Expected, unexpected, and unintended consequences of the Afirma gene sequencing classifier

Molecular testing is a diagnostic adjunct that refines cancer risk assessment of cytologically indeterminate thyroid nodules. The aim of this study was to investigate the impact of the Afirma gene sequencing classifier on clinical practice. Our hypothesis was that the gene sequencing classifier has led to an increased rate of atypia of undetermined significance.

A single-center retrospective analysis of thyroid fine-needle aspiration was performed from October 2021 to January 2025. Rates of atypia of undetermined significance for 19 months before and after the introduction of the gene sequencing classifier were compared. Cytopathologists (n = 6) were divided into those with <15 years or ≥15 years of experience.

There were 2,481 thyroid fine-needle aspirations: 1,164 pre-gene sequencing classifier and 1,317 post-gene sequencing classifier. Pre-gene sequencing classifier, 16% of fine-needle aspirations were atypia of undetermined significance compared with 20% post-gene sequencing classifier (P = .003). Following implementation of the gene sequencing classifier, there was an increase in atypia of undetermined significance from 18% to 20% for less experienced cytopathologists (P = .4), compared with 15%-20% for more experienced cytopathologists (P = .004). Pre-gene sequencing classifier, 39 patients with atypia of undetermined significance underwent thyroidectomy; 30 (77%) had benign disease and 9 (23%) had cancer. Median days to surgery was 147 (interquartile range, 89-308.5). Post-gene sequencing classifier, 51 patients with atypia of undetermined significance underwent thyroidectomy; 30 (59%) had benign disease and 21 (41%) had cancer. Median days to surgery was 151 (interquartile range, 66-249).

An unintended consequence of the gene sequencing classifier was an increase in atypia of undetermined significance, a phenomenon unexpectedly observed among more experienced cytopathologists. The gene sequencing classifier was associated with an 18% absolute increase in malignancy for patients undergoing thyroidectomy for atypia of undetermined significance without significant change in time to surgery.