Pfetin, as a Prognostic Biomarker of Pfetin, as a Prognostic Biomarker of Gastrointestinal Stromal Tumors Gastrointestinal Stromal Tumors RevRev

ealedealed by Proteomics by Proteomics

Yoshiyuki SueharaYoshiyuki Suehara1 3 41 3 4, Kunihiko Seki, Kunihiko Seki22, Kiyonaga Fujii, Kiyonaga Fujii11, Tadashi Hase, Tadashi Hasegawagawa2 2 , Tadakazu Shimoda, Tadakazu Shimoda22, Yasuo Beppu, Yasuo Beppu44, Akira Kawai, Akira Kawai44, Setsuo Hir, Setsuo Hirohashi ohashi 11,, Tadashi KondoTadashi Kondo11

1. Proteome Bioinformatics Project, National Cancer Center Research Institute, Tokyo, Japan2. Clinical Laboratory Division, National Cancer Center Research Hospital, Tokyo, Japan3. Department of Orthopedic Surgery, Juntendo University School of Medicine, Tokyo, Japan4. Orthopedic Surgery Division, National Cancer Center Hospital, Tokyo, Japan

MWMW(KDa)(KDa)

Isoelectric focuIsoelectric focusingsing

8080

5656

3636

3030

1818

pH 4.0pH 4.0 pH 7.0pH 7.0

Procedure of Proteomics StudyProcedure of Proteomics Study

1500-2000 1500-2000 spotsspots

Protein was extracted and labeled with CyDye

2D-Image

Data-MiningProtein Protein spots spots were were identifidentifiedied

High-speed/-throughput 1D-RP mLC/NSI-MS/MS Ana

lysis

Mass Spectrometry

Surgical Specimen

Back Ground (GISTs)Back Ground (GISTs)• GISTs are the most common primary mesenchymal tumor o

f the digestive tract.• It is genetically characterized by the presence of mutations a

nd overexpression of KIT and, clinically, characterized by their significant response to treatment with imatinib.

• However, there are certain populations of patients who can be treated only by simple resection and do not need imatinib treatment.

The aim of this study is to develop prognostic biomarkers for GISTs, by means of proteomic approach, and identify high-risk patients who would need further adjuvant treatment such as imatinib.

StrategyStrategy“Poor prognosis GISTs”

= Metastasis within 1 year

(8 samples)

“Good prognosis GISTs”

= No-metastasis over 2 years

Low or IM Risk group *

(9 samples)

＊ Pathological factors (Risk Classification-> Hasegawa T. et al: Human Pathology. 2002)

vs

Identify the informative Identify the informative spotsspots

Confirmed these resultsConfirmed these resultsWestern-blot, Immunochemical Western-blot, Immunochemical

studystudy

Large-scale sample set (Immunohistochemical study)Large-scale sample set (Immunohistochemical study)Verified the power of biomarkerVerified the power of biomarker

Cluster Analysis 43 spots Cluster Analysis 43 spots (p < 0.01)(p < 0.01)

Poor-prognosis GISTs

Good-prognosis GISTs PfetinPfetin

1513 spots

43 spotsWilcoxon test

Poor-prognosis GISTsMetastasis within 1 year(samples 1-8)

Good-prognosis GISTs No-metastasis over 2 years Low or IM Risk group(samples 9-17)

Immunohistochemical studyImmunohistochemical study(Pfetin antibody)

Western Western BlottingBlotting

Poor prognosis GISTs Good prognosis GISTs

(Pfetin antibody)

Good prognosis GISTs

Poor prognosis GISTs

210 cases, M0, primary samples

Metastasis-Free Survival of the M0 Metastasis-Free Survival of the M0 GISTsGISTs Patients Patients According to the Expression of Pfetin According to the Expression of Pfetin (NCC :210 cases)(NCC :210 cases)

Suehara Y et al. Clin Cancer Res (2008) 14: 1707-1717Nature Clinical Practice Oncology (2008) 5, 364-365

5-year: 93.9%

5-year: 36.2%

P < 0.0001

(Immunohistochemical Study)(Immunohistochemical Study)

Overall Survival of the M0 GISTs Patients Overall Survival of the M0 GISTs Patients According to the Expression of Pfetin According to the Expression of Pfetin (NCC :210 cases)(NCC :210 cases)

Suehara Y et al. Clin Cancer Res (2008) 14: 1707-1717Nature Clinical Practice Oncology (2008) 5, 364-365

P < 0.0001

5-year: 97.2%

5-year: 76.5%

210 cases, M0, primary samples

(Immunohistochemical Study)(Immunohistochemical Study)

Metastasis-Free Survival Curve of GISTsMetastasis-Free Survival Curve of GISTs According to Risk Classification (NCC :210 case According to Risk Classification (NCC :210 case

s)s)

N=110 N=46

N=54

High risk

Suehara Y et al. Clin Cancer Res (2008) 14: 1707-1717Nature Clinical Practice Oncology (2008) 5, 364-365

Intermediate riskLow risk

Metastasis-Free Survival Curve of GISTsMetastasis-Free Survival Curve of GISTs According to Pfetin Immunochemical Stain (NCC :210 case According to Pfetin Immunochemical Stain (NCC :210 case

s)s)

High risk

N=110 N=46

N=54

Pfetin positive (N=100)

Pfetin positive (N=27)

Pfetin positive (N=44)

Pfetin negative (N=10) Pfetin negative (N=2)

Pfetin negative (N=27)

P = 0.0002

Suehara Y et al. Clin Cancer Res (2008) 14: 1707-1717Nature Clinical Practice Oncology (2008) 5, 364-365

Intermediate riskLow risk

P<0.0001 P=0.0109

P=0.0002

Multivariate Analysis of Multivariate Analysis of Metastasis Free Survival by Metastasis Free Survival by

Cox RegressionCox RegressionMultivariate analysis of metastasis free survivl by Cox regression

Variable P value Relative risk 95% confidence intervalSite

StomachNon-stomach 0.0270 2.21 1.09-4.49

Size<5 cm

5-10cm15cm< 0.0070 2.05 1.22-3.44

DifferentationScore 1Score 2 <0.0001 10.40 3.68-29.45

PfetinPositive

Negative 0.0020 3.75 1.60-8.81

ConclusiConclusionon

• We examined the proteomic profile of GISTs accWe examined the proteomic profile of GISTs according to prognosis using both 2D-DIGE and clording to prognosis using both 2D-DIGE and clinical data.inical data.

• Pfetin expression was a significant predictor Pfetin expression was a significant predictor for metastasis and survival of patients with Gfor metastasis and survival of patients with GISTs.ISTs.

• Pfetin can be a strong candidate for a biomarkPfetin can be a strong candidate for a biomarker to predict the metastasis in GISTs patienter to predict the metastasis in GISTs patients. s.