New method can improve diagnosis of thyroid cancer

Researchers have identified a group of genes that serve as biomarkers for papillary thyroid carcinoma and high risk of lymph node involvement.

By Karina Toledo  |  Agência FAPESP – Researchers at A.C. Camargo Cancer Center’s National Institute of Science and Technology in Oncogenomics (INCITO) in Brazil have identified a group of genes that serve as biomarkers for diagnosing the most prevalent form of thyroid cancer.

The discovery has made possible the creation of a quick, accurate and inexpensive method to identify papillary thyroid carcinoma. An application has been filed for a patent.

The results of the study, conducted during the PhD research of Mateus de Camargo Barros Filho, were published in The Journal of Clinical Endocrinology & Metabolism.

The principal investigator was Luiz Paulo Kowalski, Head of INCITO, one of the National Science & Technology Institutes (INCTs) supported by the National Council for Scientific & Technological Development (CNPq), and by FAPESP in São Paulo State. Silvia Rogatto, a member of INCITO and a professor at São Paulo State University’s Botucatu Medical School (FMB-UNESP), co-coordinated the project.

“Fine needle aspiration biopsy is currently the main method of determining whether a thyroid nodule is benign or malignant. In some 20% of cases, the result of the biopsy is indeterminate, and as a precaution the patient is submitted to surgery for thyroid gland removal. Subsequent analysis shows that surgery was unnecessary in 60% of patients with an indeterminate diagnosis,” Rogatto said.

Although the hormones produced by the thyroid gland can be artificially replaced, surgical removal of the gland usually has adverse after-effects, and patients require treatment for the rest of their lives. “In some cases, the operation results in parathyroid failure, and the patient becomes dependent on calcium supplementation. Uptake of phosphorus, magnesium and other important minerals for the organism is impaired,” he added.

According to Surveillance, Epidemiology & End Results (SEER), a US National Cancer Institute program that collects incidence and survival data from cancer registries, the incidence of thyroid cancer has tripled in the past 35 years. In Brazil, the National Cancer Institute (INCA) estimated 9,200 new cases in 2014, 8,050 of them in women.

Incidence in the city of São Paulo is one of the highest in Brazil and has accelerated even faster than in the US. This is explained in part by improved diagnosis due to increased use of ultrasound scanning.

“This rise in the incidence of thyroid cancer is almost entirely driven by papillary thyroid carcinoma (PTC), the most frequent subtype. Although this tumor generally has a good prognosis, it represents a public health problem because it’s very common and requires surgical procedures,” Rogatto said.

Search for biomarkers

To enhance diagnostic accuracy and avoid unnecessary surgery, in 2011, the group at INCITO began searching for molecular markers that could help distinguish between benign and malignant lesions.

The researchers analyzed samples of thyroid tissue removed surgically from more than 350 patients and held by A.C. Camargo Cancer Center’s biobank. They included tissue from diagnosed tumors and histopathologically confirmed benign lesions, as well as samples from non-neoplastic and indeterminate cases.

Gene expression profiling was performed by microarray analysis of the protein-encoding transcripts in messenger RNA.

“Initially, we analyzed paired samples, comparing the expression profiles of normal tissue and tumor tissue from the same individual,” Rogatto said. “Then, we compared several samples of normal tissue with several unpaired tumor samples. Lastly, we compared the final profiles of the paired and unpaired samples, and the results were highly concordant.”

Next, they validated the biomarkers in a different set of thyroid tissue samples (138 pairs from PTC and surrounding normal tissue) from The Cancer Genome Atlas (TCGA, a consortium linked to the US National Cancer Institute that collects genomic and clinical data from patients in several countries) and Gene Expression Omnibus (GEO).

“The findings matched our data very closely, affording external validation for our observations, which aren’t specific to the Brazilian population,” Rogatto said.

With the aid of statistical tools, the researchers selected a list of genes that enabled them to distinguish between benign and malignant nodules. The initial validations were performed by means of computer simulation until they reached a group of five genes that displayed high selectivity and specificity for the identification of PTC.

“Three of these genes are differentially expressed in the tumor. The other two serve as a reference,” Rogatto said.

The INCITO group developed a patent-pending technique to evaluate the expression of these five target genes by means of an assay based on real-time polymerase chain reaction (PCR). According to Rogatto, the analysis also serves to identify patients who are at high risk of cervical lymph node metastasis and require more aggressive surgery.

“It’s possible to perform an assay cheaply with a small tissue sample,” Rogatto said. “We believe it’s feasible to develop a diagnostic kit that would be very useful in clinical practice.”

First, however, the researchers plan to validate the method with thyroid tissue obtained from biopsies. “We’ve applied for permission from our institution’s ethics committee to test the methodology in tissue left over from biopsy specimens after cytological analysis,” she said.

In parallel, the INCITO group are working on the validation of other biomarkers that can be used to distinguish PTC from other subtypes of thyroid cancer that are less frequent and more aggressive, such as follicular carcinoma and anaplastic carcinoma.