ThyroSeq V2 Research Articles

Clinical Validation of ThyroSeq V3 Shows High Sensitivity and Specificity

Clinical Validation of ThyroSeq V3 Shows High Sensitivity and Specificity

Evaluation of 167 Gene Expression Classifier (GEC) and ThyroSeq v2 Diagnostic Accuracy in the Preoperative Assessment of Indeterminate Thyroid Nodules: Bivariate/HROC Meta-analysis

The objective of this meta-analysis was to evaluate the performance of the Gene Expression Classifier (GEC) and ThyroSeq v2 (ThyroSeq) in the preoperative diagnosis of thyroid nodules with indeterminate fine-needle aspiration biopsy results. We searched literature databases from January 2001 to April 2018. The bivariate model analysis was performed to estimate pooled sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR−), positive predictive value (PPV), and negative predictive value (NPV). Pooled data from 1086 nodules with histopathologic confirmation from 16 GEC studies enabled calculation of diagnostic parameters (95% confidence interval): sensitivity 98% (96–99%), specificity 12% (8–20%), PPV 45% (37–53%), and NPV 91% (85–96%). Pooled data from five ThyroSeq studies assessing 459 nodules showed sensitivity of 84% (74–91%), specificity 78% (50–92%), PPV 58% (31–81%), and NPV 93% (89–97%). When both tools were compared, GEC had a significantly higher sensitivity (p = 0.003), while ThyroSeq had a significantly higher specificity (p < 0.001) and accuracy (p = 0.015). Pooled LR+ was higher for ThyroSeq: 3.79 (1.40–10.27) vs. 1.12 (1.05–1.20). Pooled LR− was higher for GEC, 0.20 (0.10–0.39) vs. 0.13 (0.05–0.31). The bivariate summary estimates of sensitivity and specificity for GEC and ThyroSeq and their pooled accuracy showed a superiority of the ThyroSeq test. The GEC with a high sensitivity and NPV may be helpful in ruling out malignancy in cases of indeterminate thyroid nodule cytology. ThyroSeq has a significantly higher specificity and accuracy with an acceptable sensitivity so that it has the potential for use as an all-round test of malignancy of thyroid nodules.

In Brief

In Brief

Benign call rate and molecular test result distribution of ThyroSeq v3.

The benign call rate (BCR) is the percentage of cytomorphologically indeterminate cases with subsequent benign or negative molecular results. For rule-out tests, the BCR is an important parameter because these molecular "negative" cases may be managed similarly to those with a benign cytology diagnosis. Although earlier versions of ThyroSeq molecular tests were less effective in excluding malignancy, the extensively expanded v3 version with a high negative predictive value is considered to represent a rule-out test. In the current study, the authors evaluated the BCR and the overall molecular result distribution of ThyroSeq v3. All indeterminate thyroid fine-needle aspiration cases (except those deemed suspicious for malignancy) with available ThyroSeq v3 results from November 2017 to June 2018 were retrieved from the cytopathology files. Because the ThyroSeq v3 genomic classifier was designed for thyroid follicular-derived lesions, cases in which parathyroid and medullary carcinoma molecular markers were detected and those that were inadequate or limited for molecular testing were excluded from the study. For the remaining cases, the indeterminate diagnoses were correlated with molecular and histologic results. Among the 224 indeterminate cases (except those deemed suspicious for malignancy), the overall ThyroSeq v3 molecular test BCR was 74.1% (166 of 224 cases). The category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) had higher BCRs compared with the other indeterminate diagnostic categories. RAS mutations were the most common positive results. The high BCR demonstrates that ThyroSeq v3 is potentially effective in sparing the large majority of indeterminate cases with "negative" results from surgery while offering risk assessment for the positive cases and guiding clinical management.

Performance of a Multigene Genomic Classifier in Thyroid Nodules With Indeterminate Cytology

Approximately 20% of fine-needle aspirations (FNA) of thyroid nodules have indeterminate cytology, most frequently Bethesda category III or IV. Diagnostic surgeries can be avoided for these patients if the nodules are reliably diagnosed as benign without surgery. To determine the diagnostic accuracy of a multigene classifier (GC) test (ThyroSeq v3) for cytologically indeterminate thyroid nodules. Prospective, blinded cohort study conducted at 10 medical centers, with 782 patients with 1013 nodules enrolled. Eligibility criteria were met in 256 patients with 286 nodules; central pathology review was performed on 274 nodules. A total of 286 FNA samples from thyroid nodules underwent molecular analysis using the multigene GC (ThyroSeq v3). The primary outcome was diagnostic accuracy of the test for thyroid nodules with Bethesda III and IV cytology. The secondary outcome was prediction of cancer by specific genetic alterations in Bethesda III to V nodules. Of the 286 cytologically indeterminate nodules, 206 (72%) were benign, 69 (24%) malignant, and 11 (4%) noninvasive follicular thyroid neoplasms with papillary-like nuclei (NIFTP). A total of 257 (90%) nodules (154 Bethesda III, 93 Bethesda IV, and 10 Bethesda V) had informative GC analysis, with 61% classified as negative and 39% as positive. In Bethesda III and IV nodules combined, the test demonstrated a 94% (95% CI, 86%-98%) sensitivity and 82% (95% CI, 75%-87%) specificity. With a cancer/NIFTP prevalence of 28%, the negative predictive value (NPV) was 97% (95% CI, 93%-99%) and the positive predictive value (PPV) was 66% (95% CI, 56%-75%). The observed 3% false-negative rate was similar to that of benign cytology, and the missed cancers were all low-risk tumors. Among nodules testing positive, specific groups of genetic alterations had cancer probabilities varying from 59% to 100%. In this prospective, blinded, multicenter study, the multigene GC test demonstrated a high sensitivity/NPV and reasonably high specificity/PPV, which may obviate diagnostic surgery in up to 61% of patients with Bethesda III to IV indeterminate nodules, and up to 82% of all benign nodules with indeterminate cytology. Information on specific genetic alterations obtained from FNA may help inform individualized treatment of patients with a positive test result.

Interinstitutional variation in predictive value of the ThyroSeq v2 genomic classifier for cytologically indeterminate thyroid nodules

BackgroundThe ThyroSeq v2 next-generation sequencing assay estimates the probability of malignancy in indeterminate thyroid nodules. Its diagnostic accuracy in different practice settings and patient populations is not well understood. MethodsWe analyzed 273 Bethesda III/IV indeterminate thyroid nodules evaluated with ThyroSeq at 4 institutions: 2 comprehensive cancer centers (n = 98 and 102), a multicenter health care system (n = 60), and an academic medical center (n = 13). The positive and negative predictive values of ThyroSeq and distribution of final pathologic diagnoses were analyzed and compared with values predicted by Bayes theorem. ResultsAcross 4 institutions, the positive predictive value was 35% (22%–43%) and negative predictive value was 93% (88%–100%). Predictive values correlated closely with Bayes theorem estimates (r2 = 0.84), although positive predictive values were lower than expected. RAS mutations were the most common molecular alteration. Among 84 RAS-mutated nodules, malignancy risk was variable (25%, range 10%–37%) and distribution of benign diagnoses differed across institutions (adenoma/hyperplasia 12%–85%, noninvasive follicular thyroid neoplasm with papillary-like nuclear features 5%–46%). ConclusionIn a multi-institutional analysis, ThyroSeq positive predictive values were variable and lower than expected. This is attributable to differences in the prevalence of malignancy and variability in pathologist interpretations of noninvasive tumors. It is important that clinicians understand ThyroSeq performance in their practice setting when evaluating these results.

Short Call Abstracts

Short Call Abstracts

Abstract #1221 Real World Experience with Thyroseq V3 Genomic Classifier in Thyroid Nodules with Indeterminate Cytology: Results of Testing of the First 3,783 Consecutive Samples

Abstract #1221 Real World Experience with Thyroseq V3 Genomic Classifier in Thyroid Nodules with Indeterminate Cytology: Results of Testing of the First 3,783 Consecutive Samples

Abstract #719 Intrathyroidal Hemorrhagic Parathyroid Cyst Diagnosed by Thyroseq V3 Molecular Testing

Abstract #719 Intrathyroidal Hemorrhagic Parathyroid Cyst Diagnosed by Thyroseq V3 Molecular Testing

Gene Expression Classifier vs Targeted Next-Generation Sequencing in the Management of Indeterminate Thyroid Nodules.

Molecular testing has reduced the need for diagnostic hemithyroidectomy for indeterminate thyroid nodules. No studies have directly compared molecular testing techniques. Compare the diagnostic performance of Afirma Gene Expression Classifier (GEC) with that of ThyroSeq v2 next-generation sequencing assay. Parallel randomized trial, monthly block randomization of patients with Bethesda III/IV cytology to GEC or ThyroSeq v2. University of California, Los Angeles. Patients who underwent thyroid biopsy (April 2016 to June 2017). Testing with GEC or ThyroSeq v2. Molecular test performance. Of 1372 thyroid nodules, 176 (13%) had indeterminate cytology and 149 of 157 eligible indeterminate nodules (95%) were included in the study. Of nodules tested with GEC, 49% were suspicious, 43% were benign, and 9% were insufficient. Of nodules tested with ThyroSeq v2, 19% were mutation positive, 77% were mutation negative, and 4% were insufficient. The specificities of GEC and ThyroSeq v2 were 66% and 91%, respectively (P = 0.002); the positive predictive values of GEC and ThyroSeq v2 were 39% and 57%, respectively. Diagnostic hemithyroidectomy was avoided in 28 patients tested with GEC (39%) and 49 patients tested with ThyroSeq v2 (62%). Surveillance ultrasonography was available for 46 nodules (45 remained stable). ThyroSeq v2 had higher specificity than Afirma GEC and allowed more patients to avoid surgery. Long-term surveillance is necessary to assess the false-negative rate of these particular molecular tests. Further studies are required for comparison with other available molecular diagnostics and for newer tests as they are developed.

Thyroseq v3 Molecular Test for Indeterminate Thyroid Nodules Has Improved Sensitivity and Specificity

Thyroseq v3 Molecular Test for Indeterminate Thyroid Nodules Has Improved Sensitivity and Specificity

Analytical performance of the ThyroSeq v3 genomic classifier for cancer diagnosis in thyroid nodules.

Molecular tests have clinical utility for thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology, although their performance requires further improvement. This study evaluated the analytical performance of the newly created ThyroSeq v3 test. ThyroSeq v3 is a DNA- and RNA-based next-generation sequencing assay that analyzes 112 genes for a variety of genetic alterations, including point mutations, insertions/deletions, gene fusions, copy number alterations, and abnormal gene expression, and it uses a genomic classifier (GC) to separate malignant lesions from benign lesions. It was validated in 238 tissue samples and 175 FNA samples with known surgical follow-up. Analytical performance studies were conducted. In the training tissue set of samples, ThyroSeq GC detected more than 100 genetic alterations, including BRAF, RAS, TERT, and DICER1 mutations, NTRK1/3, BRAF, and RET fusions, 22q loss, and gene expression alterations. GC cutoffs were established to distinguish cancer from benign nodules with 93.9% sensitivity, 89.4% specificity, and 92.1% accuracy. This correctly classified most papillary, follicular, and Hurthle cell lesions, medullary thyroid carcinomas, and parathyroid lesions. In the FNA validation set, the GC sensitivity was 98.0%, the specificity was 81.8%, and the accuracy was 90.9%. Analytical accuracy studies demonstrated a minimal required nucleic acid input of 2.5 ng, a 12% minimal acceptable tumor content, and reproducible test results under variable stress conditions. The ThyroSeq v3 GC analyzes 5 different classes of molecular alterations and provides high accuracy for detecting all common types of thyroid cancer and parathyroid lesions. The analytical sensitivity, specificity, and robustness of the test have been successfully validated and indicate its suitability for clinical use. Cancer 2018;124:1682-90. © 2018 American Cancer Society.

Molecular pathology and thyroid FNA.

This review summarises molecular pathological techniques applicable to thyroid FNA. The molecular pathology of thyroid tumours is now fairly well understood. Molecular methods may be used as a rule-in test for diagnosis of malignancy in thyroid nodules, eg BRAF V600E point mutation, use of a seven-gene mutational panel (BRAF V600E, RAS genes, RET/PTC or PAX8/PPARG rearrangement), or as a comprehensive multigene next-generation sequencing panel, eg ThyroSeq v2. Molecular methods can also be applied as rule-out tests for malignancy in thyroid nodules, eg Afirma or ThyroSeq v2 or as markers of prognosis, eg TERT promoter mutation or other gene mutations including BRAF V600E, TP53 and AKT1, and as tests for newly defined tumour entities such as non-invasive follicular thyroid neoplasm with papillary like nuclei, or as a molecular marker(s) for targeted therapies. This review describes practical examples of molecular techniques as applied to thyroid FNA in routine clinical practice and the value of molecular diagnostics in thyroid FNA. It describes the range of molecular abnormalities identified in thyroid nodules and thyroid cancers with some practical applications of molecular methods to diagnosis and prognosis of thyroid nodules and thyroid cancer.

Clinical performance of a next-generation sequencing assay (ThyroSeq v2) in the evaluation of indeterminate thyroid nodules

Molecular testing with the Thyroseq v2 next generation sequencing panel ("Thyroseq") is used to estimate the risk of cancer in indeterminate thyroid nodules. We analyzed 156 indeterminate thyroid nodules evaluated with Thyroseq, across 3 institutions. Thyroseq data and surgical pathology were matched via pathologic re-review. A result was considered Thyroseq positive if molecular alterations were annotated on the report with malignancy probability >30%. Performance characteristics were estimated using Bayes theorem. The Thyroseq-negative call rate was 65% (102/156). On surgical pathology, 16% (10/63) of nodules were malignant. The positive predictive value of a Thyroseq-positive result was 22% (8/37; if 2 noninvasive follicular thyroid neoplasm with papillary-like nuclear features are counted as malignant, 27%, 10/37). There was 1 false-negative result (negative predictive value 96%, 22/23). The most common mutation was NRAS (19/37) with a positive predictive value of 7% (1/15). The positive predictive value of all RAS mutations (HRAS, KRAS, NRAS) was 9% (2/22). The second most common mutation, BRAF V600E, had positive predictive value of 100% (3/3). We report an external analysis of Thyroseq performance in the evaluation of indeterminate thyroid nodules. These data indicate that Thyroseq is likely to offer high negative predictive value but low positive predictive value. Many genetic alterations appear to be nonspecific for malignancy, and positive results should be interpreted with caution. These findings have implications for the management of indeterminate thyroid nodules profiled with Thyroseq.

Evaluation of ThyroSeq v2 performance in thyroid nodules with indeterminate cytology.

ThyroSeq v2 claims high positive (PPV) and negative (NPV) predictive values in a wide range of pretest risks of malignancy in indeterminate thyroid nodules (ITNs) (categories B-III and B-IV of the Bethesda system). We evaluated ThyroSeq v2 performance in a cohort of patients with ITNs seen at our Academic Cancer Center from September 2014 to April 2016, in light of the new diagnostic criteria for non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Our study included 182 patients (76% female) with 190 ITNs consecutively tested with ThyroSeq v2. Patient treatment followed our institutional thyroid nodule clinical pathway. Histologies of nodules with follicular variant papillary thyroid carcinoma or NIFTP diagnoses were reviewed, with reviewers blinded to molecular results. ThyroSeq v2 performance was calculated in nodules with histological confirmation. We identified a mutation in 24% (n = 45) of the nodules. Mutations in RAS were the most prevalent (n = 21), but the positive predictive value of this mutation was much lower (31%) than that in prior reports. In 102 resected ITNs, ThyroSeq v2 performance was as follows: sensitivity 70% (46-88), specificity 77% (66-85), PPV 42% (25-61) and NPV 91% (82-97). The performance in B-IV nodules was significantly better than that in B-III nodules (area under the curve 0.84 vs 0.57, respectively; P = 0.03), where it was uninformative. Further studies evaluating ThyroSeq v2 performance are needed, particularly in B-III.

Young Investigator Challenge: Molecular testing in noninvasive follicular thyroid neoplasm with papillary-like nuclear features.

Molecular testing provides an important ancillary study for thyroid nodules with indeterminate cytology. The nomenclature shift to "noninvasive follicular thyroid neoplasm with papillary-like nuclear features" (NIFTP) will impact the performance of molecular tests. For the current study, the authors reviewed the performance of the Afirma gene-expression classifier (GEC) and the University of Pittsburgh Medical Center (UPMC) targeted mutation panel tests in thyroid nodules that were subsequently diagnosed as NIFTP on surgical resection. In total, 302 nodules were sent for molecular testing between June 2012 and June 2016. These cases were retrospectively reviewed to identify patients who underwent subsequent surgical resection and were diagnosed with follicular variant of papillary thyroid carcinoma (FVPTC). Twenty-five nodules that were diagnosed as FVPTC met the initial inclusion criteria. These cases were reviewed using strict criteria to identify NIFTP. Eight cases met criteria for NIFTP, and 4 NIFTPs underwent Afirma testing. Cytology diagnoses were all Bethesda category III, with 3 diagnosed as atypia of undetermined significance (AUS) and 1 diagnosed as follicular lesion of undetermined significance (FLUS). All of these nodules were identified as "suspicious" using GEC. Four NIFTPs underwent testing at UPMC, all using ThyroSeq V2. The cytology diagnoses for these nodules also were category III, with the exception of 1 nodule that was category IV, suspicious for follicular neoplasm. All NIFTPs were positive for mutations, all of which were RAS mutations (NRAS, KRAS). One patient who had a nodule classified as NIFTP had metastatic carcinoma identified in a lymph node. Another who had a 6-cm tumor had coexisting NRAS and TERT mutations. The current results indicate that NIFTP is a rare tumor if defined by strict criteria, that both the GEC and UPMC methods indicate abnormalities in NIFTP, and further independent study will be needed to better characterize the molecular and clinical characteristics of NIFTP. Cancer Cytopathol 2016;124:893-900. © 2016 American Cancer Society.

ThyroSeq V2 Application Study for Indeterminate Thyroid FNAs with Surgical Follow Up - Experience at a University Hospital

ThyroSeq V2 Application Study for Indeterminate Thyroid FNAs with Surgical Follow Up - Experience at a University Hospital

ThyroSeq V2 Application Study for Indeterminate Thyroid FNAs with Surgical Follow-up; Experience at a University Hospital

ThyroSeq V2 Application Study for Indeterminate Thyroid FNAs with Surgical Follow-up; Experience at a University Hospital

Molecular Characterization of Sporadic Pediatric Thyroid Carcinoma with the DNA/RNA ThyroSeq v2 Next-Generation Sequencing Assay.

The aim of this study was to test the hypothesis that our 60-gene DNA/RNA ThyroSeq v2 next-generation sequence (NGS) assay would identify additional genetic markers, including gene fusions in sporadic pediatric differentiated thyroid carcinomas (DTC) that had no known molecular alterations. Sporadic pediatric DTCs with informative molecular testing (n=18) were studied. We previously tested 15 cases by our standard 7-gene (BRAF, NRAS, HRAS, KRAS, RET/PTC1, RET/PTC3, PAX8/PPARg) mutation panel. Three cases were not tested previously. The standard 7-gene panel identified molecular alterations in 9 of 15 tumors (60%). Cases analyzed by ThyroSeq v2 NGS included the six previously negative cases by the standard 7-gene panel and three cases not previously tested. The NGS assay revealed new gene fusions in four of six previously negative cases (67%). These gene fusions included ETV6/NTRK3 (n=3) and TPR/NTRK1 (n=1). A point mutation (BRAF-V600E) was detected in one of three untested cases. While standard testing could identify only molecular alterations in 60% of cases, with the addition of the ThyroSeq v2 NGS, this increased to 87% (n=13/15). Some cases with chromosomal rearrangements, including ETV6/NTRK3, appear to be associated with an aggressive histopathologic phenotype, but had no documented history of radiation exposure. Additional work is needed to investigate if pediatric DTCs could benefit from a reclassification based on molecular subtypes, which may better reflect their underlying biologic potential. Our data support the use of broad gene panels for the molecular diagnostics of pediatric thyroid nodules to aid future classification, treatment, and clinical management recommendations.

Institutional prevalence of malignancy of indeterminate thyroid cytology is necessary but insufficient to accurately interpret molecular marker tests.

Several molecular marker tests are available to refine the diagnosis of thyroid nodules. Knowing the true prevalence of malignancy (PoM) within each cytological category is considered necessary to select the most appropriate test and to interpret results accurately. We describe our institutional PoM among cytological categories and report our experience with molecular markers. Single-center retrospective study. We calculated the institutional PoM for each category of the Bethesda system (Bethesda) on all thyroid nodules with cytological evaluation from October 2008 to May 2014. We estimated the predictive values for Afirma, miRInform, and ThyroSeq v2, based on published sensitivity and specificity. Finally, we assessed our own experience with miRInform. The PoMs for Bethesda III and IV categories were 21 and 28%, respectively. ThyroSeq v2 achieves the highest theoretical negative and positive predictive values (NPV and PPV) in Bethesda III (98 and 75%) and Bethesda IV categories (96 and 83%). At our institution, miRInform detected a mutation in 16% of 109 indeterminate nodules tested, all in Bethesda IV specimens. Histology was available in 56 (51%) nodules. The observed sensitivity and specificity in Bethesda IV specimens were 63 and 86%, yielding an NPV and a PPV of 75 and 77%, respectively. For our current Bethesda III and IV PoM, the actual performance of miRInform was worse than expected. Theoretically ThyroSeq v2 should have the best performance, but it could be affected in the same way as miRInform, given the similarities between the tests. Assessing the institutional performance of each test is necessary along with PoM individualization.