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

Funding received no funding for this work. Disclosure summary : BCS, CD, JFK, MJL, PS, SA, and SPW report no competing financial interests. MEZ reports personal fees Eli Lilly and grant support from Eli Lilly, GeneproDx, and Merck. MIH reports personal fees from Blueprint Medicine, Eli Lilly, Loxo Oncology, and Veracyte; and research support from Eli Lilly. LJW reports personal fees from Bayer, Blueprint Medicine, Eisai, Eli Lilly, Genentech, Loxo Oncology, and Merck. PWL reports personal fees from Veracyte. SGW reports personal fees from Bayer. GCK, JEB, RTK, and YH are Veracyte employees and equity owners. GCK holds relevant patents. 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. testing. 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 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.

A c c e p t e d M a n u s c r i p t 5

Background
Within the past decade it was common practice for most cytologically indeterminate thyroid nodules to undergo surgery given their 20%-30% risk of malignancy (1).
Since then, guidelines have increasingly included molecular testing as a diagnostic adjunct towards management of cytologically indeterminate thyroid nodules, rulingout cancer with a high negative predictive value test reducing the need for diagnostic hemithyroidectomy and identifying genomic alterations that are highly predictive of malignancy (2)(3)(4). Additionally, novel therapies have emerged that target specific genomic alterations to treat refractory thyroid cancer and offer the potential for increased efficacy with more tolerable side effects.
The Afirma Genomic Sequencing Classifier (GSC) is used to rule out malignancy and reclassify cytologically indeterminate (Bethesda III or IV) nodules to molecularly benign or suspicious (5). The original Afirma Xpression Atlas (XA) panel reported on 761 genomic variants and 130 fusion pairs from 511 genes (6). In March 2020, the panel was expanded to include 905 genomic variants and 235 fusion pairs from 593 genes (7). The Afirma XA intended use population includes cytologically indeterminate nodules with an Afirma GSC suspicious result, those that are cytologically suspicious for malignancy or malignant (Bethesda V or VI, respectively), and extra-thyroidal masses that are known or suspected to be thyroid cancer metastases (such as suspicious lymph nodes) (6,7).
Here, we report Afirma GSC and XA data from 50,644 Bethesda III-VI real-world clinical samples consecutively resulted in the Veracyte CLIA laboratory. These novel A c c e p t e d M a n u s c r i p t 6 data shed light on the landscape and potential clinical utility available from the preoperative genomic analysis of Bethesda III-VI thyroid nodules.

Bethesda III/IV Thyroid Fine-Needle Aspirates
Consecutive cohorts of Bethesda III/IV nodules with ALK, BRAF, NTRK or RET fusions (other than RET/PTC1 and RET/PTC3) that were submitted to Veracyte for molecular analysis were identified. Local surgical pathology diagnoses were sought with IRB approval. Only one nodule per patient was included.
Excisional surgical pathologic diagnoses were available for 64 thyroid nodules from 64 patients. No sample had a concurrent variant. Fifteen ALK fusion partners included ten STRN and five EML4. Nine (60%) were in malignant tumors ( Figure 3) whereas six (4 STRN and 2 EML4) were identified in adenomatoid or hyperplastic nodules (PPV=60%). One of these adenomatoid nodules harbored both a STRN fusion partner and an additional MRPS16/TTC18 gene fusion ( Table 2 shows Table 4.

Genomic Landscape of FNAs positive for Medullary Thyroid Carcinoma
Examination of 50,644 Bethesda III-VI FNAs revealed 152 MTC Classifier positive cases (Table 1)

Discussion
Here, we report Afirma GSC and XA data from 50,644 Bethesda III-VI real-world thyroid nodules consecutively resulted over a two-year time frame in the Veracyte These potentially PPV raising alterations were present among 31% and 39% of Afirma GSC Suspicious Bethesda III and IV FNAs, respectively. Two-thirds of the 48,952 cytologically indeterminate nodules were reported as molecularly benign (Afirma GSC benign). GSC benign results were seen in 68% of Bethesda III samples and 55% of Bethesda IV samples (Table 1). In the proper clinical context, this rate of a benign molecular result, combined with its high negative predictive value (5), supports the opportunity to avoid diagnostic surgery among many thyroid nodules with cytologically indeterminate results (2)(3)(4). Low rates of surgical resection among GSC benign nodules have been reported in multiple independent experiences (19)(20)(21)(22)(23)(24)(25)(26), including nodules with oncocytic (Hürthle cell) dominant cytology (27). While the Afirma GSC test is not clinically performed on cytologically malignant nodules, the low rate of Afirma GSC benign results (3.7%, Table 1) seen here among de-identified Bethesda VI (cytologically malignant) samples supports the high sensitivity for cancer observed during clinical validation (5).
Suspected thyroid nodules that are enlarged parathyroid glands (within or adjacent to the thyroid) are a challenge for both ultrasonography and cytopathology (28).
Nearly three quarters of Parathyroid Classifier positive samples were called Bethesda III, and almost all the remainder were called Bethesda IV (Table 1).
Overall, the Afirma Parathyroid Classifier was positive in 0.57% of cytologically indeterminate nodules. Parathyroid adenoma, parathyroid hyperplasia, and the rare parathyroid carcinoma should be considered in the differential diagnosis of a positive result. Diagnosis of MTC is also a cytological challenge (29,30). The Afirma MTC A c c e p t e d M a n u s c r i p t 13 Classifier was positive in 0.25% of cytologically indeterminate nodules, and higher prevalence was seen among Bethesda categories V and VI (  Receptor tyrosine kinase fusions are being targeted by small molecule inhibitors to treat late-stage cancers, including advanced thyroid cancer (33). Inhibitors targeting malignancies with NTRK fusions and RET alterations have received pan-or multicancer FDA approval (34)(35)(36)(37). Additionally, having these molecular data up front, even for Bethesda V/VI nodules, may provide insight for surgical management along with pre-planned therapeutic options for cancers with the potential for recurrence or aggressive behavior (38,39 Table 3). Prior studies have shown that mutations in known driver genes are typically concordant between the primary tumor and distant metastatic deposits, suggesting that molecular knowledge from the primary tumor, or the most accessible metastatic deposit, may be concordant with less accessible metastatic deposits (40)(41)(42).
The genomic landscape of MTC has been studied by others using surgical samples (43,44). Here we report that an alteration was identified in 70% of MTC Classifier positive samples via a less invasive FNA sample (Figure 4). The alterations we identified were primarily in RET, a target of several FDA-approved and investigational therapies (33,37,45). Afirma XA does not differentiate germline from somatic alterations. While family history, physical examination, and Afirma XA result may suggest a sporadic or familial origin of MTC, germline RET protooncogene testing (46) is recommended for all MTC cases regardless of the Afirma XA result.
Our study has several limitations, including that we did not correlate the surgical pathology outcome with most of the FNA samples described here. We did report surgical histology, performed locally, for small consecutive cohorts of Bethesda III/IV nodules with alterations of specific interest. There was no central blinded histopathologic review by experienced endocrine pathologists.
Another limitation is potential selection bias, especially among Bethesda V and VI samples. It is likely that a smaller fraction of Bethesda V and VI samples was submitted for molecular testing as they represented only 3% of the study cohort. This greater selectivity may increase selection bias. For example, we previously reported the incidence of the first generation Afirma (GEC) MTC Classifier positivity among small cohorts of consecutive Bethesda V and VI samples and did not limit testing to those where molecular testing was clinically requested. We did that to minimize selection bias and a progressive rise in test positivity across the Bethesda III-VI categories was seen (47). Here, however, we investigated only samples submitted to Veracyte for molecular testing and we saw a peak in MTC Classifier positivity in the Bethesda V category (Table 1). It is possible that the difference between these observations is the result of selection bias.
Strengths of our study include whole exome RNA sequencing and variant/fusion reporting on a large cohort of thyroid nodule samples consecutively submitted in real-world practice by treating physicians. We report genomic classifiers, variants, and fusions across the entire cohort, thus avoiding selection bias when some of tests may not have been requested by the submitting physician.

Conclusions
The Afirma GSC and XA assays have been analytically and clinically validated. In the current characterization of the molecular landscape of real-world Bethesda III-VI nodules, we found that two-thirds of Bethesda III/IV nodules resulted Afirma GSC benign. A genomic alteration was identified by Afirma XA in almost half of those that resulted Afirma GSC suspicious, most commonly a variant of NRAS or HRAS.