Abstract
Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of (18)O/(16)O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using (18)O/(16)O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.
Highlights
From the ‡Departments of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555; §Institute for Translational Science, University of Texas Medical Branch, Galveston, Texas 77555; ʈDepartment of Pathology, University of Texas Medical Branch, Galveston, Texas 77555; **Department of Preventative Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas 77555; ‡‡Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555; §§Department of Internal Medicine, Kelsey Seybold Clinic, Houston, Texas 77030; ¶¶Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555
The characteristics of the patients used for two-dimensional difference gel electrophoresis (DIGE) and the patients used for selected reaction monitoring (SRM) quantification of ApoA1 are presented in Table I and supplemental Table S1, respectively
We began with aptamer reduction of abundant proteins and two-dimensional DIGE, a powerful technique that can be used to identify proteins with differential expression between complex serum samples
Summary
From the ‡Departments of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555; §Institute for Translational Science, University of Texas Medical Branch, Galveston, Texas 77555; ʈDepartment of Pathology, University of Texas Medical Branch, Galveston, Texas 77555; **Department of Preventative Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas 77555; ‡‡Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555; §§Department of Internal Medicine, Kelsey Seybold Clinic, Houston, Texas 77030; ¶¶Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555. 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. AFP is a low-specificity biomarker, as it is seen in patients with cirrhosis or exacerbations of chronic hepatitis and pancreatitis [6, 7]. These limitations generate anxiety for patients and physicians alike. Biomarker Discovery for Hepatocellular Carcinoma concerns present the urgent need for a sensitive, specific, and facile screening modality for early detection, diagnosis, and monitoring of HCC that would provide significant clinical benefit.
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