Afirma Genomic Sequencing Classifier and Xpression Atlas Molecular Findings in Consecutive Bethesda III-VI Thyroid Nodules

Abstract

Context: Broad genomic analyses among thyroid histologies have been described from relatively small cohorts.

Objective: Investigate the molecular findings across a large, real-world cohort of thyroid fine-needle aspiration (FNA) samples.

Design: Retrospective analysis of RNA sequencing data files.

Setting: Clinical Laboratory Improvement Amendments laboratory performing Afirma Genomic Sequencing Classifier (GSC) and Xpression Atlas (XA) testing.

Participants: A total of 50 644 consecutive Bethesda III-VI nodules.

Intervention: None.

Main outcome measures: Molecular test results.

Results: Of 48 952 Bethesda III/IV FNAs studied, 66% were benign by Afirma GSC. The prevalence of BRAF V600E was 2% among all Bethesda III/IV FNAs and 76% among Bethesda VI FNAs. Fusions involving NTRK, RET, BRAF, and ALK were most prevalent in Bethesda V (10%), and 130 different gene partners were identified. Among small consecutive Bethesda III/IV sample cohorts with one of these fusions and available surgical pathology excision data, the positive predictive value of an NTRK or RET fusion for carcinoma or noninvasive follicular thyroid neoplasm with papillary-like nuclear features was >95%, whereas for BRAF and ALK fusions it was 81% and 67%, respectively. At least 1 genomic alteration was identified by the expanded Afirma XA panel in 70% of medullary thyroid carcinoma classifier-positive FNAs, 44% of Bethesda III or IV Afirma GSC suspicious FNAs, 64% of Bethesda V FNAs, and 87% of Bethesda VI FNAs.

Conclusions: This large study demonstrates that almost one-half of Bethesda III/IV Afirma GSC suspicious and most Bethesda V/VI nodules had at least 1 genomic variant or fusion identified, which may optimize personalized treatment decisions.

Keywords: indeterminate cytology; molecular diagnostics; personalized healthcare; thyroid cancer; thyroid nodule; variant detection.

Figure 1.

Percent of alterations among Afirma Xpression Atlas Intended Use samples: Afirma GSC Suspicious Bethesda III (n = 12,356), Afirma GSC Suspicious Bethesda IV (n = 4,238), Bethesda V (n = 837), and Bethesda VI (n = 855). The three most frequent alterations in each Bethesda category were identified. The frequency of these five alterations among each Bethesda category is shown. All other alterations are shown as “Other Alteration”. = Alteration Negative; = NRAS:p.Q61R; = BRAF:p.V600E; = HRAS:p.Q61R; = CCDC6/RET; = ETV6/NTRK3; = Other Alteration.

Figure 2.

Sankey diagram of ALK, BRAF, NTRK, and RET fusions when the partner gene was identified in at least two samples. On the left are partner genes, and on the right are the key kinase genes. The thicknesses of the vertical bars denote the number of times each partner was observed. The thicknesses of the connections reveal the number of times each partner is fused to the key kinase gene. Several partners fused with more than one key kinase gene (EMLA4, ERC1, TG, SQSTM1, VIM, TFG, and LMNA).

Figure 3.

Percent of consecutive Bethesda III/IV nodules with ALK, BRAF, NTRK, or RET fusions (other than RET/PTC1 and RET/PTC3) demonstrating carcinoma or Non-Invasive Follicular Thyroid neoplasm with Papillary-like nuclear features (NIFTP) on local surgical histopathology (positive predictive value). = Malignant; = NIFTP.

Figure 4.

Variants and fusions identified in 152 MTC Classifier positive FNA samples using the expanded Afirma Xpression Atlas.