Does addition of BRAF V600E mutation testing modify sensitivity or specificity of the Afirma Gene Expression Classifier in cytologically indeterminate thyroid nodules?

OBJECTIVE: The purpose of this study was to determine the frequency of BRAF mutation in cytologically indeterminate thyroid nodules and to investigate whether adding the BRAF test improves diagnostic accuracy of the Afirma Gene Expression Classifier (GEC). DESIGN: BRAF V600E mutational status was determined for DNA extracted from cytologically benign (n = 40), indeterminate (n = 208), and malignant (n = 48) fine-needle aspiration specimens previously categorized by GEC as molecularly Benign or Suspicious. Analytical performance of the BRAF assay was assessed to establish reproducibility and limits of detection. Molecular testing results were correlated with blinded expert histopathological diagnoses. RESULTS: The BRAF assay detected mutations reproducibly to 2.5% mutant allele frequency. The prevalence of BRAF mutations in cytologically benign specimens was 2 of 40 (5.0%, 95% confidence interval [CI], 0-16) and in cytologically malignant specimens was 36 of 48 (75.0%, 95% CI, 60-86). In the cytologically indeterminate category, 10.1% of specimens were BRAF+: 2 of 95 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (2.1%, 95% CI, 0-7); 1 of 70 as follicular neoplasm or suspicious for follicular neoplasm (1.4%, 95% CI, 0-9); and 18 of 43 as suspicious for malignancy (41.9%, 95% CI, 27-58). All BRAF+ specimens were classified as Suspicious by the GEC. CONCLUSIONS: BRAF mutations are uncommon in nodules with atypia of undetermined significance or follicular lesion of undetermined significance or follicular neoplasm or suspicious for follicular neoplasm cytology. Most cytologically indeterminate nodules that proved to be malignant were also BRAF-, and all nodules that were false-negative by GEC were also BRAF-. Similarly, all BRAF+ specimens were also GEC Suspicious. Neither GEC test sensitivity nor specificity was improved by addition of BRAF mutation testing.

Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.

OBJECTIVE: Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs). DESIGN: Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility. RESULTS: RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale). CONCLUSIONS: Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.

Preoperative diagnosis of benign thyroid nodules with indeterminate cytology.

BACKGROUND: Approximately 15 to 30% of thyroid nodules evaluated by means of fine-needle aspiration are not clearly benign or malignant. Patients with cytologically indeterminate nodules are often referred for diagnostic surgery, though most of these nodules prove to be benign. A novel diagnostic test that measures the expression of 167 genes has shown promise in improving preoperative risk assessment. METHODS: We performed a 19-month, prospective, multicenter validation study involving 49 clinical sites, 3789 patients, and 4812 fine-needle aspirates from thyroid nodules 1 cm or larger that required evaluation. We obtained 577 cytologically indeterminate aspirates, 413 of which had corresponding histopathological specimens from excised lesions. Results of a central, blinded histopathological review served as the reference standard. After inclusion criteria were met, a gene-expression classifier was used to test 265 indeterminate nodules in this analysis, and its performance was assessed. RESULTS: Of the 265 indeterminate nodules, 85 were malignant. The gene-expression classifier correctly identified 78 of the 85 nodules as suspicious (92% sensitivity; 95% confidence interval [CI], 84 to 97), with a specificity of 52% (95% CI, 44 to 59). The negative predictive values for "atypia (or follicular lesion) of undetermined clinical significance," "follicular neoplasm or lesion suspicious for follicular neoplasm," or "suspicious cytologic findings" were 95%, 94%, and 85%, respectively. Analysis of 7 aspirates with false negative results revealed that 6 had a paucity of thyroid follicular cells, suggesting insufficient sampling of the nodule. CONCLUSIONS: These data suggest consideration of a more conservative approach for most patients with thyroid nodules that are cytologically indeterminate on fine-needle aspiration and benign according to gene-expression classifier results. (Funded by Veracyte.).

Molecular classification of thyroid nodules using high-dimensionality genomic data.

OBJECTIVE: We set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA). DESIGN: We used mRNA expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology. RESULTS: Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30-fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, we demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples. CONCLUSIONS: The FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs.

The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.

In a recent clinical study, the thiazolidinedione (TZD) pioglitazone (Actos was reported to preserve cognitive function in patients with mild to moderate Alzheimer's disease and type II diabetes mellitus. TZDs are agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), are peripheral insulin sensitizers, and have recently been reported to increase mitochondrial biogenesis in the central nervous system and dendritic spine density. We report a transcriptional profile of the TZD pioglitazone and the non-TZD PPARgamma agonist GW347845 in primary cortical culture. We observed that pioglitazone, but not GW347845, increased cholesterol biosynthetic and lipogenic gene expression after 6 h, and the expression of the cholesterol efflux transporters Abca1 and Abcg1 after 24 h. Co-treatment of pioglitazone with the PPARgamma antagonist GW9662 did not significantly reduce these effects, suggesting a PPARgamma-independent mechanism. These findings suggest a novel effect of TZDs in neurons that may be of relevance as a novel approach against Alzheimer's disease.

Targeting gut T cell Ca2+ release-activated Ca2+ channels inhibits T cell cytokine production and T-box transcription factor T-bet in inflammatory bowel disease.

Prolonged Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels is crucial in activating the Ca(2+)-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca(2+) assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01-10 microM). IFN-gamma, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca(2+) influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-gamma, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cgamma2 at distinct sites.

Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCg2 (phospholipase Cg2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCg2 at Tyr753 and Tyr759, whereas GPIb-IX-V only induces significant phosphorylation at Tyr753. Both receptors stimulate tyrosine phosphorylation of Btk at the regulatory Tyr223 and Tyr551. Syk and Btk phosphorylate peptides from PLCg2 containing Tyr753 and Tyr759 respectively, suggesting that they may stimulate phosphorylation at these sites in phospholipase. Studies using PLCg2-deficient platelets demonstrated that phospholipase is not required for the activation of integrin aIIbb3 by GPIb-IX-V. Our results demonstrate fundamental differences between GPVI and GPIb-IX-V in the regulation of tyrosine phosphorylation of Syk and PLCg2 consistent with the functional impairment of phospholipase in signalling by GPIb-IX-V.

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C.

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.