Afirma Genomic Sequencing Classifier Research Articles

Outcomes of cytologically indeterminate thyroid nodules managed with Genomic Sequencing Classifier.

Molecular testing can refine the risk of malignancy in thyroid nodules with indeterminate cytology to decrease unnecessary diagnostic surgery. This study was performed to evaluate the outcomes of cytologically indeterminate thyroid nodules managed with Afirma genomic sequencing classifier (GSC) testing. Adult patients who underwent a biopsy at three major academic centers between July 2017 and June 2021 with Bethesda III or IV cytology were included. All patients had surgery or minimum follow-up of 1 year ultrasound surveillance. The primary outcomes were the sensitivity, specificity, PPV, and NPV of GSC in Bethesda III and IV nodules. The median nodule size of the 834 indeterminate nodules was 2.1 cm and the median follow-up was 23 months. GSC's sensitivity, specificity, PPV, and NPV across all institutions were 95%, 81%, 50%, and 99% for Bethesda III nodules and 94%, 82%, 65%, and 98% for Bethesda IV nodules, respectively. The overall false negative rate was 2%. The NPV of GSC in thyroid nodules with oncocytic predominance was 100% in Bethesda III nodules and 98% in Bethesda IV nodules. However, the PPV of oncocytic nodules was low (17% in Bethesda III nodules and 45% in Bethesda IV nodules). Only 22% of thyroid nodules with benign GSC results grew during surveillance. GSC is a key tool for managing patients with indeterminate cytology, including the higher-risk Bethesda IV category. GSC benign thyroid nodules can be observed similarly to thyroid nodules with benign cytology.

Use of the Afirma Xpression Atlas for cytologically indeterminate, Afirma Genomic Sequencing Classifier suspicious thyroid nodules: Clinicopathologic analysis with postoperative molecular testing.

Afirma has recently introduced its Xpression Atlas (XA) as an adjunct to its Genomic Sequencing Classifier (GSC) for risk stratification of cytologically indeterminate thyroid nodules. We evaluated the performance of Afirma XA and associated pathologic findings for Afirma GSC suspicious nodules. Intradepartmental records of thyroid fine-needle aspirations (FNAs) from January 2021 to December 2022 were identified and reviewed for patient and nodule characteristics, FNA findings, molecular test results, and final surgical pathology, if available. Material for Afirma GSC testing was collected in 624 thyroid FNAs, and 148 (24%) were classified as cytologically indeterminate. Afirma GSC testing was successful in 132 (89%) of those cases, of which 35 (27%) were Afirma GSC suspicious. Afirma XA testing was positive in 11 cases (11/35 [31%]). Eight (73%) patients underwent surgery that revealed 7 patients with papillary thyroid carcinoma and 1 patient with noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) (risk of malignancy: 100% [8/8]). Among the 24 patients with negative Afirma XA results, 19 (79%) underwent surgery, revealing 5 patients with malignancy and 3 patients with NIFTP (risk of malignancy: 42% [8/19]). Overall, the risk of malignancy for Afirma GSC suspicious nodules was 59% (16/27). Afirma XA improved risk stratification of thyroid disease with a high risk of malignancy in Afirma GSC suspicious nodules. A negative Afirma XA result, however, should not be used as a rule-out test.

Interpace, Affirma, and Thyroseq Performance Characteristics in 341 Bethesda Category III/IV Thyroid Nodules

Abstract Introduction/Objective Multiple FDA-approved molecular testing platforms are used by our institution for evaluating genomic characteristics of thyroid nodules. We aim to correlate molecular results and final histopathology (HPE) of thyroid nodules with cytologic diagnosis of The Bethesda System (TBS) Category III or IV. Methods/Case Report We included 341 cases of thyroid nodules diagnosed as TBS-III or TBS-IV with subsequent molecular analysis performed on 1 of 5 platforms (Interpace ThyraMIR Thyroid miRNA Classifier, Afirma Genomic Sequencing Classifier, Thyroseq Genomic Classifier, Next-Generation Sequencing (NGS) Fusion Panel, or RosettaGX Reveal Thyroid miRNA Classifier). All findings reported on molecular analysis and all benign or malignant neoplasms diagnosed on final HPE were considered “significant findings.” Performance characteristics of all 5 molecular platforms were analyzed. Results (if a Case Study enter NA) Of 341 cases, 80.1% (n=273) were classified as TBS-III and 19.9% (n=68) were classified as TBS-IV. The overall concordance of molecular and final HPE when available was 60.9% (n=28). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are respectively 75.0%, 60.0%, 75.0%, and 60.0% for Interpace; 65.0%, 60.0%, 85.0%, and 33.0% for Afirma; and 75.0%, 50.0%, 75.0%, and 50.0% for Thyroseq platforms. Of TBS-III cases, 66.3% (n=181), 25.6% (n=70), 4.8% (n=13), 1.5% (n=4), and 1.8% (n=5) underwent molecular testing via Interpace, Afirma, Thyroseq, NGS, and Rosetta platforms, respectively. Only 9.2% (n=25) of TBS-III cases had subsequent HPE. Concordance of molecular and final HPE for TBS-III cases was 52.0% (n=13) with a PPV of 75.0% and NPV of 40.0%. Concordances of molecular and final HPE according to molecular platform are: 57.1% (n=4) on Interpace, 53.3% (n=8) on Afirma, and 50.0% (n=1) on Thyroseq. Of TBS-IV cases, 52.9% (n=36), 36.8% (n=25), 5.9% (n=4), 2.9% (n=2), and 1.5% (n=1) underwent molecular testing via Interpace, Afirma, Thyroseq, NGS, and Rosetta platforms, respectively. Only 30.9% (n=21) of TBS-IV cases had subsequent HPE. Concordance of molecular and final HPE for TBS-IV cases was 71.4% (n=15) with a PPV of 86.0% and NPV of 43.0% Concordances of molecular and final HPE according to molecular platform are: 71.4% (n=5), 66.7% (n=6), and 75.0% (n=3) on Interpace, Afirma, and Thyroseq respectively. Conclusion Our study reports similar performance characteristics of 3 commonly used molecular platforms (Interpace, Afirma, and Thyroseq) for genomic evaluation of thyroid nodules.

DICER1-Mutant Thyroid Carcinoma of Unusual Morphology: A Follicular Carcinoma with Bizarre Nuclei or a Novel Entity?

Abstract Introduction/Objective DICER1 mutations are associated with a host of benign and malignant neoplasms, of which the thyroid ranks among those with the highest prevalence. We present a case of a DICER1-mutant thyroid carcinoma with unusual morphologic features not described in the existing literature. Methods/Case Report A 29-year old 33 weeks pregnant female presented with swelling of the left neck. The patient subsequently underwent an ultrasound, which revealed a thyroid lesion measuring 6.2cm in greatest dimension. Labs including TSH and free T4 wree within normal 3rd trimester limits. Following an unremarkable delivery, FNA revealed loosely clustered cells with marked hyperchromasia and nuclear pleomorphism and variable microfollicular architecture in a colloid-containing background. Though definitively malignant, the anomalous cytomorphology of this neoplasm prompted molecular testing by Afirma Genomic Sequencing Classifier and Xpression Atlas next-generation sequencing, which was significant for a DICER1 hotspot mutation (p.E1705K c.5113G>A). Follow-up total thyroidectomy was pertinent for a tan-red, nodular mass involving 95% of the thyroid. Histomorphology revealed a neoplasm of primarily macrofollicular architecture with scattered microfollicles and intervening zones that appeared poorly-to-undifferentiated. Cells demonstrated bizarre hyperchromatic, pleomorphic nuclei with multinucleation and Azzopardi effect. Capsular invasion was identified. Inconsistent with a poorly-differentiated or anaplastic carcinoma, the mitotic rate and Ki-67 index were low at < 1/2mm2 and 1-2%, respectively. The tumor was consequently theorized to represent a follicular carcinoma with bizarre nuclei or other entity associated with DICER1 mutations not previously described. To date, no recurrence or metastasis has occurred. Results (if a Case Study enter NA) NA Conclusion We report an enigmatic morphology of a thyroid cancer associated with a DICER1 mutation. DICER1 mutations have been hitherto described in follicular carcinoma, papillary carcinoma, poorly-differentiated carcinoma and thyroblastoma, none of which are consistent with the present case. Thus, we conjecture that this morphomolecular profile may constitute a "follicular carcinoma with bizarre nuclei" or other novel entity not previously reported. Further investigation should be pursued to determine the nature of this entity, its pathophysiology, epidemiology, possible association with germline DICER1 syndrome, and relationship between morphology and prognosis in patients with DICER1 mutations.

THU678 Real-world Performance Of Afirma Genomic Sequencing Classifier (GSC) In A Community Healthcare Setting

Abstract Disclosure: V. Master: None. J. Syeda: None. S. Charatz: None. D. Reidy: None. K. Clary: None. Introduction: Indeterminate thyroid nodules pose a significant clinical challenge due to their potential risk of malignancy. The Afirma GSC has emerged as a valuable tool for the non-invasive management of these nodules. However, its real-world performance in community healthcare settings remains poorly understood. Aim: Comparison of GSC performance in a community healthcare setting to the validation study (VS) metrics. Method: A retrospective study was conducted in a community endocrine practice, where 91 Afirma GSC samples were obtained from patients who had either suspicious cytology or atypia of undermined significance (AUS) between 1/1/2021 and 12/31/2022. Ten samples were excluded due to insufficient material. Surgical pathology was used as the gold standard for diagnosis. Molecular benign results without surgical pathology were considered true negatives, and 2 unoperated patients with suspicious results were excluded from analysis. We calculated observed positive predictive value (oPPV) and observed specificity (oSP) to account for these exclusions. Non-invasive follicular neoplasm with papillary-like features is considered malignant. The prevalence of thyroid cancer in the study population was 19%. Results are compared to previously published VS of 191 samples. Result: Our study showed a higher accuracy of 89% of the Afirma GSC in our community healthcare setting, with a sensitivity (SN) of 93%, oSP of 88%, oPPV of 63%, and negative predictive value (NPV) of 98%. In comparison, the VS had a SN of 91%, SP of 68%, PPV of 47%, NPV of 96%, and accuracy of 74%. Additionally, our study showed a higher benign call rate (BCR) of 70% compared to 54% in the VS. Conclusion: Our study provides evidence of the real-world performance of the Afirma GSC and supports its use as a non-invasive tool for the management of indeterminate thyroid nodules. Better oSP and oPPV are suggestive of higher yield of cancers for resected nodules based on suspicious GSC.

SAT630 Analytical Validation Of A Telomerase Reverse Transcriptase (TERT) Promoter Mutation Assay

Abstract Disclosure: P. Iyer: None. R. Dadu: Advisory Board Member; Self; Exelixis, Inc., Bayer, Inc. Research Investigator; Self; Merck, Exelixis, Inc., AstraZeneca. A. Barque: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. Z. Cleslei: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. H. Jiang: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. S. Walsh: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. X. Zheng: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. Y. Hao: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. J. Huang: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. J.P. Klopper: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. R.T. Kloos: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. M. Cabanillas: Advisory Board Member; Self; Exelixis, Inc., Bayer, Inc. Consulting Fee; Self; Lilly USA, LLC, Bayer, Inc. Research Investigator; Self; Genentech, Inc., Merck. TERT promoter mutations are relatively enriched in larger, aggressive thyroid cancers and these malignancies carry an independently decreased rate of disease free and disease specific survival when compared to thyroid cancers with wild type TERT promoter gene sequences. High quality analytical validation of a diagnostic test promotes confidence in the results which inform clinical decision making. TERT promoter mutation testing is now offered as a complement to Afirma Genomic Sequencing Classifier (GSC) testing in thyroid nodules that are not designated as molecularly benign. The Afirma TERT test was developed at Veracyte, Inc. based on the AmpliSeq-for-Illumina technology to detect TERT promoter variants C228T and C250T in genomic DNA (gDNA) extracted from patient thyroid fine needle aspirates (FNAs) sent for Afirma GSC testing, without any need for increased needle passes or sample volume. Herein, we describe the analytical performance of the Afirma TERT test. We examined the analytical sensitivity of the Afirma TERT test to varied input DNA amounts and the limit of detection (LOD) of variant allele frequency (VAF). We determined the negative percent agreement (NPA) and the positive percent agreement (PPA) of the Afirma TERT test against a reference primer pair. We also evaluated the analytical specificity from potential interfering substances such as RNA and blood gDNA. Further, the intra-run, inter-run and inter-laboratory reproducibility of the assay were examined. Analytical sensitivity analysis showed that the Afirma TERT test is tolerant to variation in DNA input amount (7-13 ng) and can detect expected positive TERT promoter variants down to 5% VAF LOD at 7ng DNA input with > 95% sensitivity. Both NPA and PPA were 100% against the reference primer pair. Analytical specificity studies demonstrated that the Afirma TERT test remains accurate in presence of 20% RNA or 80% blood gDNA for an average patient sample that typically has 30% VAF. The Afirma TERT test results are highly reproducible across different operators, runs, reagent lots, and laboratories. The Afirma TERT test also demonstrated a 100% confirmation rate when compared with an external NGS-based reference assay executed in a non-Veracyte laboratory. The analytical robustness and reproducibility of the Afirma TERT test support its routine clinical use among thyroid nodules with indeterminate cytology that are Afirma GSC suspicious or Bethesda V/VI nodules. Presentation: Saturday, June 17, 2023

OR32-03 mRNA Expression Based Tumor Behavior Prediction Models In Thyroid Nodules

Abstract Disclosure: A. Golding: None. G. Correa: None. E. Valenzuela Sheker: None. R. Jiang: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. Y. Hao: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. Y. Liu: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. J. Huang: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. J.P. Klopper: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. R.T. Kloos: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. G.C. Kennedy: Employee; Self; Veracyte, Inc. Stock Owner; Self; Veracyte, Inc. D. Bimston: None. The 2015 ATA thyroid cancer risk stratification system is driven primarily by the extent of vascular and extrathyroidal extension, as well as lymph node metastases. The objectives of this study were to evaluate the prevalence of invasion and lymph node metastases in a cohort enriched with thyroid malignancies and provide a risk signature with a high negative predictive value ruling out these tumor features. Histopathology reports from Afirma Genomic Sequencing Classifier (GSC) algorithm training and from thyroid cancer patients managed at an integrative endocrine surgery community care practice (total 697 and ∼50% from each) were reviewed for invasion and metastases. An integer from 0-3 was assigned based on the extent of invasion (ranging from none to extensive extra-thyroidal) or extent of lymph node metastases (ranging from none to lateral node metastases). To develop predictive signatures for invasion/metastases, over 400 literature-derived expression-based signatures were evaluated; they cover a broad spectrum of molecular processes, including cancer hallmarks such as endothelial to mesenchymal transition, tumor microenvironment, immune-oncology, metabolism, and treatment sensitivity. Further, acknowledging that multiple complex molecular processes are associated with invasion/metastases, over 200 machine-learning (ML) models were trained within the repeated and nested cross-validation (CV) framework. They utilized these literature-derived signatures as well as differentially expressed genes between high and low risk of invasion/metastases as the basic building blocks. The application of top performing signatures among Bethesda (B) V/VI nodules as well as indeterminate thyroid nodules (ITN - BIII/IV) that are GSC-suspicious, showed that 40-50% of these lesions were marked as low risk for invasion/metastases. With a starting prevalence of 10.0% of high-level invasion (scored 2 or 3), 41.3% of the cohort is ruled out for clinically relevant invasion with a negative predictive value (NPV) of 97.6%. With a starting prevalence of 11.3% of high-level metastases (scored 2 or 3), 49.8% of the cohort is ruled out for clinically relevant metastases with an NPV of 98.6%. The rule-out capability represents more than 4-fold or 7-fold reduction in high-level invasion/metastases from the baseline prevalence, respectively. Expression-based signatures that confidently rule out tumor invasion and metastasis may help personalize the surgical approach for individuals. This may reduce excess surgery and overtreatment, optimizing the initial response to therapy and decreasing the rates of surgical complications and post-operative hypothyroidism. Presentation: Sunday, June 18, 2023

Combined Afirma Genomic Sequencing Classifier and TERT promoter mutation detection in molecular assessment of Bethesda III-VI thyroid nodules.

Molecular analysis of thyroid nodules can provide diagnostic and prognostic information as well as indicate opportunities for targeted therapy. Whole RNA exome analysis offers information that can be leveraged to predict tumor behavior.

How Does the Veracyte Afirma Genomic Sequencing Classifier (GSC) Perform in the Real World for Indeterminate Thyroid Nodules?

How Does the Veracyte Afirma Genomic Sequencing Classifier (GSC) Perform in the Real World for Indeterminate Thyroid Nodules?

Performance of Afirma genomic sequencing classifier and histopathological outcome in Bethesda category III thyroid nodules: Initial versus repeat fine-needle aspiration.

There is limited data comparing the performance of Afirma Genomic Sequencing Classifier (GSC) in thyroid nodules carrying an initial versus a repeat diagnosis of atypia of undetermined significance (AUS). This study reported an institutional experience in this regard. This retrospective study included consecutive thyroid nodules that had an initial or a repeat AUS diagnosis and had a subsequent GSC diagnostic result (benign or suspicious) from 2017 to 2021. All nodules were followed by surgical intervention or by clinical and/or ultrasound monitoring. GSC's benign call rate (BCR), rate of histology-proven malignancy associated with a suspicious GSC result, and diagnostic parameters of GSC were calculated and compared between the two cohorts (initial versus repeat AUS). Statistical significance was defined with a p-value of <.05 for all analysis. A total of 202 cases fulfilled inclusion criteria, including 67 and 135 thyroid nodules with an initial and a repeat AUS diagnosis, respectively. BCR was 67% and 66% in initial and repeat AUS cohorts, respectively. Rate of histology-proven malignancy associated with a suspicious GSC result were 22% and 24% in initial and repeat AUS cohorts, respectively. Compared with the repeat AUS cohort, the initial AUS cohort showed slightly lower sensitivity (83% vs. 100%), specificity (70% vs. 73%), PPV (23% vs. 24%), NPV (98% vs. 100%), and diagnostic accuracy (72% vs. 75%). Nevertheless, these differences did not reach statistical significance. GSC demonstrated comparable performance in thyroid nodules with a repeat AUS diagnosis versus nodules with an initial AUS diagnosis.

Risk of malignancy in cytologically indeterminate thyroid nodules harboring thyroid stimulating hormone receptor mutations.

To evaluate the frequency and risk of malignancy of TSHRpI568T mutations discovered in indeterminate thyroid nodules (ITN) within the Veracyte CLIA laboratory undergoing Afirma® Genomic Sequencing Classifier (GSC) testing, and to evaluate a broader cohort of TSHR variants and their categorization as Afirma GSC benign (GSC-B) or suspicious (GSC-S). Finally, we seek to assess the risk of malignancy (ROM) of this group of TSHR mutated ITN in the GSC-S category. ITN submitted to Veracyte for Afirma GSC testing between October 2017 and February 2022 were analyzed for TSHR variants and rates of GSC-B and GSC-S were calculated based upon BIII or IV cytology, by TSHR variant codon amino acid (AA) substitution, age, and gender. For GSC-S samples, surgical pathology reports were requested, and the rate of malignancy was calculated. Five percent of the ITN samples harbored an isolated TSHR variant and 5% of those were classified as GSC-S. Among TSHRpI568T samples, 96% were GSC-B and of the GSC-S samples, 21% were malignant. Among an unselected group of TSHR, absent TSHRpI568T mutations, 16.3% of GSC-S samples were malignant, all but one with codon mutations in the transmembrane subdomains of the TSHR. This prompted a dedicated evaluation of transmembrane codons which revealed a malignancy rate of 10.7% among GSC-S nodules. In total, 13/85 (15.3%) TSHR mutated ITN with Afirma GSC-S results were found to be malignant. TSHR variants are rare in ITN, and most are categorized as benign under Afirma GSC testing which carries a < 4% risk of malignancy. For GSC-S ITN with TSHR mutations, the risk of malignancy is ≥= 15%, which is clinically meaningful and may alter treatment or monitoring recommendations for patients.

Real-World Performance of the Afirma Genomic Sequencing Classifier (GSC)-A Meta-analysis.

The Afirma® GSC aids in risk stratifying indeterminate thyroid nodule cytology (ITN). The 2018 GSC validation study (VS) reported a sensitivity (SN) of 91%, specificity (SP) of 68%, positive predictive value (PPV) of 47%, and negative predictive value (NPV) of 96%. Since then, 13 independent real-world (RW) postvalidation studies have been published. This study's objective is to compare the RW GSC performance to the VS metrics. Rules and assumptions applying to this analysis include: (1) At least 1 patient with molecular benign results must have surgery for that study to be included in SN, SP, and NPV analyses. (2) Molecular benign results without surgical histology are considered true negatives (TN) (as are molecular benign results with benign surgical histology). (3) Unoperated patients with suspicious results are either excluded from analysis (observed PPV [oPPV] and observed SP [oSP]) or assumed histology negatives (false positives; conservative PPV [cPPV] and conservative SP [cSP]) 4. Noninvasive follicular thyroid neoplasm with papillary-like nuclear features is considered malignant. In RW studies, the GSC demonstrates a SN, oSP, oPPV, and NPV of 97%, 88%, 65%, 99% respectively, and conservative RW performance showed cSP of 80% and cPPV of 49%, all significantly higher than the VS except for SN and cPPV. There was also a higher benign call rate (BCR) of 67% in RW studies compared to 54% in the VS (P < 0.05). RW data for the Afirma GSC demonstrates significantly better oSP and oPPV performance than the VS, indicating an increased yield of cancers for resected GSC suspicious nodules. The higher BCR likely increases the overall rate of clinical observation in lieu of surgery.

Performance of Afirma Genomic Sequencing Classifier in Thyroid Nodules with Preoperative Cytologic Diagnosis of AUS/FLUS

Performance of Afirma Genomic Sequencing Classifier in Thyroid Nodules with Preoperative Cytologic Diagnosis of AUS/FLUS

PSAT348 Falsely Elevated TSI Antibody Without Causing Hyperthyroidism, in a Hypothyroid Patient

Abstract Introduction Hashimoto's thyroiditis and Graves’ disease are the most common causes for Hypothyroidism and Hyperthyroidism respectively. There are case reports of co-existence of these two disorders in the same patient (1) which causes an alternating clinical course of both hypothyroidism and hyperthyroidism. The Graves’ disease specific Thyroid Stimulating Immunoglobulin (TSI) antibody is reported to have a very high sensitivity and specificity. However, the presence of TSI antibody in a long-standing hypothyroid patient without developing hyperthyroidism, is of extreme rare occurrence. Case Description Our patient is a 58-year-old Caucasian female who has a history of hypothyroidism that was diagnosed in 1990. She was initiated on levothyroxine and had maintained a stable dose of 100 mcg daily for many years. During the initial visit at our endocrinology clinic in December 2020, the patient's labs were consistent with iatrogenic thyrotoxicosis due to a low TSH 0.233 (0.30-4.00 uIU/ml), normal Free T4 1.33 (0.80-1.80 ng/dl) and normal Free T3 2.28 (2.0-4.40 pg/ml). TPO antibody was negative at 11 (0-34 IU/mL). The levothyroxine dose was reduced to 88 mcg daily, but patient did not obtain follow-up labs for many months. In August 2021, the TSH was noted to be normal at 2.29. Patient was also diagnosed with multinodular goiter with the presence bilateral sub centimeter thyroid nodules which had remained stable over the years. However, thyroid ultrasound in December 2020 showed a 1.8 cm left mid to lower pole nodule that was biopsied and reported to be Bethesda category III with benign Afirma Genomic sequencing classifier. Subsequently, patient reported experiencing swelling and pain of bilateral fingers and underwent evaluation by rheumatologist who ordered a panel of blood tests to assess for autoimmune conditions. Interestingly, thyroid antibodies were also assessed, and it came back remarkable for elevated the Graves’ disease specific TSI antibody level 221% (&amp;lt; 140%) and elevated anti-thyroglobulin (TG) antibody 40.6 (&amp;lt;4.1). Patient's medication list was verified, and she was not on any high-dose biotin supplementation, and she denied experiencing any hyperthyroid symptoms. The corresponding thyroid labs were unremarkable with normal TSH 2.2, normal free T4 1.33 and normal free T3 2.28. Patient was reassured about the normal thyroid labs and advised that her symptoms are not thyroid related. The repeat TSH level continues to remain normal and was 2.18 in December 2021. Conclusion Although the presence of TSI antibody has a high positive predictive value for diagnosing Graves’ disease, a falsely elevated TSI antibody, can rarely occur without causing any clinical consequences. Reference (1) Alvin Mathew A, Papaly R, et al. Elevated Graves’ Disease-Specific Thyroid-Stimulating Immunoglobulin and Thyroid Stimulating Hormone Receptor Antibody in a Patient With Subacute Thyroiditis. Cureus 13(11): e19448. doi: 10.7759/cureus.19448 Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Assessing Bias and Limitations of Clinical Validation Studies of Molecular Diagnostic Tests for Indeterminate Thyroid Nodules: Systematic Review and Meta-Analysis.

Background: Molecular tests for thyroid nodules with indeterminate fine needle aspiration results are increasingly used in clinical practice; however, true diagnostic summaries of these tests are unknown. A systematic review and meta-analysis were completed to (1) evaluate the accuracy of commercially available molecular tests for malignancy in indeterminate thyroid nodules and (2) quantify biases and limitations in studies that validate those tests. Summary: PubMed, EMBASE, and Web of Science were systematically searched through July 2021. English language articles that reported original clinical validation attempts of molecular tests for indeterminate thyroid nodules were included if they reported counts of true-negative, true-positive, false-negative, and false-positive results. We performed screening and full-text review, followed by assessment of eight common biases and limitations, extraction of diagnostic and histopathological information, and meta-analysis of clinical validity using a bivariate linear mixed-effects model. Forty-nine studies were included. Meta-analysis of Afirma Gene expression classifiers (GEC; n = 38 studies) revealed a sensitivity of 0.92 (confidence interval: 0.90-0.94), specificity of 0.26 (0.20-0.32), negative likelihood ratio (LR-) of 0.32 (0.23-0.44), positive LR+ of 1.24 (1.15-1.35), and area under the curve (AUC) of 0.83 (0.74-0.89). Afirma Genomic Sequencing Classifier (GSC; n = 10) had a sensitivity of 0.94 (0.89-0.96), specificity of 0.38 (0.27-0.50), LR- of 0.18 (0.10-0.30), LR+ of 1.52 (1.28-1.87), and AUC of 0.91 (0.62-0.92). ThyroSeq v1 and v2 (n = 10) had a sensitivity of 0.86 (0.82-0.90), specificity of 0.74 (0.59-0.85), LR- of 0.19 (0.13-0.26), LR+ of 3.52 (2.08-5.92), and AUC of 0.86 (0.81-0.90). ThyroSeq v3 (n = 6) had a sensitivity of 0.92 (0.86-0.95), specificity of 0.41 (0.18-0.69), LR- of 0.24 (0.09-0.62), LR+ of 1.67 (1.09-2.98), and AUC of 0.90 (0.63-0.92). Fourteen percent of studies conducted a blinded histopathologic review of excised thyroid nodules, and 8% made the decision to go to surgery blind to molecular test results. Conclusions: Meta-analyses reveal a high diagnostic accuracy of molecular tests for thyroid nodule assessment of malignancy risk; however, these studies are subject to several limitations. Limitations and their potential clinical impacts must be addressed and, when feasible, adjusted for using valid statistical methodologies.

Performance of Afirma genomic sequencing classifier and histopathological outcome are associated with patterns of atypia in Bethesda category III thyroid nodules.

Data on Afirma's genomic sequencing classifier (GSC) performance in atypia of undetermined significance (AUS) subcategories is limited. This study investigated GSC performance in AUS nodules with architectural atypia (AUS-A), cytological atypia (AUS-C), architectural and cytological atypia (AUS-AC), and predominantly Hürthle cells (AUS-HC). This study retrieved consecutive thyroid nodules having a recurrent cytologic diagnosis of AUS with qualifiers and a concurrent GSC diagnostic result. All nodules were followed by either surgical intervention or clinical and/or ultrasound monitoring (≥6 months). GSC benign call rate (BCR), rate of histology-proven malignancy, and diagnostic parameters of GSC were calculated for individual AUS subcategories. Statistical analysis was performed using the Fisher exact test. A total of 135 AUS nodules fulfilled inclusion criteria, including 79 AUS-A, 9 AUS-C, 29 AUS-AC, and 18 AUS-HC. BCR was 72.2%, 66.7%, 44.8%, and 77.8% in AUS-A, AUS-C, AUS-AC, and AUS-HC, respectively. AUS-A showed a greater BCR than AUS-AC (p &lt; .05). All GSC-benign nodules were considered benign on clinical or surgical follow-up. Among GSC-suspicious nodules, histology-proven malignancies represented 4.5% of AUS-A, 0% of AUS-C, 56.3% of AUS-AC, and 25.0% of AUS-HC cases. AUS-AC demonstrated a higher malignant rate compared with AUS-A (p &lt; .05). GSC offers 100% NPV and a wide range (5%-56%) of PPV across all AUS subcategories. AUS-AC demonstrated a greater PPV compared with AUS-A (p &lt; .05). BCR of GSC and malignant rates associated with suspicious GSC may differ in various AUS subcategories. GSC-suspicious nodules with both architectural and cytologic atypia are more likely to be malignant. These findings may improve clinical triage and/or management of patients with AUS thyroid nodules.

The Effect Modification of Ultrasound Risk Classification on Molecular Testing in Predicting the Risk of Malignancy in Cytologically Indeterminate Thyroid Nodules.

Background: Thyroid nodules with indeterminate cytology are increasingly subjected to molecular testing. We evaluated the diagnostic performances of Afirma Genomic Sequencing Classifier (GSC) and ThyroSeq v3 in thyroid nodules with high versus low/intermediate suspicion ultrasound classification. Methods: In this prospective cohort study, we analyzed all Bethesda III and IV thyroid nodules that underwent fine-needle aspiration biopsies in the University of California Los Angeles Health System from July 2017 to April 2020. All patients underwent molecular testing with Afirma GSC or ThyroSeq v3 as part of an institutional randomized trial (NCT02681328). Nodules were categorized according to the American Thyroid Association (ATA) ultrasound risk classification. The benign call rate and the positive predictive value of molecular testing were compared between ATA high suspicion versus all other categories. Results: A total of 343 patients with 375 indeterminate thyroid nodules were included. The malignancy rate in ATA high suspicion nodules was not significantly increased by a suspicious Afirma GSC result (77.8% for all ATA high suspicion nodules vs. 87.5% for nodules with ATA high suspicion and suspicious Afirma GSC results, positive likelihood ratio [LR] = 2.0, 95% confidence interval [CI 0.5-8.0], p = 1.0) or by a positive ThyroSeq v3 result (80.0% vs. 80.0%, positive LR = 1.0 [CI 1.0-1.0], p = 1.0). The rate of malignancy in ATA low/intermediate suspicion nodules increased from 21.0% to 56.3% with a suspicious Afirma GSC result (positive LR = 4.8 [CI 3.4-6.9], p < 0.0001) and decreased to 3.8% with a benign Afirma GSC result (negative LR = 0.1 [CI 0.07-0.3], p < 0.0001). Similarly, the rate of malignancy in ATA low/intermediate suspicion nodules increased from 24.3% to 66.7% with a positive ThyroSeq v3 result (positive LR = 6.2 [CI 4.0-9.7], p < 0.0001) and decreased to 2.1% with a negative ThyroSeq v3 result (negative LR = 0.07 [CI 0.02-0.3], p < 0.0001). Conclusions: Afirma GSC and ThyroSeq v3 performed well in ruling out malignancy in sonographically low/intermediate suspicion thyroid nodules but has limited diagnostic value in sonographically high suspicion nodules. Molecular testing can prognosticate more aggressive thyroid cancers, which can inform treatment decisions.

Molecular testing results as a quality metric for evaluating cytopathologists' utilization of the atypia of undetermined significance category for thyroid nodule fine-needle aspirations.

The use of the indeterminate category of atypia of undetermined significance (AUS) for thyroid fine-needle aspirations (FNAs) should be kept to a minimum. Here, we investigate the utility of combining AUS utilization rates with Afirma (Veracyte, Inc., South San Francisco, CA) genomic sequencing classifier (GSC) molecular testing results as a quality improvement metric for describing cytopathologist practice patterns. Thyroid FNAs evaluated in our laboratory by 9 cytopathologists from December 2017 to July 2021 were stratified by Bethesda diagnostic category, and Afirma GSC testing results for AUS cases were compiled and correlated with AUS call rates. Over this period, the laboratory AUS rate was 22.3% (672 of 3008), with an individual cytopathologist range of 11.6% to 39.3%. Afirma GSC testing had suspicious (GSC-S) results in 29% (48 of 167) of cases, with a cytopathologist range of 5% to 67%. Linear regression analysis of individual AUS rates versus Afirma GSC-S rates demonstrated no significant relationship between these 2 variables. However, based on the pattern of AUS use and GSC-S rates, a novel conceptual framework for understanding cytopathologist practice patterns is proposed. Combining molecular testing results with AUS call rates of thyroid nodules can provide a more nuanced explanation of cytopathologist practice patterns, and can be utilized to provide directed feedback to bring individual cytopathologist diagnostic category use in line with laboratory averages or published benchmarks.

Thyroid nodule molecular profiling: The clinical utility of Afirma Xpression Atlas for nodules with Afirma Genomic Sequencing Classifier–suspicious results

BackgroundThe Afirma Genomic Sequencing Classifier uses whole transcriptome RNA sequencing to identify thyroid nodules as benign or suspicious. The Afirma Xpression Atlas became available in 2018 and reports findings across 593 genes, including 905 variants and 235 fusions. When an alteration is identified, its risk of malignancy and associated neoplasm type is listed. We report the results of Afirma Xpression Atlas testing at our institution during its first 2 years of clinical use. MethodsAll patient charts with indeterminate thyroid nodules and Afirma Xpression Atlas results at our institution were reviewed. Thyroid nodule characteristics, cytology, Afirma Genomic Sequencing Classifier results, Afirma Xpression Atlas results, and final histopathology were reported. ResultsAfirma Xpression Atlas was performed on 136 indeterminate nodules since May 2018, and 103 met inclusion criteria. Forty-three nodules had positive Afirma Xpression Atlas results, and of these, 83.7% were follicular cell-derived thyroid cancer on surgical histopathology. This is similar to the overall 82.5% positive predictive value among Afirma Genomic Sequencing Classifier–suspicious indeterminate nodules during the same time period. Of the 60 nodules with negative Afirma Xpression Atlas, 73.3% were follicular cell-derived thyroid cancer on surgical histopathology. ConclusionAfirma Xpression Atlas positivity is predictive of follicular cell-derived thyroid cancer, but its positive predictive value is similar to that of Genomic Sequencing Classifier–suspicious results alone at our institution, which is higher than previously published. Specific mutations likely predict follicular cell-derived thyroid cancer with higher accuracy, but our current sample size of any given mutation is too small to evaluate this further. Larger studies are needed to determine whether Afirma Xpression Atlas results predictably inform the risk of malignancy and tumor characteristics in thyroid nodules.

Comparison of Afirma GEC and GSC to Nodules Without Molecular Testing in Cytologically Indeterminate Thyroid Nodules.

BackgroundAnalysis of cytologically indeterminate thyroid nodules with Afirma Gene Expression Classifier (GEC) and Genomic Sequencing Classifier (GSC) can reduce surgical rate and increase malignancy rate of surgically resected indeterminate nodules.MethodsRetrospective cohort analysis of all adults with cytologically indeterminate thyroid nodules from January 2013 through December 2019. We compared surgical and malignancy rates of those without molecular testing to those with GEC or GSC, analyzed test performance between GEC and GSC, and identified variables associated with molecular testing.Results468 indeterminate thyroid nodules were included. No molecular testing was performed in 273, 71 had GEC, and 124 had GSC testing. Surgical rate was 68% in the group without molecular testing, 59% in GEC, and 40% in GSC. Malignancy rate was 20% with no molecular testing, 22% in GEC, and 39% in GSC (P = 0.022). GEC benign call rate (BCR) was 46%; sensitivity, 100%; specificity, 61%; and positive predictive value (PPV), 28%. GSC BCR was 60%; sensitivity, 94%; specificity, 76%; and PPV, 41%. Those with no molecular testing had larger nodule size, preoperative growth of nodules, and constrictive symptoms and those who underwent surgery in the no molecular testing group had higher body mass index, constrictive symptoms, higher Thyroid Imaging Reporting and Data System and Bethesda classifications. Type of provider was also associated with the decision to undergo surgery.ConclusionImplementation of GEC showed no effect on surgical or malignancy rate, but GSC resulted in significantly lower surgical and higher malignancy rates. This study provides insight into the factors that affect the real-world use of these molecular markers preoperatively in indeterminate thyroid nodules.