Applications of proteomics to thyroid neoplasms: are we there yet?

Scientists and clinicians have long been searching for useful markers in patients with thyroid neoplasms. One major goal of this research is to use newly discovered markers to distinguish, prior to thyroid surgery, between benign and malignant thyroid neoplasms. Such markers may have other equally important uses. For example, they could serve as prognostic indicators, provide a guide to directed therapy, and help better understand the molecular heterogeneity and biology of thyroid cancer in its development, progression, and propensity to aggressive behavior. Identification and characterization of new tumor markers offers the promise to improve both the diagnosis and management of thyroid neoplasia. Biomarkers come in many forms including DNA (BRAF T1899A), RNA (RET/PTC or Pax8-PPARg rearrangements, mRNA levels of multiple transcripts), and proteins. They all have the potential to serve as markers but, of these, proteins offer some unique benefits. First, proteins are amenable to high-throughput, cost-effective, standardized methods of measurement such as immunohistochemistry, immunocytochemistry, and enzyme-linked immunosorbent assay. Second, there is important functional information that is only available at the protein level. For example, while a human cell contains only about 20,000 specific genes, downstream coand post-translational events mean that many more proteins are expressed and these incorporate functionally important modifications. Analysis of proteins therefore offers insights in cellular function (and dysfunction) that are inaccessible at the genomic level. Proteomics is the name given to a set of analytical strategies that can simultaneously identify and quantify thousands of protein components in a biological sample. There is, however, no single approach that optimally meets this demanding objective; instead, several complementary technologies have emerged, each offering distinctive strengths and weaknesses. These approaches are now facilitating new biomarker discoveries in many areas of medicine. Studies of the thyrocyte and thyroid cancer cell proteome are in their infancy compared with studies of the genome and transcriptome. In this issue of Thyroid, Sofiadis and colleagues (1) use one of these strategies, surface-enhanced laser desorption ionization–time of flight mass spectrometry (SELDI-TOF), to explore the proteome of normal thyroid tissue, papillary thyroid carcinoma, and other tumor types. SELDI utilizes protein chips with different chromatographic surfaces (hydrophobic, ionic, hydrophilic) to capture a specific set of proteins for analysis by mass spectrometry. While the approach is fast and easy to perform and has unquestionably significant strengths compared with the alternatives, SELDI offers limited coverage of the proteome. It is limited by protein charge and size, and the peaks are characterized only by their masses. Nevertheless, in their study the authors were able to perform detailed analysis of cytosolic and nuclear extracts from papillary and follicular thyroid tumors and matched normal thyroid tissue. They found 13 protein peaks between 10.1 and 13.9 kDa that differed in abundance between papillary thyroid carcinoma (PTC) and follicular tumors. They hypothesized that the peak cluster at m/z 10,200 corresponded to the Caþ2-binding protein S100A6. This was subsequently confirmed by immunoprecipitation and liquid chromatography–tandem mass spectrometry. Western blotting also confirmed a significantly higher expression of S100A6 in PTC in comparison with the other tumor groups or normal tissue. Immunohistochemical analysis on 98 tumors showed that PTC cases had significantly stronger cytosolic staining and a larger proportion of stained nuclei than follicular tumors. Finally, they showed that the BRAF mutation was not significantly associated with S100A6 protein levels but that, as had been shown previously in lung tumors and cell lines (2,3), there existed different forms of S100A6 in thyroid tumors. These findings are important because they build on and add to our slowly evolving understanding of the biochemistry of thyroid cancer. Notably, our group adopted a different proteomic strategy to examine papillary and follicular thyroid cancer (4,5). We separated intact, labeled proteins by differences in gel electrophoresis two-dimensional gel separation and then identified differentially abundant proteins by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprints. In these studies, we identified S100A6 as a potential marker of PTC along with a list of other candidates (4). The work of Sofiadis et al. (1) provides independent verification of the previous identification of S100A6 as a potential marker of PTC by our group (4) and others (6) and importantly, provides independent validation based on their immunohistochemical studies.