October 1, 2006 (Vol. 26, No. 17)

Isolating Papillary Thyroid Cancer Biomarkers

As many as one in 10 adults worldwide develops a thyroid nodule that requires clinical investigation. The incidence of thyroid cancer is rapidly increasing, particularly among women, and although the majority of thyroid nodules are benign, each one must be thoroughly analyzed.

Up to one-third of patients, however, cannot be reliably diagnosed on the basis of a fine-needle aspiration biopsy of the thyroid and they all too frequently need to undergo invasive surgery to remove the affected lobe for further analysis. The majority of these cases are still benign, and surgery could be avoided if better diagnostic tools were available.

The research group at Trinity College is trying to identify robust early-stage markers that clearly indicate a thyroid nodule’s cancerous status from just a needle biopsy.

Thyroid cancers are divided into many different types, based on their pathologies. Papillary thyroid cancer (PTC) is the most commonly occurring type and accounts for about 80% of all thyroid tumors.

The team is looking for discrete molecular events in PTC tumor samples, such as the presence or absence of certain genes or gene products that always correlate with either a good or bad prognosis. Being able to diagnose a biopsy on the basis of such markers would be much more objective than classical cytological approaches and would hugely benefit cancer patients, as well as the healthcare system.

PTC studies have extensively researched three principal genes: the ret (rearranged during transfection) proto-oncogene, ras, and BRAF. ret-ras-BRAF form part of a common cellular signaling pathway.

Mutations in ret have been associated with the more malignant PTC tumors for some time (Figure 1). After the Chernobyl accident, for example, the incidence of thyroid cancers in Belarus showed a 2,500% increase. Part of the ret gene is known to be able to translocate within its chromosome and become juxtaposed to any one of 15 other genes. Each of these is always expressed in the cell, meaning that part of the ret gene becomes inappropriately activated. Two principal genes account for probably 90% of the changes: ret-1 and ret-3.

In the Dublin project, ret has been the major genetic aberration in PTC within the Irish population. ret-1 tends to associate with classic PTC, and ret-3 activation is associated with the more malignant tumors within the tissue sample cohorts being studied.

BRAF mutations on the other hand have shown an unusual temporal association. Up until 1992, the majority of tumors involved ret aberrations, but there has been a fairly dramatic change, and since then, tumors with BRAF mutations have become the most common in the Irish population samples.


Simultaneous analysis of multiple patients’ samples and comparison of the gene-expression profiles of different tumor types was conducted using Applied Biosystems (www.appliedbioystems.com) 1700 Chemiluminescence Microarray Analyzer, which is part of the Expression Array System. Using the 1700 system significantly advanced the team’s research and the work is now dependent upon it.

Previously, experiments could typically only look at a single gene at a time, but now researchers can look at every gene possible (Figure 2). The tumor types are grouped into ret positive or negative and BRAF-mutated or not. Using software packages, such as the Panther™ Protein Classification System, molecular pathways that are affected in these samples can be identified.

So far, this approach has demonstrated a number of interesting findings. For example, the follicular variant of PTCs is particularly difficult for pathologists to accurately diagnose. Using the 1700 Analyzer, about 15 genes that are exclusively up-regulated in this particular tumor subtype have been found. In additon, the Panther system has highlighted the pathways in which those genes are involved. Thirteen of the 15 are involved in the immune system’s human leukocyte antigen molecules. This was quite an unexpected finding and the team is currently validating it at the protein level with immunocytochemistry.

The Panther System is critical to this research and is incredibly valuable for biologists. It converts lists of names and numbers into something meaningful and highlights patterns among data that might not otherwise be recognized.

The researchers in Dublin have now begun a pilot study, concentrating on a few of these 15 markers that are likely to be crucial in the follicular variant of PTCs. They are analyzing the genes in fine-needle aspiration samples using the Applied Biosystems TaqMan® Low Density Arrays and so far have identified a set of markers that effectively separate malignant nodules from benign nodules. That will also help in sub-classifying the malignant types, providing researchers useful information about prognosis and treatment.

They have also begun investigating the microRNA profiles of cell lines derived from thyroid tumors and also in vitro models of thyroid cancers where normal thyroid cells are transfected with ret-1, ret-3, or mutant BRAF.

Using the TaqMan microRNA Assays, it may be possible to identify microRNAs in paraffin-embedded material. So far, their research has shown that benign or malignant cell types have completely different microRNA profiles, as do the malignant subtypes, allowing predictions about a tumor’s malignancy, based on its microRNA profile.

Orla Sheils, Ph.D., is senior lecturer in molecular pathology, department of histopathology, Trinity College, Dublin. Web: www.tcd.ie. Phone: +353 1 608 3284. E-mail: [email protected].
This application is for research use only, not for use in diagnostic procedures. TaqMan is a registered trademark of Roche Molecular Systems.

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