Discovery and Validation of Protein Abundance Differences between Follicular Thyroid Neoplasms

Romana T Netea-Maier,Ad R Hermus,Bryan R Haugen,Brigiet M Hoevenaars,Stephen W Hunsucker,Steve M Helmke,Pieter J Slootweg,Mark W Duncan

doi:10.1158/0008-5472.can-07-5020

Abstract

Distinguishing between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC) by cytologic features alone is not possible. Molecular markers may aid distinguishing FTA from FTC in patients with indeterminate cytology. The aim of this study is to define protein abundance differences between FTC from FTA through a discovery (proteomics) and validation (immunohistochemistry) approach. Difference gel electrophoresis (DIGE) and peptide mass fingerprinting were performed on protein extracts from five patients with FTC and compared with six patients with FTA. Individual gel comparisons (i.e., each FTC extract versus FTA pool) were also performed for the five FTC patients. Immunohistochemical validation studies were performed on three of the identified proteins. Based on DIGE images, 680 protein spots were matched on individual gels. Of these, 102 spots showed statistically significant differences in abundance between FTC and FTA in the individual gel analyses and were therefore studied further. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify 54 of these protein spots. Three candidates involved in protein folding (heat shock protein gp96, protein disulfide isomerase A3, and calreticulin) were studied by immunohistochemistry. Moderate calreticulin immunohistochemical staining was the best single marker with a high negative predictive value (88%); combining all three markers (any marker less than moderate staining) had the best positive predictive value (75%) while still retaining a good negative predictive value (68%). With DIGE, we identified 54 proteins differentially abundant between FTC and FTA. Three of these were validated by immunohistochemistry. These findings provide further insights into the diagnosis, prognosis, and pathophysiology of follicular-derived thyroid neoplasms.

Highlights

Thyroid cancer is the most common endocrine malignancy and its most frequent clinical presentation is as a thyroid nodule, eitherNote: Supplementary data for this article are available at Cancer Research Online.It is challenging to distinguish between thyroid neoplasms of the follicular type [i.e., benign follicular thyroid adenoma (FTA), malignant follicular thyroid carcinoma (FTC), and follicular variant of papillary carcinoma] based on cytologic examination alone
Recent data suggest that the so-called atypical FTA, which is characterized by high cellular density, mitoses, and a less regular cytologic pattern, may share genetic features with both FTC and papillary thyroid carcinomas (PTC) [5], but the progression of thyroid adenoma to carcinoma has not been clearly shown
Defining the differences in protein levels that distinguish between FTA and FTC will provide additional insight in the earliest steps of follicular neoplasia transformation and might deliver a clinical tool that could improve the diagnostic accuracy of Fine-needle aspiration biopsy (FNAB) in patients with indeterminate cytology

Summary

Introduction

It is challenging to distinguish between thyroid neoplasms of the follicular type [i.e., benign follicular thyroid adenoma (FTA), malignant follicular thyroid carcinoma (FTC), and follicular variant of papillary carcinoma] based on cytologic examination alone. All these tumors have similar cytologic features and surgery is usually required to obtain a definitive tissue sample. Defining the differences in protein levels that distinguish between FTA and FTC will provide additional insight in the earliest steps of follicular neoplasia transformation and might deliver a clinical tool that could improve the diagnostic accuracy of FNAB in patients with indeterminate cytology

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