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Indeterminate thyroid nodules form a heterogenous group of lesions that constitute 5% to 30% of thyroid cytology diagnoses. We introduce a triple immunostaining protocol for subtyping. Galectin-3, HBME-1, and p27 triple immunostaining, performed on destained cytology slides and formalin-fixed paraffin-embedded tissue, was developed and applied to 51 patients retrospectively with preoperative cytologic diagnoses of follicular lesion of undetermined significance (n=40), atypia of undetermined significance (n=6), and suspicious for follicular neoplasm (n=5). The malignant rate in this series was 43.1% (22/51). A hierarchal evaluation algorithm was generated based on digital image quantitation of triple-stained histologic sections, and applied to both cytology and histology specimens. Fifty of 51 cytology cases have triple staining validated by internal controls. In cytology specimens, the individual sensitivities and specificities of p27, Galectin3, and HBME1 for cancer with 95% confidence interval are: 86.2% (0.674, 0.955)/66.7% (0.431, 0.845); 77.3% (0.542, 0.913)/72.4% (0.525, 0.866); and 72.7% (0.496, 0.884)/93.1% (0.758, 0.988), respectively. Sensitivity is increased to 95.5% (0.751, 0.998), but specificity is decreased to 69.0% (0.490, 0.840), if Galectin3 and HBME1 are both used in combination as markers for malignancy. However, the level of specificity is increased to 86.2% (0.674, 0.955) and sensitivity remains high 100% (0.808, 1) if in addition, using the Galectin3/HBME1:p27 ratio (ratio ≥2 indicating malignancy) for 2 or 3 markers positive cases. Thus, the triple staining method on cytology slides and histology sections shows a similar sensitivity/specificity/positive predictive value/negative predictive value of 100.0%/86.2%/84.0%/100.0% and 95.5%/86.2%/84.0%/96.2%, respectively (P=0.92). Overall, p27 is the most frequent single positive marker (19/50, 38% in cytology), consistent with benign nature of most indeterminate thyroid nodules. Galectin-3 and HBME-1 colocalization (positive in the same cell) was demonstrated in thyroid cancer in 45.5% (10/22) of histology sections, but in none of the normal thyroid tissues and benign thyroid lesions. This supports the notion that synchronous activation of Galectin-3 and HBME-1 occurs in thyroid malignancy and is highly specific for malignancy. We have demonstrated the performance and pattern of triple immunostaining for subtyping indeterminate thyroid nodules. Further studies and validation in different larger populations are warranted.

applied immunohistochemistry and molecular morphology

A Pilot Study of Galectin-3, HBME-1, and p27 Triple Immunostaining Pattern for Diagnosis of Indeterminate Thyroid Nodules in Cytology With Correlation to Histology