Jiekai Yu

Discovery and identification of potential biomarkers of papillary thyroid carcinoma.

BackgroundThyroid carcinoma is the most common endocrine malignancy and a common cancer among the malignancies of head and neck. Noninvasive and convenient biomarkers for diagnosis of papillary thyroid carcinoma (PTC) as early as possible remain an urgent need. The aim of this study was to discover and identify potential protein biomarkers for PTC specifically.MethodsTwo hundred and twenty four (224) serum samples with 108 PTC and 116 controls were randomly divided into a training set and a blind testing set. Serum proteomic profiles were analyzed using SELDI-TOF-MS. Candidate biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays.ResultsA total of 3 peaks (m/z with 9190, 6631 and 8697 Da) were screened out by support vector machine (SVM) to construct the classification model with high discriminatory power in the training set. The sensitivity and specificity of the model were 95.15% and 93.97% respectively in the blind testing set. The candidate biomarker with m/z of 9190 Da was found to be up-regulated in PTC patients, and was identified as haptoglobin alpha-1 chain. Another two candidate biomarkers (6631, 8697 Da) were found down-regulated in PTC and identified as apolipoprotein C-I and apolipoprotein C-III, respectively. In addition, the level of haptoglobin alpha-1 chain (9190 Da) progressively increased with the clinical stage I, II, III and IV, and the expression of apolipoprotein C-I and apolipoprotein C-III (6631, 8697 Da) gradually decreased in higher stages.ConclusionWe have identified a set of biomarkers that could discriminate PTC from non-cancer controls. An efficient strategy, including SELDI-TOF-MS analysis, HPLC purification, MALDI-TOF-MS trace and LC-MS/MS identification, has been proved successful.

SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK are prognosis-related in colorectal cancer.

AIM To investigate the expression of markers that are correlated with the prognosis of colorectal cancer (CRC) patients. METHODS One hundred and fifty-six CRC patients were followed up for more than 3 years after radical surgery. Immunohistochemical (IHC) analysis was performed to detect the expression of 14 pathway-related markers (p53, APC, p21ras, E-cadherin, endothelin-B receptor, Shp2, ADCY-2, SPARCL1, neuroligin1, hsp27, mmp-9, MAPK, MSH2 and rho) in specimens from these patients. Bioinformatics analysis involving a Support Vector Machine (SVM) was used to determine the best prognostic model from combinations of these markers. RESULTS Seven markers (SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK) were significantly related to the prognosis and clinical pathological features of the CRC patients (P < 0.05). Prognostic models were established through SVM from combinations of these 7 markers and proved able to differentiate patients with dissimilar survival, especially in stage II/III patients. According to the best prognostic model, the p53/SPARCL1 model, patients having high p53 and low SPARCL1 expression had about 50% lower 3-year survival than others (P < 0.001). CONCLUSION SPARCL1, Shp2, MSH2, E-cadherin, p53, ADCY-2 and MAPK are potential prognostic markers in CRC. A p53/SPARCL1 bioinformatics model may be used as a supplement to tumor-nodes-metastasis staging.

Proteomic analysis of primary colon cancer-associated fibroblasts using the SELDI-ProteinChip platform *

ObjectiveCancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts.MethodsPrimary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform.ResultsMost primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin β-4-like protein 3, and tight junction-associated protein 1.ConclusionsUsing the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.

Association between Oxidative DNA Damage and Risk of Colorectal Cancer: Sensitive Determination of Urinary 8-Hydroxy-2′-deoxyguanosine by UPLC-MS/MS Analysis

Oxidative DNA damage plays crucial roles in the pathogenesis of numerous diseases including cancer. 8-hydroxy-2′-deoxyguanosine (8-OHdG) is the most representative product of oxidative modifications of DNA, and urinary 8-OHdG is potentially the best non-invasive biomarker of oxidative damage to DNA. Herein, we developed a sensitive, specific and accurate method for quantification of 8-OHdG in human urine. The urine samples were pretreated using off-line solid-phase extraction (SPE), followed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. By the use of acetic acid as an additive to the mobile phase, we improved the UPLC-MS/MS detection of 8-OHdG by 2.7−5.3 times. Using the developed strategy, we measured the contents of 8-OHdG in urine samples from 142 healthy volunteers and 84 patients with colorectal cancer (CRC). We observed increased levels of urinary 8-OHdG in patients with CRC and patients with tumor metastasis, compared to healthy controls and patients without tumor metastasis, respectively. Additionally, logistic regression analysis and receiver operator characteristic (ROC) curve analysis were performed. Our findings implicate that oxidative stress plays important roles in the development of CRC and the marked increase of urinary 8-OHdG may serve as a potential liquid biomarker for the risk estimation, early warning and detection of CRC.

Combining proteomics, serum biomarkers and bioinformatics to discriminate between esophageal squamous cell carcinoma and pre-cancerous lesion

ObjectiveBiomarker assay is a noninvasive method for the early detection of esophageal squamous cell carcinoma (ESCC). Searching for new biomarkers with high specificity and sensitivity is very important for the early detection of ESCC. Serum surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS) is a high throughput technology for identifying cancer biomarkers using drops of sera.MethodsIn this study, 185 serum samples were taken from ESCC patients in a high incidence area and screened by SELDI. A support vector machine (SVM) algorithm was adopted to analyze the samples.ResultsThe SVM patterns successfully distinguished ESCC from pre-cancerous lesions (PCLs). Also, types of PCL, including dysplasia (DYS) and basal cell hyperplasia (BCH), and healthy controls (HC) were distinguished with an accuracy of 95.2% (DYS), 96.6% (BCH), and 93.8% (HC), respectively. A marker of 25.1 kDa was identified in the ESCC patterns whose peak intensity was observed to increase significantly during the development of esophageal carcinogenesis, and to decrease obviously after surgery.ConclusionsWe selected five ESCC biomarkers to form a diagnostic pattern which can discriminate among the different stages of esophageal carcinogenesis. This pattern can significantly improve the detection of ESCC.

Identification of a new protein biomarker for colorectal cancer diagnosis.

As one of the most common cancers, colorectal cancer (CRC) is a major public health issue worldwide. Thus, the identification of novel biomarkers to aid in the early diagnosis of CRC is crucial. The aim of the present study was to identify a novel protein biomarker for CRC, and to identify its structure. In this study, a total of 99 serum samples from 73 CRC patients and 26 healthy controls were collected and analyzed by SELDI-TOF-MS. The biomarkers were separated using HPLC and detected with MALDI-TOF-MS. The qualified peaks were ranked by p-value of non-parametric tests and the top 10 peaks displaying significant differences were selected. Among the 10 protein biomarkers, the concentration of a 3.9‑kDa protein in the serum of the CRC patients was much lower than that in the healthy controls. Therefore, the 3.9‑kDa protein was selected as a biomarker for CRC and its separation and purification were performed. The structure of the 3.9-kDa protein biomarker was determined by LC-MS/MS, and was confirmed to be a fragment of serine/theonine kinase 4 (MST1/STK4). The 3.9‑kDa protein biomarker had high sensitivity and specificity for CRC, and its potential clinical application warrants further investigation.

Detection and Significance of Serum Protein Marker of Hirschsprung Disease

OBJECTIVE. The objective of this study was to identify a specific fingerprint chromatogram model of serum proteins for early screening and diagnosis of Hirschsprung disease. METHODS. To detect the protein mass spectrograms of 78 serum specimens (42 specimens of Hirschsprung disease, 16 specimens of adhesive ileus including appendicitis and Meckel diverticulum after operation and inflammatory bowel disease, and 20 specimens of normal control subjects), we used surface-enhanced laser desorption/ionization time of flight mass spectrometry technology, combined with bioinformatics methods (support vector machine) to develop and compare protein mass spectrograms from serum samples. RESULTS. We identified 3 protein markers, the mass-to-charge ratio of which is positioned at 3221.7, 5639.2, and 6884.2 from the fingerprint chromatogram model of serum protein for early screening and diagnosis of Hirschsprung disease. The markers had 100% sensitivity and specificity. CONCLUSION. The fingerprint chromatogram model of serum protein using surface-enhanced laser desorption/ionization time of flight mass spectrometry technology combining support vector machine is a new method of early screening and diagnosis of Hirschsprung disease that is worthy of additional research and application.

Identification of Apolipoprotein C-I as a Potential Wilms’ Tumor Marker after Excluding Inflammatory Factors

Wilms’ tumor is one of the most common malignant tumors observed in children, and its early diagnosis is important for late-stage treatment and prognosis. We previously screened and identified protein markers for Wilms’ tumor; however, these markers lacked specificity, and some were associated with inflammation. In the current study, serum samples from children with Wilms’ tumors were compared with those of healthy controls and patients with systemic inflammatory response syndrome (SIRS). After exclusion of factors associated with inflammation, specific protein markers for Wilms’ tumors were identified. After comparing the protein peak values obtained from all three groups, a protein with a m/z of 6438 Da was specified. Purification and identification of the target protein using high-pressure liquid chromatography (HPLC) and two-dimensional liquid chromatography-linearion trap mass spectrometry(2D-LC-LTQ-MS) mass spectrometry, respectively, revealed that it was apolipoprotein C-I (APO C-I). Thus, APO C-I is a specific protein marker for Wilms’ tumor.

Identification of MST1 as a potential early detection biomarker for colorectal cancer through a proteomic approach

Colorectal cancer (CRC) is a common malignant neoplasm worldwide. It is important to identify new biomarkers for the early detection of CRC. In this study, magnetic beads and the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) platform were used to analyse CRC and healthy control (HC) serum samples. The CRC diagnosis pattern was established to have a specificity of 94.7% and sensitivity of 92.3% in a blind test. The candidate biomarker serine/threonine kinase 4 (STK4, also known as MST1) was identified by Tandem mass spectrometry (MS/MS) and verified with western blotting and enzyme-linked immunosorbent assay (ELISA). The results indicated that there was a higher concentration of MST1 in HC subjects than stage I CRC patients for the early detection of CRC and a lower concentration in stage IV patients than in other CRC patients. The sensitivity and specificity of MST1 combined with carcinoembryonic antigen (CEA) and faecal occult blood test (FOBT) in diagnosis of colorectal cancer were 92.3% and 100%, respectively. Additionally, low MST1 expression was associated with the poor prognosis. These results illustrate that MST1 is a potential biomarker for early detection, prognosis and prediction of distant metastasis of CRC.

Discriminating patients with early-stage breast cancer from benign lesions by detection of oxidative DNA damage biomarker in urine

Breast cancer is one of the most commonly diagnosed and death-related cancers in women worldwide. Mammography is routinely used for screening and invasive examinations such as painful tissue biopsies were recommended for patients with abnormal screening outcomes. However, a considerable proportion of these cases turn out to be benign lesions. Thus, novel non-invasive approach for discriminating breast cancer from benign lesions is desirable. Herein, we applied a high-throughput ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis to determine the oxidative DNA damage biomarker, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in urine samples from 60 patients with early-stage breast cancer (stage I, II), 51 patients with benign breast diseases and 73 healthy volunteers. We demonstrated that the concentration of urinary 8-oxodG in patients with early-stage breast cancer was significantly higher not only than that in healthy controls, but also than that in patients with benign breast diseases, whereas no significant difference of urinary 8-oxodG level was observed between benign breast diseases group and healthy control group. Moreover, there was significant difference between early-stage breast cancer group and non-cancerous group which consisted of benign breast diseases patients and healthy controls. Besides, logistic regression analysis and receiver operator characteristic (ROC) curve analysis were also performed. Our findings indicate that the marked increase of 8-oxodG in urine may serve as a potential biomarker for the risk estimation, early screening and detection of breast cancer, particularly for discriminating early-stage breast cancer from benign lesions.