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Surgical operation

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Surgical operation

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  1. PROTEOMIC APPROACH REVEALED NOVEL PROGNOSTIC BIOMARKERS IN GASTROINTESTINAL STROMAL TUMOR (GIST)Tadashi KondoDaisuke Kubota, Hiroshi Ichikawa, Takashi Tajima, Kenta Mukaihara, Yutaka Sugihara, Yoshiyuki Suehara, KazutakaKikuta, Akira KawaiDivision of PharmacoproteomicsNational Cancer Center Research Institute17th Annual MeetingConnective Tissue Oncology SocietyNovember 14-17, 2012

  2. GastroIntestinalStromalTumor, GIST Sarcoma in GI Neural cell, Cajal c-kit mutation Surgical operation

  3. GastroIntestinalStromalTumor, GIST Sarcoma in GI Neural cell, Cajal Imatinibmesylate, Gleevec c-kit mutation Tyrosine kinase inhibitor

  4. Tyrosine kinase inhibitor, imatinib, for GIST Before treatment 1 month 16 months N Engl J Med. 2002;347(7):472-80

  5. Adjuvant imatinib suppressed metastasis Br J Cancer (2007) 96(11), 1656-1658

  6. Problems of imatinib $4000 /month JAMA 2012 307 (12) 1265 Lancet Oncol 2012; 13: 265-74 JAMA 2012 307 (12) 1265

  7. We need a prognostic biomarker

  8. Metastasiswithin one year after surgery8 casesｖｓNo metastasismore than two years9 cases Protein expression profiling of primary tumors No adjuvant treatments SueharaClin Cancer Res 2008

  9. Two-dimensional difference gel electrophoresis2D-DIGE

  10. Single internal control for multiple gels

  11. Results of 2D-DIGE experiments Tissue 2D-DIGE Metastasis No metastasis SueharaClin Cancer Res 2008

  12. Eight protein spots of pfetin Potassium channel tetramerization domaincontaining protein Spot 1303 Spot 1314 Spot 1376 Spot 1371 Spot 1378 Spot 1372 Spot 1413 Spot 1421 Metastasis within 1 year No metastasis more than 2 years Spot 1421 Spot 1303 Spot 1314 Spot 1376 Spot 1372 Spot 1378 Spot 1371 Spot 1413 SueharaClin Cancer Res 2008

  13. JARO 5, 185-202, 2004

  14. pfetin pfetin

  15. Validation by western blotting No metastasis more than 2 years Metastasis within 1 year pfetin SueharaClin Cancer Res 2008

  16. Validation by immunohistochemistry No metastasis more than 2 years pfetin Metastasis within 1 year SueharaClin Cancer Res 2008

  17. 210 cases in the National Cancer Center Hospital 1.0 pfetin positive 171cases 0.8 0.6 Probability of Disease Free Survival 0.4 pfetin negative 39cases 0.2 P < 0.0001 0.0 0 240 60 120 180 300 360 SueharaClin Cancer Res 2008 Disease Free Survival (months)

  18. Original monoclonal antibody 1 2 3 4 kDa kDa 75 50 96 37 52 156 cases at the NCC 37 25 29 20 2D western • 1. pfetin-GST • 2. GST alone 3. poor GIST 4. good GIST Kikuta et al, JJCO 2010

  19. 100 cases the Niigata University Hospital 1.0 pfetin positive 74cases 0.8 0.6 Probability of Disease Free Survival 0.4 pfetin negative 26cases 0.2 P = 0.0001 0.0 240 60 120 180 300 0 Disease Free Survival (months) Kikuta et al, JJCO 2010

  20. 40 cases the Juntendo University Shizuoka Hospital 1.0 pfetin positive 32cases 0.8 0.6 Probability of Disease Free Survival 0.4 pfetin negative 8 cases 0.2 P = 0.0006 0.0 240 0 60 120 180 Disease Free Survival (months) Kubota et al 2011 JJCO

  21. 72 cases the Juntendo University Hospital 1.0 pfetinpositive N=57 0.8 0.6 Probability of Disease Free Survival pfetinnegative N=15 0.4 0.2 P < 0.0001 0.0 240 360 300 0 60 120 180 Disease Free Survival (months) Kubota et al 2012JJCO

  22. 422cases 1.0 pfetin positive 334 cases 0.8 0.6 Probability of Disease Free Survival 0.4 pfetin negative 88 cases 0.2 P < 0.0001 0.0 0 60 120 180 240 300 360 Disease Free Survival (months)

  23. Independent prognostic biomarker

  24. Risk classification and prognosis in the GIST Low risk, N=228 1.0 Intermediate risk, N=97 0.8 0.6 Probability of Disease Free Survival High risk, N=103 0.4 P < 0.0001 0.2 0.0 240 0 60 120 180 300 360 Disease Free Survival (months) Kubota et al JJCO 2012

  25. Low risk group Intermediate risk group 1.0 1.0 pfetin positive N=194 pfetin positive N=79 0.8 0.8 pfetin negative N=34 0.6 0.6 Probability of Disease Free Survival Probability of Disease Free Survival 0.4 0.4 97 cases 228 cases pfetin negative N=18 0.2 0.2 P< 0.0001 P< 0.0001 0.0 0.0 0 60 120 180 300 60 120 180 300 360 0 240 240 360 Disease Free Survival (months) Disease Free Survival (months) High risk group 1.0 pfetin positive N=49 0.8 In each risk classification group, pfetin had prognostic potentials. 0.6 Probability of Disease Free Survival 103 cases 0.4 pfetin negative N=54 0.2 P< 0.0001 0.0 60 120 180 300 360 0 240 Kubota et al JJCO 2012 Disease Free Survival (months)

  26. pfetin may have growth suppressive effects P < 0.05 Control siRNA1 siRNA2 siRNA3 P < 0.01 Cell growth pfetin GAPDH Control siRNA2 siRNA3 GIST T1 cell

  27. Large format 2D gel device Large gradient gel maker 24x33 cm gel Up to 5000 spots Large electrophoresis apparatus Kondo et al, Nat Protoc 2007

  28. Unfavorable prognostic biomarker in GIST Poor prognosis cases Good prognosis cases ATP-dependent RNA helicase DDX39 DDX39 weak DDX39 strong Kikuta et al, J Proteomics 2012

  29. ATP-dependent RNA helicase DDX39 N = 72 Kikuta et al, J Proteomics 2012

  30. Multi-biomarker: pfetin with DDX39 pfetin positive & DDX39 negative N=46, 93.48% 1.0 pfetin positive & DDX39 negative or pfetin negative & DDX39 positive N=16, 87.50% 0.8 P<0.0001 0.6 N = 72 P=0.0002 Probability of Disease Free Survival 0.4 pfetin negative & DDX39 positive N=10, 0% 0.2 0.0 120 160 20 40 60 0 80 Kubota et al JJCR 2012 Disease Free Survival (months)

  31. Conclusions • Proteomic study identified pfetin and DDX39 as novel prognostic biomarkers in GIST. • Immunohistochemical validation was successful Pfetin, N = 422; DDX39, N=72; pfetin+DDX39, N=72 • Further validation studies will be performedfor the clinical applications.

  32. Y Nakamura M Sakumoto M Kikuchi K Sakamoto Y. Takai F Kito Dr. K Kikuta Dr. T Tajima Dr. D Kubota T Kondo Dr. H Ichikawa Dr. N Hosoya Dr. K Mukaihara Y Sugihara T. Yamaka K Arai A Haga H Yonemori R Sakamoto

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