Significance Of Serum Protein Fingerprints Of Different Phases During The Carcinogenesis And Progression Of Gastric Carcinoma

Significance of serum protein fingerprints of different phases during the carcinogenesis and progression of gastric carcinomaIntroductionGastric cancer is one of the most common malignancy in China,The inci-dence is about 20/100 thousand and mortality is the first in the county and sec-ond in the city.The main reason of poor prognosis of gastric carcinoma is that gastric cancer can't be diagnosed in the early stage and most cases diagnosed in clinic are advanced gastric carcinoma with distant metastasis.At present,it is a effective way to discover gastric cancer by gastroscopy,but it also has deficit for spread examination.So it is important to find a simple,sensitive and specific method to diagnose early gastric cancer.As reported in domestic,the metastasis rate of gastric cancer is 64.2%,among these cases,the most in male are liver metastasis and ovary metastasis in female.The average survival time of these pa-tients are 5.5 months and 6 months,respectively.How to find a effective meth-od to early diagnose for liver and ovary metastasis from gastric cancer has be-come a big challenge to enhance the patients'five-year-survival rate.Very recently,a new technique,surface-enhanced laser desorption ioni-zation/time-of-flight mass spectrometry(SELDI/TOF-MS),has been de-veloped,whereby small amounts of proteins are bound to a biochip carrying spots with different types of chromatographic material,i.e,hydrophobic,hydro-philic,cation-exchanging,and anion-exchanging characteristics.SELDI/ TOF-MS can be apphed to analyze differential peptide and protein expression patterns in cancer and noncancer patients in various biological fluids. ObjectiveTo investigate the identification of serum protein fingerprints between the healthy,patients with gastric cancer in different clinico-pathological stages and histological differentiation using SELDI/TOF-MS and Protein-Chip technolo-gy,and find out the specific serum protein biomarkers related to the carcinogen-esis and progression of gastric carcinoma.Materials and Methods1.Clinical MaterialsFifteen patients with gastric cancer were involved in this study,male;fe-male 39;18,ages 42-84,average age 60 15.3.Clinico-pathological staging; ealy 19 cases,advanced 38,among the advanced group,3 without any metasta-sis;11 cases with distant metastasis;9 metastasizing to liver;1 to the ovary;1 to the kidney.There were 7 cases with benign lesion of gastric mucosa diagnosed through gastroscope.Thirty cases of the healthy from the normal people perform-ing health examination,male;female 18;12,age 35-78,average age 49 18.2.Each sample of limosis blood was collected from patients before accepting any treatments,centrifuged for 5mins in 2500 rpm after congealed.The sera were stored in refrigerator at-70℃for later use.2.Detection of the serum protein fingerprintsIn present study,Surface-enhanced laser desorption ionization/time-of -flight mass spectrometry(SELDI/TOF-MS,Ciphergen Com,USA)and protein-chip(WCX2)were used to detect the serum protein fingerprints.Time-of-flight spectra were generated by laser shots collected in the pos-itive mode of WCX2.The laser intensity was set to 190,detector sensitivity to 8,and 60 laser shots per average spectrum were performed.A mixture of mass standard calibrant proteins was used for calibration of mass accuracy.Peak de-tection was performed using the Ciphergen Protein-Chip Software 3.0.Spectra ranging from 1000 to 50 000 m/z were selected for the analysis.Smaller masses were not used,since artifacts with EAMS and other contaminants could not be excluded.The spectra were normalized according to the intensity of total ion cur-rent.Automatic peak detection was performed in the range of 1000 to 20000 M/ Z,following baseline subtraction.3.Statistical analysisStatistical analysis was automatically performed using Biomarker Wizar soft-ware.P value less than 0.05 was considered as significant.4.The construction of decision-tree classification for screening gastric cancerThirty patients with gastric cancer(10 in early stage,14 advanced,6 with distant metastasis)and 30 normal persons were included to construct the deci-sion-tree classifier model.The specificity of model was tested with the blind set including 27 gastric cancer,7 gastric benign lesion and 19 healthy cases.Results1.The changes.of serum protein fingerprints among the healthy,patients with gastric benign lesion and gastric canerThere were 26 different proteins between gastric benign lesion patients and the healthy,while only 6 different proteins between gastric benign lesion and early gastric cancer.Between early gastric cancer and the healthy,there were 42 different proteins,among them 27 proteins lowly expressed and the others highly expressed in early gastric carcinoma.Proteins 8367M/Z and 31851M/Z expressed higher in early gastric cancer than in gastric benign lesion group, while proteins 4799M/Z and 6196M/Z lower in early gastric cancer than in gas-tric benign lesion group.2.The changes of serum protein fingerprints among the groups of different histological differentiation of gastric cancersThere were 10 proteins,which were 2091M/Z,2197M/Z,2281M/Z, 2298M/Z,2310M/Z,2520M/Z,3105M/Z,4162M/Z,4479M/Z,8147M/Z., highly expressed in poorly differentiated gastric cancer,while in well differentia-ted gastric cancer only one protein 34927M/Z highly expressed. 3.The changes of serum protein fingerprints among the groups of gastric cancer in different clinico-pathological stages.The expression of five different proteins showed a different changing tenden-cy with the progressing of gastric cancer,proteins 4078 M/Z,3895 M/Z and 4289M/Z expressed gradually lower,while proteins 11687M/Z and 36997M/Z gradually higher with progression of the cancers.4.The changes of serum protein fingerprints btween liver metastases from gastric cancer and from other digestive tract cancerBetween the liver metastases derived from gastric cancers and from other di-gestive tract cancers,16 different proteins were obtained.Nine proteins 2923M/ Z,2024M/Z,10260M/Z,3887M/Z,3102M/Z,9288M/Z,2637M/Z, 2952M/Z,8149M/Z expressed higher in liver metastasis from gastric cancer than from the other digestive tract cancers;Proteins 4329M/Z,5836M/Z, 5085M/Z,4887M/Z,5067M/Z,6196M/Z,5705M/Z expressed lower in liver metastasis from gastric cancer than from the other digestive tract cancers.Inter-estingly,expression of proteins 4887M/Z,6196M/Z,10260M/Z,9288M/Z were significantly different between gastric cancer and the healthy cases.5 The construction and evaluation of decision-tree classifier for screening early gastric cancerThree decision tree classifier models had been constructed by our study.The modelⅠwas consisted of 6196M/Z protein.The results of blind screen test for modelⅠshowed that 26(96.3%)patients with gastric cancers were cor-rectly classified,only one was falsely recognized as non-cancer.Seventeen (89.5%)healthy caseses were classified correctly,only two were recognized as cancer falsely.Three(42.9%)patients with gastric benign lesion were correct-ly classified.The overall sensitivity of modelⅠwas 96.3%,specificity 76.9%, negative predicting value 95.2%,positive predicting value 81.3%,ffective rate 86.8%.The modelⅡwas consisted of 6366M/Z protein.The results of blind screen test for modelⅡshowed that 26(96.3%)patients with gastric cancers were correctly classified,only one was falsely recognized as non-cancer.All 19(100%)healthy cases were correctly classified.Three(42.9%)patients with gastric benign lesion correctly classified.The overall sensitivity of the mod-elⅡwas 96.3%,specificity 84.6%,negative predicting value 95.7%,posi-tive predicting value 86.7%,total effective rate 90.6%.The modelⅢwas consisted of 3 different proteins,13738M/Z,8931 M/Z and 2048M/Z.The results of blind screen test for modelⅢⅠshowed that 26 (96.3%)patients with gastric cancers were correctly classified,only one was falsely recognized as non-cancer.Eighteen(94.7%)healthy cases were clas-sifted correctly,only one was recognized as cancer falsely.One(14.3%)pa-tients with gastric benign lesion was correctly classified.The overall sensitivity of modelⅢwas 96.3%,specificity 73.1%,negative predicting value 94.7%, positive predicting value 78.8%,total effective rate 84.9%.Nine patients with early stage gastric cancer of the blind set were accurately recognized as cancer by these three decision tree classifier models.Conclusions1.Compared with the healthy,the serum protein fingerprint of early gastric cancer was significantly different.This finding suggested that the serum protein fingerprinting has potential value for screening patients at high risk for gastric cancer.The proteins 6366 M/Z and 6196M/Z first found in this study have not been presents in the relevant literatures so far,and may be the new biomarkers for predicting and the early diagnosis of gastric cancer.2.There were different serum protein fingerprints between different grades of histological differentiation of gastric cancers.That poorly diffrentiated adeno-carcinoma possessed most distinct protein fingerprint pattern suggests that the abnormality of genes and proteins related to poorly diffrentiated cancer may be much more complicated than usually understood.3.Between the liver metastases derived from gastric cancers and from other digestive tract cancers,16 different proteins were obtained.The proteins 6196 M/Z,4887 M/Z,10260 M/Z and 9288M/Z are more likely to be the new bio-markers identifying the liver metastasis derived from gastric cancer.4.Among 3 decision-tree classification models constructed in the study, the most efficient one(modelⅡ)was consist of single protein 6366M/Z.the re-sult of test with blind set display that its sensitivity is 96.3%,specificity 84. 6%,negative predicting value 95.7%,positive predicting value 86.7%,total effective rate 90.6%.Further study is needed to explore the potential value of clinical application in predicting occurrence of gastric cancer.However,there is still some extent limitation for thses three decision-tree classification models to identifying the gastric cancer from gastric benign lesion.