Use of the Ion AmpliSeq Cancer Hotspot Panel in clinical molecular pathology laboratories for analysis of solid tumours: With emphasis on validation with relevant single molecular pathology tests and the Oncomine Focus Assay

Targeted application of next-generation sequencing (NGS) technology allows detection of specific mutations that can provide treatment opportunities for cancer patients. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel V2 (CHV2) using formalin-fixed, paraffin-embedded (FFPE) tissue of clinical specimens.Thirty-five FFPE tumour samples with known mutational status were collected from four different hospitals and sequenced with CHV2 using an Ion Chef System and Ion S5 XL system. Out of 35 cases, seven were sequenced with Oncomine focus Assay Panel for comparison. For the limit of detection test, we used an FFPE reference standard, a cell line that included an engineered 50% EGFR T790?M in an RKO cell line background. Coverage analysis results including number of mapped reads, on target percent, mean depth, and uniformity were not different according to hospitals. Sensitivity for mutation detection down to 3% was demonstrated. NGS results showed 100% concordance with the results from single molecular pathology tests Assay in 30 cases with 24 known positive mutations and 14 known negative mutations, and another NGS panel of the Oncomine focus in seven cases.The CHV2 NGS test for solid tumours using Ion chef system and S5 XL system in clinical molecular pathology laboratories for analysis of solid tumours could be routinely used and could replace some single molecular pathology tests after a stringent and thorough validation process.     

Catalpol protects against 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a well‐known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells. Catalpol inhibited TCDD‐induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3‐E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD‐treated cells and significantly inhibited TCDD‐induced increases in the levels of cytochrome P450 1A1 and extracellular signal‐regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal‐regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD‐induced damage in MC3T3‐E1 osteoblastic cells.     

Xanthohumol ameliorates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin–induced cellular toxicity in cultured MC3T3‐E1 osteoblastic cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is an environmental contaminant. Xanthohumol is a prenylated flavonoid found in hops (Humulus lupulus) and beer. The aim of the current study was to explore the role of xanthohumol in modulating the toxicity of TCDD in MC3T3‐E1 osteoblastic cells. In cells treated with TCDD alone, intracellular Ca²⁺ concentrations, mitochondrial membrane potential disruption, reactive oxygen species production, cardiolipin peroxidation, nitric oxide release and cytochrome P450 1A1 expression were significantly increased. TCDD treatment increased the mRNA levels of extracellular signal‐regulated kinase 1 and nuclear factor kappa B, and significantly decreased the level of protein kinase B (AKT) in MC3T3‐E1 osteoblastic cells. However, the presence of xanthohumol alleviated the pathological effects of TCDD. In addition, xanthohumol treatment significantly increased the expression of genes associated with osteoblast differentiation (alkaline phosphatase, osteocalcin, osteoprotegerin and osterix). We conclude that xanthohumol has a beneficial influence and may antagonize TCDD toxicity in osteoblastic cells.     

Interobserver agreement in emergency department triage

STUDY OBJECTIVES: We measure the interobserver reliability of the triage process, examine the effect of vital signs on the triage process, and provide a context for the prior observation of poor interobserver agreement between in-person and telephonic interviews. METHODS: We performed a prospective observational study using a randomized crossover design at a university teaching hospital emergency department. Patients were eligible if they spoke English, were not presenting for a reevaluation, and were unlikely to be harmed by the delay created by a second triage interview. Every eligible patient underwent 2 independent, sequential, in-person ED intake interviews conducted by experienced ED triage nurses. After taking a history, each nurse chose 1 of 5 hypothetic triage designations (ED by 911, ED within 2 hours, see a physician within 8 hours, see a physician within 24 hours, or home care-see a physician in >24 hours) and, after being told the patient's vital signs, again selected a designation. RESULTS: Three hundred sixty-three patients presented during the study period: 113 were ineligible, 34 were missed by the investigators, and 15 refused to participate. Nineteen nurses participated in the triage of the 201 study patients. Agreement between 2 in-person designations made without knowledge of vital signs was poor (percent agreement 53%; kappa=0.30; tau(b)=0.50). Knowledge of vital signs did not improve agreement (percent agreement 49%; kappa=0.25; tau(b)=0.45). CONCLUSION: There was poor interobserver agreement between certified triage nurses using a 5-item triage scale designed for telephonic triage. These findings suggest that only a small portion of the poor interobserver agreement observed in a prior study of telephonic versus in-person triage can be attributed to the use of the telephone.