC, 48.7% for CR PC, 46.7% for BPH, and 7.1% for normal prostates.
OBJECTIVES
Goal #1 will validate the detection of anti-tNASP antibodies as serum-based biomarkers of prostate cancer. We will address the hypothesis that tNASP-specific antibodies will be present in the blood of prostate cancer patients, but remain absent in Benign Prostate Hyperplasia (BPH) patients or otherwise healthy men.
Goal #2 will validate the utility of tNASP protein as a tissue-based diagnostic and prognostic biomarker of prostate cancer. We will address the hypothesis that expression of tNASP in prostate tissue samples obtained during needle biopsies can be used to diagnose prostate cancer and differentiate its prognosis from BPH.
CONCLUSIONS
ELISA demonstrated markedly elevated concentrations of anti-tNASP antibodies in the sera of PC patients versus healthy patients. Presence of tNASP specific antibodies detected in serum of prostate cancer patients together with high level of PSA could be used as a screening method for early detection in high-risk population groups.
Advanced and more aggressive prostate cancers show higher expression of tNASP protein compared to the earlier stage tumors. Therefore, high levels of tNASP expression detected in prostate tissue samples obtained during needle biopsies can be used for prostate cancer diagnostics and differentiation between BPH and malignant tumor. In addition, tNASP expression could be used as a prognostic marker for prostate cancer tumor staging.
Limitations of the current project: We did not have the opportunity to observe the development of patient’s disease over time, as well as obtain a full set of clinical information for the patients sampled. These limitations did not allow us to draw any prognostic values from the set of data obtained. Testing the tNASP protein in the clinical setting is necessary for further evaluation.
Most prostate cancer patients demonstrated serum concentration of anti-tNASP antibody exceeding 600 ng/ml, whereas normal patients were mostly below this level.
Spearman rank correlation demonstrated that levels of anti-tNASP antibody co-vary with the levels of Prostate Specific Antigen (PSA).
MATERIALS and METHODS
Expression of tNASP protein in different stages of prostate cancer
Hematoxillin-eosin (A) and anti-tNASP antibody staining (B) of the normal prostate. Only a few cells in the stroma have a positive staining for t-NASP. No positive epithelial cells detected. Corpus amylaceous is located in the lumen of the gland. A B
REFERENCES
Richardson e.a. (2000). Characterization of the histone H1-binding protein, NASP, as a cell cycle-regulated somatic protein. J Biol Chem, 275(39),
Batova e.a. (2000). Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides
Ali-Fehmi e.a. (2010) Analysis of the expression of human tumor antigens in ovarian cancer tissues. Cancer Biomarkers, 6, 33-48
tNASP specific fragment (red line) of Homo Sapiens nuclear autoantigenic sperm protein (histone-binding), transcript variant 2 (NM_ ) was PCR amplified from human testis C-DNA library and cloned in pEXP5-CT-TOPO vector for protein expression. This fragment was used for production of tNASP specific antibody and for the detection of anti-tNASP antibody as bait for ELISA assay.
Specific anti-tNASP antibody was purified from anti human NASP antibody (recognizes both tNASP and sNASP) by using tNASP specific protein fragment as a bait in aminolink plus column purification.
Anti-tNASP antibody staining of the prostatic intraepithelial neoplasia (PIN). Numerous epithelial cells are positive. Large atypical epithelial cells with prominent nucleoli demonstrate homogeneous distribution of tNASP protein.
ACKNOWLEDGEMENTS
This project was supported by Campbell University School of Osteopathic Medicine (CUSOM) and a research grant from the US Department of Defense. First and second authors contributed equally to this project.