| Malignant neoplasms of digestive system constitute a major health problem around the world. It is estimated that approximately 10.9 millian new cases of digestive carcinomas and 6.7 millian deaths occured in the word in 2002. By some estimates, gastric cancer and primary hepatic carcinoma (PHC) is the second and third most common malignant disorder worldwide. In our country, the high incidence and the poor prognosis of gastric carcinoma and PHC have made people's life and heath in serious danger. So it is very important field to prevent and treat digestive tumor.In this study we focus on gastric cancer and primary hepatic carcinoma (PHC) by reasons of their high incidence and representative of digestivesystem neoplasms. Gastric cancer is a debilitating disease associated with a high mortality. Gastric carcinoma is rampant in many countries around the world. In 2002, more than 876, 000 new cases of gastric cancer are estimated to occur in the word. It remains the second leading cause of cancer death. The incidence of gastric carcinoma is extremely high and its incidence has been on the increase for less than 40 years in China. Its successful treatment relies on early diagnosis, but this remains a challenge since the progression of the malignancy is usually silent until it reaches a more advanced stage which prognosis is poor. Certainly, early detection can drastically facilitate treatment and improve the long-term survival of the patient. Thus, gastric carcinoma continues to pose a major challenge for epidemiologists, gastroenterologists, surgical oncologists, radiation oncologists, and medical oncologists. PHC is also rampant in many countries around the world. According to the statistic of enregister in Shanghai, the incidence of the liver cancer is 39.86 in each 100, 000 population, in which female is 16.45 each 100, 000 persons. PHC has become the third most common malignant disorder in China. PHC is often diagnosed at an advanced stage and remain a poor prognosis.Primary hepatic carcinoma (PHC) presents at a late clinical stage in more than 80% of patients and the 1,3,5-year survival of PHC in this population is 66.1%,39.7% and 32.5%. By contrast, the 1,3,5-year survival for patients with early stage liver cancer exceeds 93.5%,70.1% and 59.1%. Therefore, increasing the number of patients diagnosed with early stage disease should have a direct effect on the mortality and economics of this cancer without the need to change surgical or chemotherapeutic approaches.Early diagnosis improves the long-term survival chances of patients withcancers. However, one of the features of nearly all human cancers is that there is molecular heterogenicity, meaning that screening for a single diagnostic marker is not efficient, with some patients not being correctly diagnosed. A logical development to improve the early diagnosis of cancer is to therefore simultaneously screen for multiple biomarkers to increase the probability of detection. The sensitivity of the current single biomarkers in tumor diagnosis is low (usually less than 40%) and complicated by a high return of 'false-positives. Further, none of the existing serum markers can be used individually for screening for cancer. It would be highly desirable to have a new rapid and sensitive diagnostic test for cancer. And new technologies for the detection of early stage cancer are urgently needed.Clearly , the use of single biomarkers to diagnose cancer has disadvantages. It is a logical step, therefore, to explore the possibility that a use of a combination of biomarkers could improve diagnostic power. Pathological changes within an organ might be reflected in proteomic patterns in serum. Proteomics is the large-scale study of proteins, or the simultaneous measurement of a large number of expressed proteins. Proteomic profiling enables a new approach to the discovery of biomarkers in disease. It has recently been shown to be useful in identifying biomarkers for the diagnosis of bladder, prostate, ovary, breast, liver malignancies and other cancers.With the development in genomic research, protemics has become an extremely active and increasingly important part of the worldwide life science research and the core of the functional genomics era or the "postgenomicera". Several new technologies in proteomics developed in the past few years greatly boosted the discovery-based research of tumor markers beyond thedisadvantages of traditional approaches.Generation of the mass spectra requires only a small serum sample that could be obtained by fingerprick, and results are obtained in less than 30 min. Cost-effective, high-throughput screening is feasible. The concept and tool are flexible, and can be applied to any biological state and to data derived from future mass spectrometry platforms with higher resolution, sensitivity, and mass accuracy than the platform used herein. Samples can be applied to mass spectroscopy chips in the local laboratory and then transported to a central laboratory that houses the analytical software. Moreove , transportation of the raw spectra via the internet to a central site that incorporates an ever-expanding training set is feasible. By this approach, the pattern itself, independent of the identity of the proteins or peptides, is the discriminator, and might represent a new diagnostic paradigm.Modern proteomic profiling involves ProteinChip technology that sometimes utilizes an enhanced surface laser desorption/ionization (SELDI) time of flightmass spectrometry system. Clinical proteomics involves the analysis of protein expression profiles in samples for de novo discovery of disease-related biomarkers and for gaining insight into the biology of disease processes. Analysis of samples through SELDI has tremendous potential because of its ability to conveniently resolve a wide spectrum of proteins. The technology utilizes patented biochip arrays to capture time of flight mass spectrometry data on individual proteins, or groups of proteins, with common biochemical properties such as hydrophobicity, from complex mixtures. It is numerous advantages over other analytic methods such as 2D-gel electrophoresis. For example, although 2D-gel electrophoresis is capable ofresolving several thousands of proteins, it is labor intensive, requires a large sample volume And needs an additional evaluation assay system. Conversely, SELDI has a much higher throughput capacity, requires significantly lower amounts of samples, has sub-femtomol range sensitivity and enables higher resolution over a lower mass range. There are also several ProteinChip arrays available that have different chromatographic properties, such as anion exchange, cation exchange, immobilized-metal affinity, with hydrophilic or hydrophobic surfaces. These are directly compatible with serum and urine, which are the commonly used body fluids to screen for biomarkers. Recent studies have successfully implemented the use of SELDI on sera to identify unique proteomic patterns that discriminate between malignant and benign growth in the ovary.The SELDI assay and detection system has not been used for the profiling of protein clusters in gastric cancer or liver cancer. In the present studies, therefore, we employed SELDI to probe for serum proteins that may be expressed in patients with gastric cancer and liver cancer. We addressed the possibility that serum proteins critical to the progression of gastric cancer and liver cancer can be identified through the changes in peptide/proteinmass spectral patterns. We also investigated if such potential changes in spectral patterns are discriminatory of patients with gastritis compared to healthy volunteers.Part I Serum proteomic patterns for gastric lesions as revealed by SELDI mass spectrometryAim: SELDI-TOF mass spectrometry was used to investigate protein expression in sera of patients with gastric cancer and gastritis compared to normal volunteers. To establish a serum protein pattern model for screening gastric cancer, and used it to identify proteomic patterns in serum that distinguish neoplastic from non-neoplastic disease within the gastric cancer. Material: Type protein chip reader (PBS II, Ciphergen Biosystems, Inc., Fremont , USA); strong anionic exchanger (SAX2) Chips (Ciphergen Biosystems, Inc., Fremont, USA); the BiomarkerWizard software (version 3.0, Ciphergen Biosystems, Fremont, CA, USA). Approximately 5 ml of blood was withdrawn via vein puncture from each patient. Training group: gastric cancer patients 33 samples (confirmed by pathology), gastritis patients 30 samples (confirmed by gastroscopy and pathology), healthy volunteers 31 samples; independent group: gastric cancer 15 samples, gastritis 10 samples, healthy volunteers 10 samples. Methods: SELDI-TOF mass spectrometry was used to investigate protein expression in sera of patients with gastric cancer and gastritis compared to normal volunteers. A preliminary "training" set of spectra derived from analysis of serum from 33 patients with gastric cancer and 30 patients with and 31 healthy volunteers were analyzed by an iterative searching algorithm that identified a proteomic pattern that completely discriminated cancer from noncancer. The discovered pattern was then used to classify an independent set of 35 masked serum samples: 15 from gastric cancer, and 10 from gastritis, and 10 from healthy volunteers. Results: A comparison of protein mass spectra obtained from the sera of the gastric cancer group and the healthy volunteer group revealed 45 protein peaks. There were statistical differences between 3 protein peaks located at 5910Da, 5084 Da and 8691 Da (P<0.05); the intensity of protein peaks at 5910 Da in the sera from patients with gastric cancer was clearly higher than that of the healthy controls (P<0.05). Bi-peak or tri-peaks at 5084 Da were also observed in the sera from patients with gastric cancer. Further, the protein peak at 8691 Da in the sera frompatients with gastric cancer was down-regulated compared to normal healthy volunteers. Using the above profiles, 30 of 33 patients diagnosed pathologically with gastric cancer were correctly identified by SELDI. Twenty-nine of 31 healthy volunteers were correctly identified as normal. The sensitivity of neoplastic identification was 90.91 % (30/33) for patients, whereas the specificity of control verification was 93.55% (29/31). The mass spectral patterns with three special protein peaks, were used on the double-blinded sera. Using these criteria, gastric cancerwas correctly identified in 14 out of 15 samples, gastritis was correctly identified in 10 out of 11 patients and all 10 out of 10 normal volunteers were identified. The biomarkers therefore had an accuracy of 93.3%and 90.9%for the identification of gastric cancer and gastritis, respectively. The biomarkers had a 100% accuracy to identify healthy volunteers that did not have gastric cancer or gastritis. Conclusion: Use the SELDI-TOF mass spectrometry to establish a serum protein pattern model for screening gastric cancer, and identify proteomic patterns in serum that distinguish gastric cancer from healthy volunteers. The importance of our studies has been to demonstrate the usefulness of SELDI in the discovery of potential tumor markers in serum samples. The comparison of protein expression profiles from serum appears to provide an effective approach to identifying unique biomarkers for gastric cancer and gastritis. Larger scale studies appear warranted to confirm theability of SELDI as a proteomic screen in the clinical setting.Part II Screening Serum Biomarkers of gastric cancer by Two-dimensional Electrophoresis and Mass SpectrometryAim: To study the proteins related to the Gastric carcinoma occurrence and development in the blood plasma of Gastric carcinoma patients. Methods: To establish two-dimensional eletrophoresis profiles with serum protein of patients before or after gastric carcinoma operation and paired normal serum protein.After argent nitrate staining and diferential expression protein spots screening via protein blotched increase and decrease and color shade of 2-DE atlas, we selected these distinction proteins via Agfa DUOSCAN gel scanning and measured the peptides'finger printings after enzymolysis analysis by MALDITOF.Then searched SWISS2PROT data base to screen these distinction proteins. Results: When comparing the sera proteins of these three groups by dimensional gel electrophoresis, we found 5 protein spots in serum protein of patients before gastric carcinoma operation were different significantly from after gastric carcinoma operation or normal group The five proteins undertook situ enzymolysis, we harvested successfully five finger printings via peptides finger printing analysis, the data base searching showed that the five proteins were Serpin B6 (Placental thrombin inhibitor) (Cytoplasmic antiproteinase) (CAP) (Protease inhibitor6) (PI-6) , Septin-I(LARP) (Serologically defined breast cancer antigen NY-BR-24), Kallikrein-6 precursor(Protease M) (Neurosin) (Zyme) (SP59), Hemoglobin beta chain and Beta-defensin 108 precursor (Beta-defensin 8) (DEFB-8). Conclusion: We successfully screened gastric carcinoma related proteins,these proteins might be symbol protein for gastric carcinoma, so the success of this experiment undoubtedly could serve as a basis for the gastric carcinoma earlier detection and therapy.Part III Serum proteomic patterns for primary hepatic carcinoma as revealed by SELDI mass spectrometryAim: SELDI-TOF mass spectrometry was used to investigate protein expression in sera of patients with Primary hepatic carcinoma (PHC) and hepatocirrhosis compared to normal volunteers. To establish a serum protein pattern model for screening liver cancer, and used it to identify proteomic patterns in serum that distinguish neoplastic from non-neoplastic disease within the PHC. Material: Type protein chip reader (PBS II, Ciphergen Biosystems, Inc., Fremont, USA); Strong anionic exchanger (SAX2) Chips (Ciphergen Biosystems, Inc., Fremont, USA); The Biomarker Wizard software (version 3.0, Ciphergen Biosystems, Fremont, CA, USA). Approximately 5 ml of blood was withdrawn via veinipuncture from each patient. PHC patients 40 samples (confirmed by type-B ultrasonic, CT,AFP mensuration, pathology), hepatocirrhosis patients 46 samples ( confirmed by liver puncture), healthy volunteers 60 samples. Methods: SELDI-TOF mass spectrometry was used to investigate protein expression in sera of patients with PHC and hepatocirrhosis compared to normal volunteers. A preliminary "training" set of spectra derived from analysis of serum from 40 patients with PHC and 46 patients with hepatocirrhosis and 60 healthy volunteers were analysed by aniterative searching algorithm that identified a proteomic pattern that completely discriminated cancer from noncancer. Results: A comparison of protein mass spectra obtained from the sera of the PHC group and the healthy volunteer group revealed 68 protein peaks. There were statistical differences between 5 protein peaks located at 4477 Da,8943 Da,5181 Da,8617 Da, 13761 Da; the intensity of this 5 protein peaks from patients with PHC was clearly higher than that of the healthy controls (P<0.05). Using the above profiles, 18 of 20 patients diagnosed pathologically with PHC were correctly identified by SELDI. Sixty of 60 healthy volunteers were correctly identified as normal. The sensitivity of neoplastic identification was 90% (18/20) for patients, whereas the specificity of control verification was 100% (60/60). A comparison of protein mass spectra obtained from the sera of the PHC group and the hepatocirrhosis group revealed 46 protein peaks. There were statistical differences between 3 protein peaks located at 4477 Da,13761 Da,4097 Da; the intensity of the 4477 Da and 13761 Da protein peaks from patients with PHC was clearly higher than that of hepatocirrhosis. Meanwhile, the intensity of the 4097 Da protein peaks from patients with PHC was clearly lower than that of hepatocirrhosis. Using the 4477 Da, we can distinguish PHC from hepatocirrhosis. A comparison of protein mass spectra obtained from the sera of the hepatocirrhosis group and the healthy volunteer group revealed 85 protein peaks. There were statistical differences between 3 protein peaks located at 8943 Da,13761 Da> 4097 Da; the intensity of the 8943 Da,13761 Da protein peaks from patients with hepatocirrhosis was clearly higher than that of the healthy volunteer. Meanwhile, the intensity of the 4097 Da protein peaks from patients with hepatocirrhosis was clearly lower than that of thehealthy volunteer. Using the this 3 protein peaks, we can distinguish hepatocirrhosis from the healthy volunteer. Conclusion: Use the SELDI-TOF mass spectrometry to establish a serum protein pattern model for screening PHC and hepatocirrhosis and the healthy volunteer, and identify proteomic patterns in serum that distinguish PHC from noncancer. The importance of our studies has been to demonstrate the usefulness of SELDI in the discovery of potential tumor markers in serum samples. The comparison of protein expression profiles from serum appears to provide an effective approach to identifying unique biomarkers for PHC and hepatocirrhosis. Larger scale studies appear warranted confirming the ability of SELDI as a proteomic screen in the clinical setting.
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