Analytical Validation of a Novel MicroRNA Panel for Risk Stratification of Cognitive Impairment.

We have been developing a novel approach to identify cognitive impairment-related biomarkers by profiling brain-enriched and inflammation-associated microRNA (miRNA) in plasma specimens of cognitively unimpaired and cognitively impaired patients. Here, we present an analytical validation of the novel miRNA panel, CogniMIR®, using two competing quantitative PCR technologies for the expression analysis of 24 target miRNAs. Total RNA from the plasma specimens was isolated using the MagMAX mirVana Kit, and RT-qPCR was performed using stem-loop-based TaqMan and LNA-based qPCR assays. Evaluation of RNA dilution series for our target 24 miRNAs, performed by two operators on two different days, demonstrated that all CogniMIR® panel miRNAs can be reliably and consistently detected by both qPCR technologies, with sample input as low as 20 copies in a qPCR reaction. Intra-run and inter-run repeatability and reproducibility analyses using RNA specimens demonstrated that both operators generated repeatable and consistent Cts, with R2 values of 0.94 to 0.99 and 0.96 to 0.97, respectively. The study results clearly indicate the suitability of miRNA profiling of plasma specimens using either of the qPCR technologies. However, the LNA-based qPCR technology appears to be more operationally friendly and better suited for a CAP/CLIA-certified clinical laboratory.

A Retrospective Evaluation of the Diagnostic Performance of an Interdependent Pairwise MicroRNA Expression Analysis with a Mutation Panel in Indeterminate Thyroid Nodules.

Background: The addition of genetic analysis to the evaluation of thyroid nodule fine-needle aspiration biopsy samples improves diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs) with Bethesda III or IV cytopathology. We previously reported the performance of a multiplatform molecular test, referred to in this study as MPTXv1, that includes a mutation panel (ThyGeNEXT®) plus an algorithmic microRNA (miRNA) risk classifier (ThyraMIR®). Complex interactions of growth-promoting and -suppressing miRNAs affect the phenotype. We previously demonstrated that accounting for these interactions with pairwise miRNA expression analysis improves the diagnosis of medullary thyroid carcinoma. In this study, we assess the impact of pairwise miRNA expression analysis on risk stratification of ITNs. Methods: Pairwise expression analysis of 11 miRNAs was performed on a training cohort of histopathology-proven benign nodules (n = 50) to define the mean and standard deviation of each pairwise analysis and create a Benign/Malignant Profiler (MPTXv2), deviations from which predicted the malignancy risk. Clinical validation of MPTXv2 was assessed using a cohort of 178 ITN (Bethesda III and IV) samples from a multicentered, blinded retrospective study, previously evaluated by MPTXv1. Results: Compared with MPTXv1, MPTXv2 significantly improved the test performance. The receiver operating characteristic (ROC) areas under the curve (AUC) increased from 0.85 to 0.97 (p < 0.001), and the diagnostic accuracy at the positive threshold increased significantly (p < 0.05) from 83% [95% confidence interval (CI) = 76-88] to 93% [CI = 89-96]. The significant improvement in the ROC AUC and the diagnostic accuracy was due to a strong statistical trend for improvement in specificity at the positive threshold. At the positive threshold, the specificity for MPTXv1 was 90% [CI = 84-95] and improved to 98% [CI = 94-99] for MPTXv2. Using the MPTXv2, the Moderate-Risk cohort decreased from 50 samples (28% of the cohort) to 24 samples (13% of the cohort). This 52% decrease is statistically significant (p < 0.001) and clinically meaningful. Conclusion: As compared with MPTXv1, pairwise miRNA expression analysis used in MPTXv2 significantly improved the diagnostic accuracy of ITN risk stratification and reduced the size of the Moderate-Risk group. Prospective trials are indicated to confirm these findings in a clinical practice setting.

Analytical and clinical validation of pairwise microRNA expression analysis to identify medullary thyroid cancer in thyroid fine-needle aspiration samples.

BACKGROUND: Medullary thyroid carcinoma (MTC) is an aggressive malignancy originating from the parafollicular C cells. Preoperatively, thyroid nodule fine-needle aspiration cytology (FNAC) and pathogenic gene mutations are definitive in approximately one-half of cases. MicroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs that regulate gene expression, a characteristic that confers the potential for identifying malignancy. In the current study, the authors hypothesized that differential pairwise (diff-pair) analysis of miRNA expression levels would reliably identify MTC in FNA samples. METHODS: The relative abundance of 10 different miRNAs in total nucleic acids was obtained from ThyraMIR test results. Diff-pair analysis was performed by subtracting the critical threshold value of one miRNA from the critical threshold values of other miRNAs. Next-generation sequencing with the ThyGeNEXT panel identified oncogenic gene alterations. The discovery cohort consisted of 30 formalin-fixed, paraffin-embedded benign and malignant thyroid neoplasms, including 4 cases of MTC. After analytical validation, clinical validation was performed using 3 distinct cohorts (total of 7557 specimens). RESULTS: In the discovery cohort, 9 diff-pairs were identified as having significant power using the Kruskal-Wallis test (P < .0001) to distinguish MTC samples from non-MTC samples. The assay correctly classified all MTC and non-MTC samples in the analytical validation study and in the 3 clinical validation cohorts. The overall test accuracy was 100% (95% confidence interval, 99%-100%). In indeterminate FNAC samples, the sensitivity of the diff-pair analysis was greater than that of the MTC-specific mutation analysis (100% vs 25%; P = .03). CONCLUSIONS: Pairwise miRNA expression analysis of ThyraMIR results were found to accurately predict MTC in thyroid FNA samples, including those with indeterminate FNAC findings.

Multiplatform molecular test performance in indeterminate thyroid nodules.

BACKGROUND: Approximately 25% of thyroid nodule fine-needle aspirates (FNAs) have cytology that is indeterminate for malignant disease. Accurate risk stratification of these FNAs with ancillary testing would reduce unnecessary thyroid surgery. METHODS: We evaluated the performance of an ancillary multiplatform test (MPTX) that has three diagnostic categories (negative, moderate, and positive). MPTX includes the combination of a mutation panel (ThyGeNEXT®) and a microRNA risk classifier (ThyraMIR®). A blinded, multicenter study was performed using consensus histopathology diagnosis among three pathologists to validate test performance. RESULTS: Unanimous consensus diagnosis was reached in 197 subjects with indeterminate thyroid nodules; 36% had disease. MPTX had 95% sensitivity (95% CI,86%-99%) and 90% specificity (95% CI,84%-95%) for disease in prevalence adjusted nodules with Bethesda III and IV cytology. Negative MPTX results ruledout disease with 97% negative predictive value (NPV; 95% CI,91%-99%) at a 30% disease prevalence, while positive MPTX results ruledin high risk disease with 75% positive predictive value (PPV; 95% CI,60%-86%). Such results are expected in four out of five Bethesda III and IV nodules tested, including RAS positive nodules in which the microRNA classifier was useful in rulingin disease. 90% of mutation panel false positives were due to analytically verified RAS mutations detected in benign adenomas. Moderate MPTX results had a moderate rate of disease (39%, 95% CI,23%-54%), primarily due to RAS mutations, wherein the possibility of disease could not be excluded. CONCLUSIONS: Our results emphasize that decisions for surgery should not solely be based on RAS or RAS-like mutations. MPTX informs management decisions while accounting for these challenges.

Development and Analytical Validation of an Expanded Mutation Detection Panel for Next-Generation Sequencing of Thyroid Nodule Aspirates.

Molecular analysis is used to evaluate the risk of malignancy for thyroid fine-needle aspirates, identified as indeterminate by microscopic cytology, on the basis of the detection of various oncogenic DNA mutations and fusion transcripts or on the use of various mRNAs or miRNA-based classifier algorithms. Our approach has been to use a combination test using the detection of oncogenic mutations/fusion transcripts and an miRNA expression-based classifier algorithm. To improve the performance of the combination test, the next-generation sequencing (NGS)-based mutational panel was expanded from the detection of 5 oncogenes to 10 oncogenes and tumor suppressor genes and the detection of fusion transcripts was increased from 6 to 38. Herein, we describe the assay development of the expanded panel NGS test and optimization of various steps for the library preparation of multiplexed target genes to maintain quality parameters for sequencing and to improve the robustness of the test for use in clinical testing in a College of American Pathologists/Clinical Laboratory Improvements Amendments-certified laboratory. Technical hurdles in NGS library preparation for the sequencing of both normal and high guanine-cytosine-rich regions, and balanced amplification of various amplicons in highly multiplexed PCRs, were successfully overcome. Analytical validation as a laboratory-developed test (ThyGeNEXT) included the demonstration of assay reproducibility, lower limit of detection, as well as other fundamental quality parameters.

Utility of microdissected cytology smears for molecular analysis of thyroid malignancy.

BACKGROUND: Molecular testing of thyroid fine-needle aspirates has demonstrated value in cases of indeterminate cytology (Bethesda categories III, IV, and V) enabling optimized individual patient management leading to better outcomes with health economic benefits. For most molecular testing modalities, including mutational panels and classifier analyses, part or all of a dedicated needle aspiration pass is required to obtain an adequate sample for testing. Our analysis, which is based on a combination approach (mutation detection and microRNA classifier status), has documented clinical validity and utility when performed on thyroid fine-needle aspirates placed directly into RNA preservative fluid. Here we show that the combination approach can be extended to microdissected stained cytology slides provides the physician greater opportunity to resolve cytological indeterminacy. METHODS: Extracted nucleic acid from needle aspirate and corresponding cytology preparations of 47 thyroid nodules were analyzed using identical methodology and results were compared. RESULTS: Of 94 molecular analyses (47 mutational analyses, 47 microRNA classifier assessments based on a validated 10 marker panel) only 5 samples showed discordant results. CONCLUSION: These findings, together with supplementary work using archival specimens shows that the combination approach can be effectively applied to both direct aspirated thyroid nodule aspirates or to nucleic acid extracted from macrodissected and microdissected cytology slide smears, with the expectation of equivalent results. The advantages of both specimen sources, direct aspirate, and cytology slide smears are discussed.

Mutational analysis of metastatic lymph nodes from papillary thyroid carcinoma in adult and pediatric patients.

BACKGROUND: Limited data are available on the analysis of somatic mutations in metastatic lymph nodes in adult and pediatric patients with papillary thyroid carcinomas. METHODS: A total of 92, microdissected, formalin-fixed, paraffin-embedded tissue specimens from 39 patients were analyzed for the presence of somatic mutations utilizing the ThyGenX next-generation sequencing test. RESULTS: Somatic mutations were detected in 67% of papillary thyroid carcinoma specimens. The majority of patients with synchronous and all 6 patients with radioactive iodine-resistant (metachronous) metastatic lymph nodes contained BRAF mutations. Four patients had mutations detected in their metastatic lymph nodes that were not detected in their primary tumors. For the most part, BRAF mutations were seen in adults, and RAS mutations were seen in children. CONCLUSION: Findings of different mutations in metastatic lymph nodes compared with the primary papillary thyroid carcinomas are probably the result of tumor heterogeneity. The presence of the BRAF mutation in metastatic lymph nodes might be responsible for the recurrence of papillary thyroid carcinomas and resistance to radioactive iodine therapy. The good prognosis observed in papillary thyroid carcinomas found in pediatric and young adult patients might be explained by the predominance of RAS rather than BRAF mutations.