Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples

Abstract

Context: Thyroid nodules are common in adults, but only a small fraction of them is malignant. Fine-needle aspiration (FNA) cytology provides a definitive diagnosis of benign or malignant disease in many cases, whereas about 25% of nodules are indeterminate, hindering most appropriate management.

Objective: The objective of the investigation was to study the clinical utility of molecular testing of thyroid FNA samples with indeterminate cytology.

Design: Residual material from 1056 consecutive thyroid FNA samples with indeterminate cytology was used for prospective molecular analysis that included the assessment of cell adequacy by a newly developed PCR assay and testing for a panel of mutations consisted of BRAF V600E, NRAS codon 61, HRAS codon 61, and KRAS codons 12/13 point mutations and RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements.

Results: The collected material was adequate for molecular analysis in 967 samples (92%), which yielded 87 mutations including 19 BRAF, 62 RAS, 1 RET/PTC, and five PAX8/PPARγ. Four hundred seventy-nine patients who contributed 513 samples underwent surgery. In specific categories of indeterminate cytology, i.e. atypia of undetermined significance/follicular lesion of undetermined significance, follicular neoplasm/suspicious for a follicular neoplasm, and suspicious for malignant cells, the detection of any mutation conferred the risk of histologic malignancy of 88, 87, and 95%, respectively. The risk of cancer in mutation-negative nodules was 6, 14, and 28%, respectively. Of 6% of cancers in mutation-negative nodules with atypia of undetermined significance/follicular lesion of undetermined significance cytology, only 2.3% were invasive and 0.5% had extrathyroidal extension.

Conclusion: Molecular analysis for a panel of mutations has significant diagnostic value for all categories of indeterminate cytology and can be helpful for more effective clinical management of these patients.

Figures

Fig. 1.

Evaluation of FNA sample adequacy before mutational analysis using the expression of the GAPDH and KRT7 genes detected by RT-PCR. A, KRT7 and GAPDH were expressed at similar levels in normal thyroid tissues (NT) (n = 10), follicular carcinomas (FC) (n = 10), and papillary carcinomas (PC) (n = 40) (mean ΔCt = −0.41, where ΔCt is CtGAPDH − CtKRT7; P = 0.90). However, KRT7 was expressed at a significantly lower level in white blood cells (WBC), which are a frequent contaminant of thyroid FNA samples (mean ΔCt = 13; P < 0.001). B, Serial dilution of RNA from thyroid papillary carcinoma tissue in RNA derived from normal WBC demonstrated a linear decrease in KRT7 expression compared with stable GAPDH expression, with the ΔCt of 3.5 corresponding to approximately 10% of thyroid epithelial cells within the sample. The ΔCt of 3.5 was used as a cutoff for sample adequacy.

Fig. 2.

Correlation between the results of mutational analysis in FNA samples and outcome in specific groups of indeterminate cytology. PTC, Papillary thyroid carcinoma; PTC, FV, papillary thyroid carcinoma, follicular variant; FTC, follicular thyroid carcinoma; FA, follicular adenoma; HN, hyperplastic nodule.

Fig. 3.

Proposed clinical algorithm for management of patients with cytologically indeterminate thyroid FNA applying the results of mutational analysis.