Discovery and identification of potential biomarkers of papillary thyroid carcinoma

Abstract

Background: Thyroid 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.

Methods: Two 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.

Results: A 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.

Conclusion: We 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.

Figure 1

A representative mapping of SELDI-TOF-MS analysis of sera from PTC patients and healthy controls. Differentially expressed proteins with potential diagnostic significance are arrowed. Top-group denotes sera from patients with PTC, in which the protein with m/z of 9190 Da was over-expressed. Bottom-group denotes sera from healthy individuals, in which the proteins with m/z of 6631 and 8697 Da were up-regulated.

Figure 2

A representative mapping of SELDI-TOF-MS analysis from different stages of PTC patients and non-cancer controls. The level of the 9190 Da protein progressively increased with the clinical stage I, II, III and IV, and the expression of 6631 and 8697 Da proteins gradually decreased in higher stages.

Figure 3

MALDI-TOF-MS spectra of three purified potential protein markers.

Figure 4

Results of the identification of protein (8697 Da) by LC-MS/MS. (A) Chromatogram of peptide mixture. (B) MS/MS spectrum of one peptide.

Figure 5

Representative spectra from ProteinChip array with specific immobilized antibodies. (A) Representative spectra of the negative control (nonspecific rabbit IgG). (B) Representative spectra from ProteinChip array with anti-haptoglobin alpha chain antibody. (C) Representative spectra from ProteinChip array with anti-apolipoprotein C-I. (D) Representative spectra from ProteinChip array with anti-apolipoprotein C-III.