Towards a single-assay approach: a combined DNA/RNA sequencing panel eliminates diagnostic redundancy and detects clinically-relevant fusions in neuropathology.

Since the introduction of integrated histological and molecular diagnoses by the 2016 World Health Organization (WHO) Classification of Tumors of the Nervous System, an increasing number of molecular markers have been found to have prognostic significance in infiltrating gliomas, many of which have now become incorporated as diagnostic criteria in the 2021 WHO Classification. This has increased the applicability of targeted-next generation sequencing in the diagnostic work-up of neuropathology specimens and in addition, raises the question of whether targeted sequencing can, in practice, reliably replace older, more traditional diagnostic methods such as immunohistochemistry and fluorescence in-situ hybridization. Here, we demonstrate that the Oncomine Cancer Gene Mutation Panel v2 assay targeted-next generation sequencing panel for solid tumors is not only superior to IHC in detecting mutation in IDH1/2 and TP53 but can also predict 1p/19q co-deletion with high sensitivity and specificity relative to fluorescence in-situ hybridization by looking at average copy number of genes sequenced on 1p, 1q, 19p, and 19q. Along with detecting the same molecular data obtained from older methods, targeted-next generation sequencing with an RNA sequencing component provides additional information regarding the presence of RNA based alterations that have diagnostic significance and possible therapeutic implications. From this work, we advocate for expanded use of targeted-next generation sequencing over more traditional methods for the detection of important molecular alterations as a part of the standard diagnostic work up for CNS neoplasms.

Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions.

Recently, the US Food and Drug Administration approved several targeted therapies directed against oncogenic fusions. One of the most effective such targeted therapies is Vitrakvi (larotrectinib), highly specific oral tropomyosin receptor kinase inhibitor indicated for the treatment of patients with any solid tumor harboring a fusion involving one of the neurotrophic receptor tyrosine kinase (NTRK) genes. Although several diagnostic approaches can be used to detect these NTRK fusions, RNA-based next-generation sequencing remains one of the most sensitive methods, as it can directly detect the transcribed end product of gene fusion at the mRNA level. In this study, performance characteristics of three RNA-based next-generation sequencing assays with distinct mechanisms and chemistries were investigated: anchored multiplex PCR, amplicon-based multiplex PCR, and hybrid capture-based enrichment method. Analytical sensitivity analysis shows that the amplicon-based multiplex PCR method has the lowest limit of detection. However, both hybrid-capture and anchored multiplex PCR methods can detect NTRK fusions with uncommon or novel fusion partners, which is challenging for the amplicon-based multiplex method. As for clinical sensitivity, all three methods were highly concordant in detecting NTRK fusions in patient samples. Additionally, they all presented equivalent high-level performance in specificity, suggesting that all three platforms can detect NTRK fusions in clinical samples with similar performance characteristics.

Preanalytic variables in quality and quantity of nucleic acids extracted from FNA specimens of thyroid gland nodules collected in CytoLyt: Cellularity and storage time.

BACKGROUND: Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated. METHODS: Thyroid fine-needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity. RESULTS: Fifty-six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P = .01), and storage time (median, 43 days; range, 7-77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P = .01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0-23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0-42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell-free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false-negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics. CONCLUSIONS: Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors.

Recurrent genetic alterations and biomarker expression in primary and metastatic squamous cell carcinomas of the vulva.

Using a comprehensive next-generation sequencing pipeline (143 genes), Oncomine Comprehensive v.2, we analyzed genetic alterations on a set of vulvar squamous cell carcinomas (SCCs) with emphasis on the primary and metastatic samples from the same patient, to identify amenable therapeutic targets. Clinicopathologic features were reported and genomic DNA was extracted from 42 paraffin-embedded tumor tissues of 32 cases. PD-L1 expression was evaluated in 20 tumor tissues (10 cases with paired primary and metastatic tumors). Fifteen (88%) of 17 successfully analyzed HPV-unrelated SCCs harbored TP53 mutations. 2 different TP53 mutations had been detected in the same tumor in 4 of 15 cases. Other recurrent genetic alterations in this group of tumors included CDKN2a mutations (41%), HRAS mutations (12%), NOTCH1 mutations (12%) and BIRC3 (11q22.1-22.2) amplification (12%). Six HPV-related tumors harbored PIK3CA, BAP1, PTEN, KDR, CTNNB1, and BRCA2 mutations, of which, one case also contained TP53 mutation. Six cases showed identical mutations in paired primary site and distant metastatic location and four cases displayed different mutational profiles. PD-L1 expression was seen in 6 of 10 primary tumors and all 6 paired cases showed discordant PD-L1 expression in the primary and metastatic sites. Our results further confirmed the genetic alterations that are amenable to targeted therapy, offering the potential for individualized management strategies for the treatment of these aggressive tumors with different etiology. Discordant PD-L1 expression in the primary and metastatic vulvar SCCs highlights the importance of evaluation of PD-L1 expression in different locations to avoid false negative information provided for immunotherapy.

Breast Tumor Resembling the Tall Cell Variant of Papillary Thyroid Carcinoma: Molecular Characterization by Next-Generation Sequencing and Histopathological Comparison With Tall Cell Papillary Carcinoma of Thyroid.

Recent publications have brought attention to the histopathological, immunohistochemical, and molecular aspects of the rare breast tumor resembling the tall cell variant of papillary thyroid carcinoma (BrTC). Nine archived cases of this entity were retrieved, reviewed, and compared with randomly selected tall cell variants of papillary thyroid carcinoma (ThTC). Seven of the BrTC cases as well as 5 cases of solid papillary carcinoma of breast were analyzed by Oncomine next-generation sequencing. BrTC and ThTC were histologically distinguishable by the presence of solid architecture, luminal histiocytes, and reverse polarity in the former, and psammoma bodies, giant cells, and optically clear nuclei in the latter. Sequencing revealed IDH2 R172 single-nucleotide variants in all 7 BrTCs, 6 of which had concurrent PIK3CA mutations. None of the conventional solid papillary carcinomas demonstrated IDH2 mutation. BrTC bears superficial resemblance to other papillary tumors but is unique in terms of histology and molecular profile.

Promoter hypomethylation upregulates Na+-K+-2Cl- cotransporter 1 in spontaneously hypertensive rats.

The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is one of several transporters that have been implicated for development of hypertension since NKCC1 activity is elevated in hypertensive aorta and vascular contractions are inhibited by bumetanide, an inhibitor of NKCC1. We hypothesized that promoter hypomethylation upregulates the NKCC1 in spontaneously hypertensive rats (SHR). Thoracic aortae and mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The expression levels of nkcc1 mRNA and protein in aortae and heart tissues were measured by real-time PCR and Western blot, respectively. The methylation status of nkcc1 promoter region was analyzed by combined bisulfite restriction assay (COBRA) and bisulfite sequencing. Phenylephrine-induced vascular contraction in a dose-dependent manner, which was inhibited by bumetanide. The inhibition of dose-response curves by bumetanide was much greater in SHR than in Wistar Kyoto (WKY) normotensive rats. The expression levels of nkcc1 mRNA and of NKCC1 protein in aortae and heart tissues were higher in SHR than in WKY. Nkcc1 gene promoter was hypomethylated in aortae and heart than those of WKY. These results suggest that promoter hypomethylation upregulates the NKCC1 expression in aortae and heart of SHR.

Effects of 3',4'-dihydroxyflavonol on vascular contractions of rat aortic rings.

1. 3',4'-Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti-oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions. 2. Contractile and relaxation responses were determined in rat endothelium-denuded aortic rings mounted in organ baths. In addition, the phosphorylation of myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17) was determined using western blot analysis. Levels of GTP RhoA, as a marker of RhoA activation, were also measured. 3. Cumulative addition of increasing concentrations of NaF (3.0-12.0 mmol/L) or U46619 (1.0-1000 nmol/L) concentration-dependently increased vascular tension. These responses were inhibited by pretreatment of aortic rings with DiOHF (10, 30 or 100 micromol/L), which dose-dependently decreased vascular contractions induced by 8.0 mmol/L NaF, 30 nmol/L U46619, 0.1 micromol/L phenylephrine and 50 mmol/L KCl. 4. The K(+) channel blockers 4-aminopyridine (3 mmol/L), charybdotoxin (10 nmol/L), apamin (500 nmol/L) and glibenclamide (10 micromol/L) had no effect on vascular relaxation induced by DiOHF (1-30 micromol/L). 5. At 30 micromol/L, DiOHF decreased the activation of RhoA and subsequent phosphorylation of MYPT1, CPI-17 and MLC(20) to almost basal levels. 6. In conclusion, DiOHF decreases vascular contraction at least partly by inhibition of the RhoA/Rho-kinase pathway in rat endothelium-denuded aorta. These results suggest that DiOHF may have therapeutic potential in the treatment of cardiovascular diseases.

Effects of glyceollin I on vascular contraction in rat aorta.

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K(+) channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC(20). Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.

Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas.

We hypothesized that fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway in isolated rat aortas. Rat aortic rings were mounted in organ baths and contracted with sodium fluoride (NaF). We measured the amount of GTP-RhoA as well as vascular tension. We also determined the level of phosphorylation of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17kDa (CPI17). In both physiological salt solution and Ca(2+)-free solution, NaF increased vascular tension and MLC(20) phosphorylation in dose-dependent manners. NaF increased not only phosphorylation level of MYPT1(Thr855) and CPI17(Thr38), but also the amount of GTP-RhoA. Both H1152 and Y27632, inhibitors of Rho kinase, but not Ro31-8220, an inhibitor of PKC, attenuated NaF-induced contraction and phosphorylation level of MLC(20), MYPT1(Thr855) and CPI17(Thr38). In conclusion, fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway.

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 microM) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC(20)), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC(20) phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1(Thr855) and CPI17(Thr38), the downstream effectors of Rho-kinase. In regards to the Ca(2+)-free solution, flavone inhibited the phosphorylation of MYPT1(Thr855) and CPI17(Thr38), as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

17beta-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.

We hypothesized that 17beta-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway. Rat aortic rings were contracted with cumulative addition of U46619, NaF, KCl or PDBu 30 min after pretreatment with 17beta-estradiol (10, 30, and 100 microM) or vehicle. We measured the amount of GTP RhoA and the level of phosphorylation of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Pretreatment with 17beta-estradiol dose-dependently inhibited the concentration-response curves in response to U46619, NaF or KCl, but not to PDBu. 17beta-Estradiol decreased not only the level of phosphorylation of MYPT1(Thr855) and CPI17(Thr38) as well as MLC(20), but also the activity of RhoA induced by U46619 or NaF. However, 17beta-estradiol did not affect the level of phosphorylation of CPI17 induced by PDBu. 17beta-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.

A role for Rho-kinase in Ca-independent contractions induced by phorbol-12,13-dibutyrate.

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.

Isoflavone attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway.

Isoflavones decrease blood pressure, improve lipid profiles, and restore vascular function. We hypothesized that isoflavone attenuates vascular contraction by inhibiting RhoA/Rho-kinase signaling pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with 11,9 epoxymethano-prostaglandin F(2alpha) (U46619), a thromboxane A2 analog, or KCl 30 min after the pretreatment with genistein (4',5,7-trihydroxyisoflavone), daidzein (4',7-dihydroxyisoflavone), or vehicle. We determined the phosphorylation level of the myosin light chain (MLC(20)), myosin phosphatase-targeting subunit 1 (MYPT1), and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17) by means of the Western blot. We also measured the amount of GTP RhoA as a marker regarding RhoA activation. The cumulative additions of U46619 or KCl increased vascular tension in a concentration-dependent manner, which were inhibited by pretreatment with genistein or daidzein. Both U46619 (30 nM) and KCl (50 mM) increased MLC(20) phosphorylation levels, which were inhibited by genistein and daidzein. Furthermore, both genistein and daidzein decreased the amount of GTP RhoA activated by either U46619 or KCl. U46619 (30 nM) increased phosphorylation of the MYPT1(Thr855) and CPI17(Thr38), which were also inhibited by genistein or daidzein. However, neither genistein nor daidzein inhibited phorbol 12,13-dibutyrate-induced vascular contraction and CPI17 phosphorylation. In conclusion, isoflavone attenuates vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.

Heat shock augments myosin phosphatase target-subunit phosphorylation.

Our previous study demonstrated that heat shock augmented vascular contraction. In the present study, we hypothesized that heat shock augments myosin phosphatase target-subunit (MYPT1) phosphorylation resulting in augmented vascular contraction. Endothelium-denuded rat aortic rings were mounted in organ baths, exposed to heat shock (42 degrees C for 45 min), and subjected to contraction 4 h after the heat shock followed by Western blot analysis for MLC(20) (the 20 kDa light chains of myosin II) or MYPT1. The contractile responses in both control and heat shock-treated aorta were inhibited by Y27632, an inhibitor of Rho-kinase. The level of the MLC(20) and MYPT1(Thr855) phosphorylation in response to KCl was higher in heat shock-treated aorta than that in timed-control. The increased MYPT1(Thr855) phosphorylation was inhibited by Y27632 (1.0 microM) in parallel with inhibition of MLC(20) phosphorylation and vascular contraction. These results indicate that heat shock augments MYPT1 phosphorylation resulting in augmented vascular contraction.

Vasorelaxation by Samhwangsasim-tang, an herb medicine, is associated with decreased phosphorylation of the myosin phosphatase target subunit.

Samhwangsasim-tang (SST) is a widely used herbal medicine with vasodilatory actions in oriental countries. We hypothesized that SST modulates vascular contractility by decreasing phosphorylation of the myosin phosphatase target subunit. Rat aortic ring preparations were mounted in organ baths and subjected to contractions or relaxations. Phosphorylation of 20kDa myosin light chains (MLC(20)) and MYPT1, a target subunit of myosin phosphate 1, were examined with immunoblots. SST relaxed aortic ring preparations precontracted with phenylephrine whether endothelium was intact or denuded. Treatment of aortic rings with N(ω)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelial nitric oxide synthase or methylene blue, an inhibitor of guanylyl cyclase, did not affect the relaxing action of SST. Furthermore, SST inhibited vascular contractions induced by NaF or phenylephrine, but not by phorbol dibutyrate. SST also decreased vascular tension precontracted by 8.0mmol/L NaF or 1.0µmol/L phenylephrine, but not by 1.0µmol/L phorbol dibutyrate. In vascular strips, SST decreased the phosphorylation level of both MLC(20) and MYPT1 induced by 8.0mmol/L NaF. In conclusion, SST inhibited vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit.