| ObjectiveGastric cancer(GC) is a major public health burden nowadays. Globally, it is the fourth most common cancer and the second leading cause of cancer-related death, with 700,000 deaths every year. Almost two-thirds of the cases occurred in developing countries and 42% in China alone. Although GC is declining in frequency in almost all populations, it remains the most common malignancy in many countries worldwide and more than 930,000 new cases were diagnosed annually. Therefore, the early diagnosis and therapy of gastric cancer are very important. At present, endoscopic evaluation is the gold standard for gastric cancer screening and diagnosing gastric cancer. Though rapid progress has been made in endoscopy for gastric cancer, it is still an invasive procedure with a risk of complication and unpleasant for the patient. There are lack of sensitivity and specificity of serology screening biomarkers such as CEA, CA199, CA125, CA50 and CA72-4 which have only better prediction of recurrence, not giagnosis. In recent years, lots of progresses have been made in proteomics with leads a new approach to identify novel biomarkers for cancer diagnosis. In our researches surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) chip system with CM10 chip was employed to screen novol serum protein biomarker of gastric cancer.Methods1,Screening the difference protein peaks of gastric cancer by SELDI system1.1 Serum samples were collected from thirty patients with gastric cancer (GC), sixteen patients with chronic benign gastric disease (5 gastric ulcer and 11 chronic erosive gastritis with intestinal metaplasia) and 30 normal control were tested by CM10 SELDI chips. The sera tumor marker of CEA, CA199, CA72-4 of these people also were tested using a radioimmunoassay method.1.2 The information of all serum protein peak information was acquired using a bioinformatics approach.1.3 The protein profilings of thirty patients with gastric cancer (GC), sixteen patients with chronic benign gastric disease(5 gastric ulcer and 11 chronic erosive gastritis with intestinal metaplasia) and 30 normal control were compared in pairs to find significantly different protein peaks for gastric cancer diagnosis.1.4 Diagnostic Indexes (sensitivity, specificity and area under the receiver operative curve) of the significant protein peaks were evaluated to find the most valuable one.1.5 The relationship of the significantlly different protein peaks with tumor lymphatic metastasis was analyzed. The relationship of the significantlly different proteins with early gastric cancer (Phaseâ… -â…¡) and later gastric cancer (Phaseâ…¢-â…£) was analyzed.2 Clinical validation of the different protein peaks of gastric cancer2.1 Validation the different protein peaks used for blind group with ten gastric cancer patients, five chronic benign gastric disease (chronic erosive gastritis with intestinal metaplasia) and ten normal control.2.2 Evaluated the diagnostic Indexes of the validation group to further confirm the gastric specific protein peaks.3 Identification of the most significantly different protein peak of gastric cancer3.1 Serum proteins were enriched and purified from the mixture of the sera of ten gastric caner patients with high 5910 peak by using the WCX Clinprot magnetic beads.3.2 The purified protein was analyzed by using a LC-MS/MS system. Database searching was performed with the program IPI Human (3.45).4 Validation of the most significantly different protein peak of gastric cancerThe fraction of Fibrinogen alpha chain was validated by immunoprecipitation using antibody against 5910 peptids made by ourselves and antibody against Fibrinogen alpha chain in order to find which is really evaluated in gastric cancer patients the fraction 5910 or Fibrinogen alpha chain.Results1,Screening difference protein peaks of gastric cancer using SELDI system.1.1 There are 54 significantly different protein peaks among the three groups by using principle that the 30% percent cases have the peak signal to noise ratio higher than five.1.2. Compared the proteomic profilings of the thirty patients with gastric cancer and sixteen patients with chronic benign gastric disease, there are sixty-six significant protein peaks. Compared the proteomic profilings of the thirty patients with gastric cancer and thirty normal control, there are fifteen significant protein peaks(P<0.05). Compared the proteomic profilings of this three group together, there are nineteen significant protein peaks (P<0.05). There are five protein peaks significantly different among these three groups, they are protein peaks with mass to charge ratio 5910,5342,6439,3268 and 3476. Among the five peaks, 5910 was with the most significantly difference. The diagnostic Indexes of this peak were 83.3% of sensitivity, 91.3% of specificity, and the area under ROC 0.89. The diagnostic Indexes of 5342 were 83.3% of sensitivity, 82.6% of specificity, and the area under ROC 0.85. The diagnostic Indexes of 6439 were 73.3% of sensitivity, 67.3% of specificity, and the area under ROC 0.75.1.3 There are significant differences between sera tumor markers CEA and CA125 between patients with gastric cancer and patients with chronic benign gastric diseas(eP<0.05), also between patients with gastric cancer and normal control(P<0.05). Using 8 as the cut off value, the diagnostic Indexes of CEA were sensitivity 20.3%, specificity 97.8%, and the area under ROC 0.52. Using the 35 as cut of value, the diagnostic Indexes of CA125 were sensitivity 23.3%, specificity 100.0%, and the area under ROC 0.83. .1.4 Compared the peaks 5910 with CEA, we found that 5910 SELDI peak had a better diagnostic value than CEA (P=0.00), and 5910 peaks has a diagnostic Indexes with for diagnose gastric cancer patients with CEA negative.1.5 There is no relationship of 5910 with progress of the gastric cancer development and its lymphatic metastasis2 Validation of the different protein peaks of gastric cancer2.1. Validation the different protein peaks used for blind group with ten gastric cancer patients five chronic benign gastric disease (chronic erosive gastritis with intestinal metaplasia) and ten normal control was according with the training group in three peaks 5910, 5342, 6439. The peaks of 3268 and 3476 were not consistent between training group and validation group. So the peaks of 3268 and 3476 were not the gastric specific protein peaks. The diagnostic Indexes of 5910 in the validation group were sensitivity 100.0%, specificity 93.3%.3. Identification of the most significantly different protein peak.3.1 Serum proteins were enriched and purified from the mixture of ten sera of gastric caner patients with high 5910 peak by using the WCX Clinprot magnetic beads.3.2 The purified protein was analyzed by using a LC-MS/MS system. Database searching with the program IPI Human (3.45) identified the 5910 peak protein to be the C-terminal fraction of fibrinogen alpha chain or called fibrinopeptide A. 4. Validation of the most significantly different protein peakThe result immunoprecipitation showed that the fraction 5910 of fibrinogen alpha chain is evaluated in the serum diagnosis of gastric cancer paptients.Conclusion1. SELDI protein chip system was valuable for identifying serum protein biomarkers for the diagnosis of gastric cancer.2. 5910, 5342, and 6439 protein peaks in mass-to-charge ratio (M/Z) are gastric cancer specific protein peaks. For the diagnosis of gastric cancer, 5910 protein is better than CEA, CA199 and CA125, but the 5910 peak is not related with tumor lymphatic metastasis and tumor progress.3. The 5910 peptids is the C-terminal fragment of fibrinogen alpha chain, or called fibrinopeptide A, and a potential gastric cancer biomarker in serum.
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