Clinical Performance of the Afirma Genomic Sequencing Classifier (GSC) in Pediatric Patients With Cytologically Indeterminate Thyroid Nodules

Introduction

• Cytologically indeterminate thyroid nodules (ITN) in pediatrics carry a higher risk of malignancy (~30-60%) compared with adult ITN (13-34%).¹
• The 2015 ATA pediatric thyroid nodule guidelines recommend surgical resection for ITN.¹
• The Afirma GSC is an exome-enriched RNAseq assay that was validated in patients > 21 yrs with a 96% negative predictive value (NPV) for malignancy when benign (GSC-B) in lesions with indeterminate cytology, allowing patients to avoid unnecessary thyroid surgery.²

Study Aim

The goal of this study was to evaluate the performance of the Afirma GSC in pediatric patients with ITN.

Methods

• Forty-nine ITN from patients < 21 yrs sent for Afirma GSC testing had histopathology or clinical follow-up data collected.
  • These outcomes were collected on consecutive patients and were not known prior to inclusion in the study.
• Afirma GSC ensemble classifier and Xpression Atlas (XA – the GSC molecular variant and fusion panel) data were analyzed for all samples (though XA data is not reported clinically for GSC-B cases).
• For Afirma GSC-B cases that avoided surgery, a two-year clinical follow-up with exam and/or ultrasound was a surrogate for a true negative (TN) result.

Results

• Nine males and 40 females (ages 12-20 yrs, median 18.5 [IQR 17.3-19.8]) had 30 GSC-B and 19 GSC-suspicious (S) cases from nodules ranging from < 1cm to > 7cm in size (Table 1 and Figure 1).
• Of GSC-S cases that were resected, histopathology determined that 14 (73.7%) were malignant (true positive, TP) and five (26.3%) were benign (false positive, FP) (Table 1).
• All GSC-B cases were either histologically (n=9) or clinically benign (TN) (Table 1).
• All 14 malignancies were GSC-S (sensitivity 100% [95% CI 77-100%]); 30/35 clinically or histologically benign cases were GSC-B (specificity 86% [95% CI 70-95%]). The NPV for a GSC-B result was 100% [95% CI 88-100%] (Figure 2).
• Genomic alterations were not detected by XA in the 30 Afirma benign samples and there was a DICER1 alteration in one of the five false positive lesions (Table 2).
• Among the 14 TP samples, there were 7 papillary thyroid carcinomas (PTC; n=1 with FGFR2::VCL fusion and n=1 PPP1R21::ALK fusion (Fig 3)); 1 oncocytic carcinoma (PAX8::PPARG fusion); 1 NIFTP; 2 infiltrative follicular subtype of PTC; and 3 follicular thyroid carcinomas (n=1 with PAX8::PPARG fusion, n=2 with NRAS p.Q61K) (Table 2).
  • The FGFR2::VCL fusion was recently reported in 4 thyroid tumor samples from a cohort of 5030 sequenced thyroid malignancies and was reported in a 21-year-old with BIII cytology who had a PTC on final histopathology.⁴
  • The PPP1R21::ALK fusion has previously been reported in 6 thyroid tumor samples from the Afirma DB without histologic correlate.⁵
  • The PPP1R21::ALK fusion has been described when testing a resected tumor in a 12-year-old female with a classic PTC and T2N1b histopathology findings that had a Bethesda VI cytology result.⁶
  • To our knowledge, this is the first report of detection of the PPP1R21::ALK fusion in a pre-operative thyroid tumor specimen with Bethesda III cytology and a histologic correlate.
  • Figure 3 shows a representative schematic of the FGFR2::VCL (3A) and PPP1R21::ALK (3B) fusions.

Conclusion

• In older adolescents with ITN, the Afirma GSC showed excellent performance when defining a true negative result by histology or clinical follow-up.
• Our findings indicate that an Afirma GSC-B result appears reassuring and may allow for a more conservative management strategy in younger patients with ITN.
• The exome-enriched RNAseq Afirma GSC platform additionally allows for the discovery of rare and targetable alterations such as ALK and FGFR2 fusions. More data is required to fully understand the clinical significance of pediatric thyroid tumors harboring these rare fusions.