Prognosis Of Liver Transplantation Biomarker Screening And Serum Diagnostic Prediction Model Created

Orthotopic liver transplantation (OLT) is the most useful therapeutic option for patients with hepatocellular carcinoma (HCC), because it removes the multifocal potential of HCC in many patients that limits the success and applicability of resection, and also treats the underlying liver disease. Due to a lack of appropriate selection of candidates, the results obtained in the early series were disappointing. This discouraging experience and the scarcity of donor liver grafts have forced the medical community to establish specific selection criteria with an aim to predict post-transplant survival. When several criteria from different centers are applied for candidate selection, post-transplant survival is comparable to recipients without malignancy. However limitations of clinical staging systems based solely on the results of the pre-transplantation imaging techniques and contradictory results when comparing different criteria become evident.High throughput genomics and proteomics techniques have facilitated a better understanding of diseases such as cancer by deciphering the unique molecular signature that predicts clinical outcomes and therapeutics. The nascent field of proteomics—the complete description of all the proteins encoded by the genome—promises to rapidly expand our understanding of cancer. An important goal of clinical proteomics is to develop robust, sensitive, and specific methodologies for the simultaneous analysis of all the proteins expressed by the human genome, and to establish "bio-signature" profiles that discriminate between disease states.One of these technological advances in proteomics is the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Applications of this technology have suggested great potential for the early detection of various cancers. The objective of this study is to identify biomarkers indicative of prognosis in lysated cells and pre-transplantation serum derived from HCC subjects. Then a clinical diagnostic and proteomic predictive pattern on recurrence is established and tested.Part one: Analysis of risk factors on the prognoses of patients after OLT for hepatocellular carcinoma and the strategy for preventing recurrenceThe objective of this part of research was to analyze the impact of risk factors on the prognoses of patients after OLT for HCC and sum up the strategy for preventing recurrence in the diagnosis and therapy. Clinicopathologic features were examined in the 254 patients by using univariate and multivariate analysis.RESULTS:1. 0.5-year, 1-year, 2-year survival in 254 patients was 89.3%, 79.4%, 66.7% respectively, and disease free survival (DFS) was 85.6%, 76.4%, 71.6% respectively.2. Univariate analysis revealed tumor size, the presence of vascular invasion, Edmondson grade, TNM classification, and preoperative alpha-fetoprotein (AFP) were significantly related to DFS. The foregoing four factors were also related to survival rates.3. Cox regression analysis suggested that the presence of vascular invasion was an independent prognostic factor on patients' survival and DFS.Part Two: Establishment of the platform of protein chip SELDI-TOF- MSThe objective of this part of research was to determine the best conditions for SELDI-TOF-MS platform. Pre-transplantation serum and tumor lysate samples from eight cases of patients were collected respectively. The mass-to-charge ratio (M/Z) of each of the proteins captured on the array surface was determined according to externally-calibrated standards (Ciphergen Biosystems). Protein or peptide spectra were produced by SELDI-TOF-MS measurement after testing the sample onto four types of Protein Chips. They were weak cation exchange (WCX) chip, strong anion exchange (SAX) chip, immobilized metal affinity capture (IMAC) chip and hydrophobic surface (H4) chip. Through the protein profiling results, proper parameters and protein chips were selected respectively, and the reliability and stability of the platform were evaluated.RESULTS:1. Most of the proteins detected by protein chip ranged from 2000 to 30000 at the M/Z value.2. These preliminary assays resulted in the selection of WCX2 chip as the most effective chip array in detecting serum protein fingerprints.3. Analysis of tumor lysate protein fingerprints also indicated WCX2 Chip combined the most proteins.4. The coefficient variation (CV) of protein intensity and M/Z value was 0.152 and 0.0001 within the same protein chip.5. The CV of protein intensity and M/Z value was 0.157 and 0.0004 among the protein chips.Part Three: Screen tissue biomarkers with relation to recurrence on the patients with liver transplantation for HCCThe objective of this part of research was to screen lysated cells biomarkers with relation to recurrence on the patients undergoing liver transplantation for HCC, supply the research basis of the molecular mechanisms involved in recurrence and micro-metastasis, and provide the control data for part four. A cohort of tissue samples were obtained from 20 patients with recurrence and also 22 patients with DFS. After lysated cells extracted from tumor, special protein spectra were determined by SELDI-TOF-MS measurement. WCX2 chips were analyzed under the following settings: laser intensity 185, detector sensitivity 8, molecular mass range 2000～30000 Da. The protein fingerprints between two groups were compared by Biomarker Wizard software package.RESULTS:1. According to protein fingerprints, a total of 101 protein peaks were identified at the M/Z value ranging from 2000 to 30000, and 35 proteins were significantly associated with recurrence when compared with controls.2. Between the two groups, 26 proteins were up regulated with the M/Z value of 2308, 2495, 2990, 5994, 3246, 4231, 2762, 3746, 6791, 2948, 2972, 3112, 3277, 4263, 4131, 1690, 6747, 4560, 6721, 1791, 2401, 2551, 7097, 1854, 3172, 4046 in recurrence group.3. 9 proteins were down regulated with the M/Z value of 6171,1683,12323,7707,1994, 5361, 9621, 10226, 6332 in recurrence group.4. These discriminated proteins may correspond with the malignant character of tumor and the existence of micrometastasis outside the liver.Part Four: Screen serum biomarkers with relation to the recurrence on the patients with liver transplantation for HCC and establish the serum diagnostic and predictive proteomic pattern pre-operationThe aim of this study was to appraise and compare protein expression profiles in sera of patients without or with recurrence in the liver transplantation patients for HCC using SELDI-TOF-MS technology, and find if there were common discriminated proteins both in serum and in lysated cells from cancer tissue. The mass spectra of proteins were generated by using the detection size range between 2000 and 30000Da, detector sensitivity set at 8 and laser intensity at 185. Spectra were analyzed with Ciphergen ProteinChip software (version 3.1) and normalized. Then intensity values for each peak were input into Biomarker Patterns Software for classification tree analysis and the best performing tree was chosen for testing. Second, the randomly selected samples were categorized with the decision tree being tested to ensure that the decision tree was valid.RESULTS:1. According to serum protein fingerprints, a total of 115 protein peaks were identified at the M/Z value ranging from 2000 to 30000, 19 significant differential proteins were found between the groups of HCC with recurrence and those with DFS (P<0.05) .2. Among 19 significant differential proteins, 5 proteins were down regulated with the M/Z value of 28123, 2197, 2476,2293 and 2236 in recurrence group, 14 proteins were up regulated with the M/Z value of 4897, 4659, 2146, 2780, 6784, 5758, 5530,4878, 2832, 2938, 5634, 6443, 3517 and 3574 respectively in recurrence group.3. Serum protein profiles obtained from 64 HCC patients were generated using classification trees by Biomarker Patterns Software, which combined with 4 candidate proteins with the M/Z value of 2146, 2780, 4659, and 4897 with a sensitivity of 93.33% (28/30) and specificity of 82.35% (28/34).4. The validity of the decision tree was then challenged with a test set of 43 samples. As a result of blind assessment, an sensitivity of 89.47% (17/19) and specificity of 83.33% (20/24) were obtained.CONCLUSIONS1. Among clicopathological parameters that influence tumor-free survival after liver transplantation, vascular invasion played a leading role in evaluating the patients' prognoses. With current cancer biomarkers, much was left to be desired in terms of clinical applicability. We need new cancer biomarkers that will further enhance our ability to diagnose and predict therapeutic response in the transplantation patients with HCC. Looking for sensitive methods to exclude the micrometastasis pre-transplantation and early detection of disease will be the chief challenge in the future.2. Utilizing SELDI-TOF and the same bioinformatics software package is an ideal technological platform for proteomic research. These technology allows researchers to draw a proteomic profile rapidly from little quantity because of high reproducibility and stability. WCX2 protein chip was not only suitable for the research of serum proteomics in HCC patients pre-transplantation, but also for cellular proteomics. Quality control and standardization of analysis conditions could be key issue for the reliability of outcome.3. Some of differential proteins screened by SELDI-TOF technique in the serum and lysated cells may correlate with the prognoses of the liver transplantation patients with HCC. The decision tree promised to well understand the prognoses of the transplantation and detect micrometastasis in the circulation pre-transplantation. Through the research, we can exclude the high-risk patients more sensitively and afford neo-adjuvant therapy to reduce the recurrence rate.NOVELTY1. Establishment of lysated cells and pre-transplantation serum protein fingerprints in transplantation patients with HCC.2. Establishment of clinical serum diagnostic and predictive proteomic pattern for screening HCC patients for liver transplantation.3. The discriminated protein would supply us with more sensitive and specific index to select HCC patients for liver transplantation and well understand their prognoses. Among these proteins, we would find and identify some new molecular biomarkers for micrometastasis outside the liver pre-transplantation.