Serum fibronectin distinguishes the early stages of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death, necessitating the discovery of serum markers for its early detection. In this study, a total of 180 serum samples from liver cirrhosis (LC), hepatocellular carcinoma (HCC) patients and paired samples of HCC patients who recovered (Recovery) were analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) to verify biomarkers. The three-fold crossvalidation was repeated 100 times in the training and test sets to evaluate statistical significance of 124 candidate proteins. This step resulted in 2 proteins that had an area under the receiver operating curve (AUROC) values ≥0.800 in the training (n = 90) and test sets (n = 90). Specifically, fibronectin (FN1, WCGTTQNYDADQK), distinguished HCC from LC patients, with an AUROC value of 0.926 by logistic regression. A FN1 protein was selected for validation in an independent sample (n = 60) using enzyme-linked immunosorbent assay (ELISA). The combination of alpha-fetoprotein (AFP) and FN1 improved the diagnostic performance and differentiated HCC patients with normal AFP levels. Our study has examined candidate markers for the benign disease state and malignancy and has followed up on the consequent recovery. Thus, improvement in the early detection of HCC by a 2-marker panel (AFP + FN1) might benefit HCC patients.


Experimental design
To verify the protein markers that complemented the early diagnosis of HCC, we quantitated candidate markers in the serum of patients with liver cirrhosis, HCC, and recovery from HCC. All serum samples were collected retrospectively from the same institution. A list of previously discovered proteins from the LiverAtlas database was measured by multiple reaction monitoring-mass spectrometry (MRM-MS), a linear mixed effects model (LMM), and logistic regression (LR) methods were used to select significant markers of HCC. The performance of the targets was based on their area under the receiver operating curve (AUROC). Differentially expressed proteins were also verified by western blot. The final target, fibronectin (FN1), was validated by enzyme-linked immunosorbent assay (ELISA) to ensure its differential expression.
The number of samples was based on an AUROC values ≥ 0.800 with 95% confidence interval (CI), because the AUROC value of AFP has been reported to be 0.50 -0.76 1-3 .
The α (significance) and β (1-power) were 0.05. The ratio of cases to controls was set to 1:1, and a total of 40 samples, composed of 20 cases and 20 controls, were needed (Supplementary Table S4). In this study, 240 samples, comprising 80 cases and 160 controls, were collected. We negated the subjectivity of experimenters by blocked randomization. Equal numbers of control and case samples were assigned in each randomly ordered block using our best effort using Excel, ver.
2013 (Microsoft Corp., WA, USA). Random allocation was processed using Random Allocation Software, ver. 1.00 (Isfahan University of Medical Sciences, Isfahan, Iran). We block-randomized the 3 steps (depletion order, digestion order, order of analysis by MRM-MS) to prevent experimental bias.

Clinical sample preparation for MRM-MS analysis
The high-abundance proteins albumin, IgG, IgA, haptoglobin, transferrin, and alpha-1antitrypsin were depleted using a Multiple Affinity Removal System Human-6 (MARS Hu-6, 4.6 mm × 100 mm, Agilent, CA, USA) affinity column on a high-performance liquid chromatography (HPLC) instrument to remove any masking effects and discover low-abundance targets. Serum sample were thawed on ice and centrifuged at 14,000 g for 10 min at 4°C. For each sample, 40 μL of each serum was diluted with 160 µL MARS buffer A (Agilent, CA, USA) and passed through 0.22 µm Spin-X filters (Corning Costar, NY, USA). The diluted serum protein was then processed using the recommended column run cycle. The specific process was as follows: After equilibration with buffer A (load/wash buffer), the MARS column was loaded with 200 μL of the diluted serum at a flow rate of 0.5 mL/min for 10 min. Flow-through fractions, representing depleted serum, were eluted at 3 min. The bound proteins were released with 100% buffer B (elution buffer) at 16.5 min with a flow rate of 1.0 mL/min. Each depletion cycle took 28 min of total run time.
The depleted serum was then concentrated on 3000 Da molecular weight cutoff (MWCO) centrifugal filter units (Amicon Ultra-4 3K, Millipore, MA, USA). One hundred micrograms of concentrated serum, as measured by bicinchoninic acid assay, was denatured with 6 M urea. The reduction and alkylation were performed with 20 mM dithiothreitol (Merk, Darmstadt, Germany), 0.1 M Tris, pH 8.0 at 37°C for 30 min and 50 mM iodoacetamide (Sigma, MO, USA) in the dark at room temperature for 30 min, respectively. To enhance the digestion with sequencing-grade trypsin (Promega, WI, USA) at a 1:50 enzyme-to-substrate ratio, the serum was diluted 10-fold with 0.1 M Tris, pH 8.0 and incubated for 16 h at 37°C. To cease the enzymatic reaction, neat formic acid was added until a final concentration of 2%.
The digested sample was loaded onto the cartridge after equilibration with 3 mL 0.1% formic acid.
The cartridge was then washed with 3 mL 0.1% formic acid and eluted with 1 mL 80% acetonitrile in 0.1% formic acid. The collected sample was lyophilized on a vacuum centrifuge. The serum was stored at -80°C and resolubilized in 0.1% formic acid to 2 µg/µL prior to the MRM-MS analysis.