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Moscow Research Institute of Medical Ecology

Moscow Research Institute of Medical Ecology. Elaboration of new method for the early non-invasive prostate cancer diagnostic. Disadvantages of the PSA diagnostics. “Grey zone” – 4-10 ng/ml Biopsy verification. Hepsin structure. Normal prostate Benign prostate

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Moscow Research Institute of Medical Ecology

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  1. Moscow Research Institute of Medical Ecology Elaboration of new method for the early non-invasive prostate cancer diagnostic

  2. Disadvantages of the PSA diagnostics • “Grey zone” – 4-10 ng/ml • Biopsy verification

  3. Hepsin structure Normal prostate Benign prostate hyperplasia Prostate cancer Prostate cancer gr. II gr. IV-V purple – protease domain, blue - SRCR domain, green – active senter Immunohistochemical detectionof hepsin in tissue sections Antibodies Neutralizing Hepsin Protease Activity Do Not Impact Cell Growth but Inhibit Invasion of Prostate and Ovarian Tumor Cells in Culture J. Xuan et al . Cancer Research 2006; 66(7): 3611-3619

  4. Principle of the method • Kinetic enzymatic assay • Urea cells after prostate massage • Chromogen staining

  5. Kit composition PBS – 50tab. Lysis buffer – 25ml. Protease inhibitors cocktail – 1 bot. Incubation buffer solution – 10ml. Substrate – 100 mg. Hepsinrecombinant– 1,5nmol/l*min +/- 10%. • Bacterial strain producing human recombinant hepsin was deposited in Russian National Collection of Industrial Microorganisms (VKPM), accession number B-10098 • Patents: • Bacterial strain producing human recombinant hepsin soluble form (HPNTM-A) • Prostate cancer diagnostic kit and prostate cancer diagnostic technique.

  6. Groups of patients

  7. Determination of prostate pathology with PSA concentration or HEPSIN activity

  8. 1 - Tricoli JV, Schoenfeldt M, Conley BA. Detection of prostate cancer and predicting progression: current and future diagnostic markers. Clinical Cancer Research 2004; 10(12 Pt 1): 3943-53. 2 -Sardana G. Dowell B., Diamandis P. Emerging biomarkers for the diagnosis and prognosis of prostate cancer. Clinical Chemistry 2008; 54:12, pp. 1951-1960

  9. Thank you for your attention!

  10. Supplementary slides

  11. Prognostic value of the method p<0.05

  12. Specificity of the method Tumor cell lines

  13. Linearity and sensitivity of the method Minimaldetecting activity – 0,05 nmol/l*min

  14. Recombinant hepsin plasmid DNApETM-HPNTM-A

  15. Hepsin purification 2nd step – ion-exchange chromatography 1st step - affinity chromatography М 1 2 3 4 1 – E.coli lysate 2 – 1st step chromatography, peak #2 3 – 2nd step chromatography, peak #3 4 - 2nd step chromatography, peak #3 after incubation with thrombin

  16. Substrate specificity of PSA and Hepsin PSA hydrolyzes substrate in high efficacy (2200-3100 М-1s-1) in: Ser SerTyr Ser Gly Gln His Asp Leu Ser Asn Hepsin hydrolyzes substrate in: Lys Asn Lys ArgSer

  17. Substrate selection KPR AAR (H-D-Lys-(γ-Cbo)-Pro-Arg-pNA.2AcOH (Cbo- Ala-Ala-Arg-pNA.2AcOH)

  18. Inhibition of hepsin activity – the possible direction of anti-cancer therapy Anthralin (dithranol) Recombinant hepsin activity according to anthralin consentration

  19. Specifications of the techniques

  20. Conclusions • Hepsin is a marker of high specificity for the prostate cancer • Sensitivity of the method – 0,05 nmol/l*min and linearity of the method is between 0,05 nmol/l*min and 4,5 nmol/l*min • Hepsin activity in prostate cancer patients and other patients with benign prostate disorders has statistically significant difference • Hepsin activity has very poor correlation with the age of patients

  21. Deficiency in PSA diagnostics • Prostate-Specific Antigen Screening: Friend or Foe? M. Linn et al. Urol Nurs. 2007;27(6):481-489. • Operating characteristics of prostate-specific antigen in men with an initial PSA level of 3.0 ng/ml or lower. I.Thompson et al. JAMA. 2005;294(21):2698. • Prevalence of prostate cancer among men with a prostate-specific antigen level less than/equal 4.0ng/mL. I.Thompson et al. The New England Journal of Medicine 2004; 350: 2239-2246. • Screening for prostate cancer: Opportunities and challenges.I.Thompson et al. Surgical Oncology Clinics of North America 2005; 14: 747-760.

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