Search

Purpose: To create an in vivo model of PDGFRA D842V-mutant gastrointestinal stromal tumor (GIST) and identify the mechanism of tumor persistence following avapritinib therapy. Experimental Design: We created a patient-derived xenograft (PDX) of PDGFRA D842V-mutant GIST and tested the effects of imatinib, avapritinib, and ML-7, an inhibitor of myosin light-chain kinase (MYLK). Bulk tumor RNA sequencing and oncogenic signaling were evaluated. Apoptosis, survival, and actin cytoskeleton were evaluated in GIST T1 cells and isolated PDX cells in vitro. Human GIST specimens were analyzed for MYLK expression. Results: The PDX was minimally responsive to imatinib but sensitive to avapritinib. Avapritinib therapy increased tumor expression of genes related to the actin cytoskeleton, including MYLK. ML-7 induced apoptosis and disrupted actin filaments in short-term cultures of PDX cells and decreased survival in GIST T1 cells in combination with imatinib or avapritinib. Combined therapy with ML-7 improved the antitumor effects of low-dose avapritinib in vivo. Furthermore, MYLK was expressed in human GIST specimens. Conclusions: MYLK upregulation is a novel mechanism of tumor persistence after tyrosine kinase inhibition. Concomitant MYLK inhibition may enable the use of a lower dose of avapritinib, which is associated with dose-dependent cognitive side effects.

BACKGROUND: Follicular thyroid carcinoma (FTC) and Hurthle cell carcinoma (HCC) are rare and aggressive thyroid cancers with limited published data comparing their outcomes or regarding their subtypes. The aim of this study was to describe clinicopathological features and compare clinical outcomes of patients with FTC and HCC based on the 2017 World Health Organization definition and extent of vascular invasion (VI). METHODS: We retrospectively studied 190 patients with HCC and FTC primarily treated with surgery at Memorial Sloan Kettering Cancer Center between 1986 and 2015. Patients were classified as minimally invasive (MI), encapsulated angioinvasive with focal VI (EA-FVI), encapsulated angioinvasive with extensive VI (EA-EVI), and as widely invasive (WI). To compare clinical outcomes, patients were grouped as follows: group 1 = FTC-MI and FTC EA-FVI, group 2 = FTC EA-EVI and FTC-WI, group 3 = HCC-MI and HCC EA-FVI, group 4 = HCC EA-EVI and HCC-WI. Outcomes of interest were overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS), locoregional recurrence-free survival (LRRFS), and distant recurrence-free survival (DRFS). Outcomes were determined using the Kaplan–Meier method and compared with log-rank test. RESULTS: Patients with HCC (n = 111) were more likely to be older than 55 years old (59% vs. 27%, p

Supplemental Figure 5.Short term culture of cells derived from PDGFRA D842V PDXs are tumor cells.

Supplemental Figure 1.

Supplemental Figure 8.Relative MYLK mRNA expression by qPCR in GIST PDGFRA D842V PDXs treated with vehicle(Veh) or imatinib (IM) for 2 weeks.

Supplemental Figure 3. GIST PDGFRA D842V PDXs are not sensitive to crenolanib.

Supplemental Figure 7.

Supplemental Figure 4.

Purpose:To create an in vivo model of PDGFRA D842V-mutant gastrointestinal stromal tumor (GIST) and identify the mechanism of tumor persistence following avapritinib therapy.Experimental Design:We created a patient-derived xenograft (PDX) of PDGFRA D842V-mutant GIST and tested the effects of imatinib, avapritinib, and ML-7, an inhibitor of myosin light-chain kinase (MYLK). Bulk tumor RNA sequencing and oncogenic signaling were evaluated. Apoptosis, survival, and actin cytoskeleton were evaluated in GIST T1 cells and isolated PDX cells in vitro. Human GIST specimens were analyzed for MYLK expression.Results:The PDX was minimally responsive to imatinib but sensitive to avapritinib. Avapritinib therapy increased tumor expression of genes related to the actin cytoskeleton, including MYLK. ML-7 induced apoptosis and disrupted actin filaments in short-term cultures of PDX cells and decreased survival in GIST T1 cells in combination with imatinib or avapritinib. Combined therapy with ML-7 improved the antitumor effects of low-dose avapritinib in vivo. Furthermore, MYLK was expressed in human GIST specimens.Conclusions:MYLK upregulation is a novel mechanism of tumor persistence after tyrosine kinase inhibition. Concomitant MYLK inhibition may enable the use of a lower dose of avapritinib, which is associated with dose-dependent cognitive side effects.

Supplemental Figure 6.Cell survival of GIST T1 cells treated with imatinib or avapritinib for 72h in complete media.Each data point represents an average of 10 wells.

The adaptive immune system plays an essential anti-tumor role through immunosurveillance and response to immunotherapies. Characterizing phenotypic features and mechanisms of dysfunction of tumor-specific T cell populations may uncover novel immunotherapeutic targets and biomarkers of response. To study tumor-specific T cell responsesin vivo, a tumor model must express a known neoantigen. While transplant models with known neoantigen expression are widely available, autochthonous tumor models in which the tumor coevolves with the immune system are limited. In this study, we combined CRISPR/Cas9 and sleeping beauty transposase technology to develop an autochthonous orthotopic murine sarcoma model with oncogenic KrasG12D, functionally impaired p53, and expression of known MHCI and MHCII sarcoma neoantigens. Using MHC tetramer flow cytometry, we identified a tumor-specific immune response in the peripheral blood as early as 10 days after tumor induction leading to tumor clearance. Tumors developed at high penetrance after co-depletion of CD8 and CD4 T cells, but depletion of either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These results suggest that CD8 and CD4 T cells can independently contribute to immunosurveillance leading to clearance of sarcomas expressing MHCI and MHCII neoantigens.

Periocular skin cancers require both effective and tissue-sparing treatment to minimize morbidity and preserve eyelid and lacrimal system function. We aim to define outcomes of periocular tumors treated with Mohs micrographic surgery (MMS) and factors associated with poor outcomes after surgery. This is a retrospective cohort study of all periocular tumors treated with MMS at an academic, large metropolitan-based referral center from January 1, 2013 to December 31, 2018. For 316 tumors from 307 patients, 75.3% of cases were basal cell carcinoma (BCC) (n = 238), 20.9% were squamous cell carcinoma (SCC) (n = 66), 2.5% were melanoma (n = 8), and 1.3% were primary adnexal carcinoma (n = 4). Over a mean follow-up of 47 months (range 12-108 months), local recurrence of two BCCs was observed. There were no recurrences for SCC, adnexal carcinoma, or melanoma. For BCC, previously treated tumors had higher risk for recurrence after MMS. AJCC 8 T stage was not associated with poor outcomes after MMS for periocular carcinoma or melanoma. Mohs micrographic surgery offers excellent cure rates for periocular cutaneous tumors. For basal cell carcinoma, previously treated lesions were associated with additional recurrence after MMS.

Background: Mitochondrial genome sequences have become critical to the study of biodiversity, though the cost of doing so with genome skimming or other methods has been difficult to reduce beyond ~$15–20 per sample making it costly to scale up. Here, we report on an approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION as a proof-of-concept. We implemented several cost-saving measures to reach a per-sample cost as low as $7 per mtGenome on the PacBio platform. Results: With the PacBio data we recovered at least one of the two fragments for 96% of samples (~80% of input amplicons) with mean coverage ~1,500x. The ONT data recovered less than 50% of input fragments due to the use of PacBio Barcoded Universal Primers (BUPs) and low throughput, though we are confident results will improve with ONT-optimized BUPs. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, significant improvement in tree support with longer alignments. Conclusions: This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a Kit

In Canada, recreational use of cannabis was legalized in October 2018. This policy change along with recent publications evaluating the efficacy of cannabis for the medical treatment of epilepsy and media awareness about its use have increased the public interest about this agent. The Canadian League Against Epilepsy Medical Therapeutics Committee, along with a multidisciplinary group of experts and Canadian Epilepsy Alliance representatives, has developed a position statement about the use of medical cannabis for epilepsy. This article addresses the current Canadian legal framework, recent publications about its efficacy and safety profile, and our understanding of the clinical issues that should be considered when contemplating cannabis use for medical purposes.

Introduction: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit receptor. While highly effective, tyrosine kinase inhibitors like imatinib are not curative. The natural ligand for the Kit receptor is Kit Ligand (KitL), which exists in both soluble and membrane-bound forms. We sought to determine the role of KitL in the oncogenic Kit signaling of GIST. Methods: KitV558Δ/+ mice, which spontaneously develop an intestinal GIST, were treated with imatinib or vehicle for 1 week, and sorted non-immune (CD45-) cells from tumors were submitted for single-cell RNA sequencing (n=3/group). GIST T1 cells were treated with KitL with or without imatinib and oncogenic Kit signaling was evaluated. KitV558Δ/+ mice were crossed with various KitL mutant and inducible models and tumor weights were measured (n=3-4/group). Human GISTs were stained for KitL expression by IHC, and an existing bulk RNA sequencing human GIST dataset with matched human serum was analyzed. Results: Unsupervised clustering revealed endothelial, smooth muscle, and tumor cells had high expression of KitL RNA in untreated tumors from KitV558Δ/+ mice. Imatinib therapy increased KitL RNA in all 3 cell types, suggesting that extra-tumoral KitL expression is dependent on oncogenic signaling (p Citation Format: Andrew D. Tieniber, Ferdinando Rossi, Andrew Hanna, Mengyuan Liu, Mark Etherington, Kevin Do, Laura Wang, Ronald P. DeMatteo. Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A017.

Limited evidence on the relationship between antiepileptic drug (AED) tapering and the likelihood of a seizure during an Epilepsy Monitoring Unit (EMU) admission is available, and no evidence specific to the pediatric population has been published. Our study sought to determine whether AED tapering leads to increased seizure likelihood in a pediatric EMU setting.We performed a retrospective chart review of children admitted to the pediatric EMU at the Hospital for Sick Children in Toronto between June 1, 2014 and June 1, 2016. Data collected included demographics, reason for EMU referral, and epilepsy and medical characteristics. Among those with nondaily seizures, Kaplan-Meier curves were fit to compare probability of EMU seizure in those who were tapered fully from at least one AED to those not tapered. A Cox proportional hazards model was fit to evaluate this relationship after adjustment for subject sex, distance traveled to hospital, epilepsy duration, seizure frequency, time since last seizure, whether EMU referral was part of presurgical planning, magnetic resonance imaging (MRI) findings, and number of prescribed AEDs. An interaction between medication taper and number of prescribed AEDs was also included. Terms not significant at p 0.3 were removed from the model, and the reduced model was recomputed.Of the 281 children included in the study, 159 had nondaily seizures. Kaplan-Meier curves indicated fully tapering at least one AED was associated with increased likelihood of seizure during EMU; however, after adjustment for confounding variables, this association was not preserved. Abnormal MRI findings, referral for presurgical evaluation, and shorter (≤3 months) time since last seizure were associated with increased likelihood of seizure during EMU.Short-term AED tapering in pediatric patients may not be effective for increasing seizure likelihood in the EMU.

Although critics have debated the political topicality of Robert Henryson’s Morall Fabillis for several decades, they have left the Fabillis’ relationship to political theory largely underexplored. This essay examines the Fabillis’ creative engagement with and critique of fifteenth-century Scottish political theory, particularly its underpinnings in natural law. The prominent political treatises of fifteenth-century Scotland use nature and natural imagery to affirm human dignity and set a high ethical standard for princely governance. In contrast, Henryson’s Fabillis convey that the world is not governed by a benevolent Nature or natural law, as the political works would have it, but rather by the relentless force of bestial appetite in humans and animals alike. This critique is enabled by both Henryson’s relatively humble social position and his experimental mixture of beast fable with Reynardian beast epic and neo-Augustinian theology.

Background: Estimation of the intake of oily fish at a population level is difficult. The measurement of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in biological samples may provide a useful biomarker of intake. Objective: We identified the most appropriate biomarkers for the assessment of habitual oily fish intake and changes in intake by elucidating the dose- and time-dependent response of EPA and DHA incorporation into various biological samples that represent roles in fatty acid transport, function, and storage. Design: This was a double-blind, randomized, controlled intervention trial in 204 men and women that lasted 12 mo. EPA and DHA capsules were provided in a manner to reflect sporadic consumption of oily fish (ie, 1, 2, or 4 times/wk). EPA and DHAwere assessed at 9 time points over 12 mo in 9 sample types (red blood cells, mononuclear cells, platelets, buccal cells, adipose tissue, plasma phosphatidylcholine, triglycerides, cholesteryl esters, and nonesterified fatty acids). Results: A dose response (P , 0.05) was observed for EPA and DHA in all pools except for red blood cell EPA (P = 0.057). EPA and DHA measures in plasma phosphatidylcholine and platelets were best for the discrimination between different intakes (P , 0.0001). The rate of incorporation varied between sample types, with the time to maximal incorporation ranging from days (plasma phosphatidylcholine) to months (mononuclear cells) to .12 mo (adipose tissue). Conclusions: Plasma phosphatidylcholine EPA plus DHAwas identified as the most suitable biomarker of acute changes in EPA and DHA intake, and platelet and mononuclear cell EPA plus DHAwere the most suitable biomarkers of habitual intake. This trial was registered at Current Controlled Trials (www.controlled-trials.com) as ISRCTN48398526. Am J Clin Nutr 2012;96:748‐58.

Although Robert Henryson's Testament of Cresseid has attracted plenty of critical commentary, relatively little of it has focused on the pivotal moment near the end of the poem, when Henryson portrays Cresseid writing her testament. This essay aims to refocus critical attention on this moment and its significance. Drawing on the contexts of medieval literary and legal history, this essay argues that Henryson treats Cresseid more humanely than is commonly thought, for he portrays her producing, via her testament, both a beautiful work of art and a moving attempt to defend herself against future slander. As a work of art, Cresseid's testament departs from the conventional satirical and devotional modes of testamentary writing in favour of the tragic, to powerful effect. Moreover, like many real medieval women's wills, it reflects a conscious act of self-revision, of shaping a posthumous reputation. Considering Cresseid's plight as an exiled leper and divorcee, and the legal constraints on such people in the...

This present work describes the development of a novel high throughput comparative matrix-assisted laser desorption ionization (MALDI) mass spectrometry profiling technique for endogenous compounds using a new isotope-coded label for relative quantitation of carboxylic acids. The key new aspect of this technique was a differential label, 3-hydroxymethyl-1-ethylpyrididinium iodide (HMEP), designed specifically for simultaneous quantitative MALDI analysis of two physiological states. The HMEP-d(0) and HMEP-d(5) variants of the label were applied to profiling endogenous fatty acid levels during a fish oil intervention study, using the metabolite profile of every individual person in the study as their own personal analytical reference standard. Initially, analytical figures of merit such as sensitivity, linear dynamic range, limit of quantitation, and precision were determined from the comparative quantitation experiments. Importantly, the permanently charged HMEP mass tag not only increased the ionization efficiency of the studied fatty acids but also ensured that the solution phase properties of the analytes became more similar, allowing the use of CHCA as a single MALDI matrix compound for the entire range of analytes. The label exhibited a further very unique feature; it provided complete suppression of MALDI matrix-related ions. The MALDI assay was able to generate the data much faster than conventional gas chromatography (GC) methods for fatty acids. It is shown in this study that analyzing a single sample took less than 10 s with analytical results of comparable quality to those obtained by GC.

Stormwater best management practices have helped to address environmental concerns regarding peak water flows and accumulation of sediment and contamination by pollutants, but often overlook potential public health repercussions such as creating habitat for virus-carrying mosquitoes. Retention ponds frequently hold standing water for more than two weeks during the mosquito breeding season, creating development habitat for the aquatic stages of West Nile virus vectors. To avoid this problem, knowledge of vector ecology should be incorporated in the design of best management practices and integrated stormwater management plans. An integrated approach that incorporates hydrological management of water depth, flow, and surface agitation combined with manipulation of native biota and habitats based on the unique ecology and behaviour of the vector species can deter egg-laying mosquitoes by mimicking natural processes. Such an approach could help prevent disease transmission and alleviate the need for widespread application of pesticides.

To explore whether differences in vitamin K nutrition might, at least in part, underlie differences in fracture incidence between Asian and European populations, the vitamin K status of older individuals in Shenyang, China (eighty-six men, ninety-two women) and in Cambridge, UK (sixty-seven men, sixty-seven women) was compared. Dietary information was collected by food questionnaire in Shenyang and food diary in Cambridge and used to estimate the intake and sources of phylloquinone. Fasting blood was analysed for phylloquinone, triacylglycerol, total osteocalcin (tOC) and undercarboxylated osteocalcin (expressed as percentage of tOC; %ucOC). The mean intakes of green leafy vegetables were 127 (SD 90) g/d in Shenyang and 39 (SD 48) g/d in Cambridge. The estimated phylloquinone intakes (geometric means) were 247 (95% CI 226, 270) μg/d in Shenyang and 103 (95% CI 94, 112) μg/d in Cambridge. Plasma phylloquinone concentrations (geometric means) were significantly higher in the Shenyang subjects (2·17 (95% CI 1·95, 2·42) nmol/l) than in the Cambridge subjects (0·69 (95% CI 0·63, 0·76) nmol/l; PP=0·03) and Cambridge subjects (coefficient 0·29 (SE 0·10); P=0·005). tOC concentration and %ucOC (after adjusting for tOC) were significantly lower in the Shenyang than in the Cambridge subjects (tOC 25·2 (SE 4·2) % and %ucOC 68·5 (SE 10·0) % lower respectively; PP=0·0003) and Cambridge subjects (coefficient −0·17 (SE 0·07); P=0·02). The present study demonstrates that older individuals in northern China have a better vitamin K status compared with their British counterparts in Cambridge, UK.

A modified high-performance liquid chromatography (HPLC) method, based on Davidson and Sadowski [Meth. Enzymol. 282 (1997) 408], with fluorescence detection after zinc postcolumn reduction was developed and validated for the analysis of phylloquinone (vitamin K1) in plasma or serum samples. Compensation for procedural losses of vitamin K1 was made by the method of internal standardization using a proprietary vitamin K derivative. Increased sensitivity of detection by the use of a high-sensitivity Waters 440 fluorescence detector and optimized chromatography conditions increased the sensitivity to 4 fmol vitamin K1. The response was linear and free from interfering peaks and from baseline drift. It is therefore adequately sensitive for 0.25 ml or less of plasma sample. Long-term reproducibility of quality assurance (QA) samples was verified over a period of 4 months. The intraassay precision estimates of the QA samples within-run with mean vitamin K1 concentrations of 0.4, 1.4 and 3.4 nmol/l were 5.2% (n=6), 8.2% (n=6) and 3.0% (n=12), respectively, while interassay precision estimates between runs were 16% (n=22), 12% (n=21) and 8.1% (n=15), respectively. The assay accuracy was validated by comparing the results we obtained for 14 samples from the Vitamin K External Quality Assessment Scheme (KEQAS) with the consensus of the results from the other participating laboratories. Good agreement was obtained, with y=1.06x-0.09, R2=0.99. Validation also included linearity of response, absence of interference and confirmation of vitamin K1 peak purity.

Vicky Papaioannou MClSc, AUD(C), Reg. CASPLO3, Elizabeth Harvey MD, FRCPC4, Susan Richardson MD, FRCPC5, Ari Bitnun MD, MSc, FRCPC6 and Winnie Seto BScPhm, PharmD, MSc, ACPR, RPh1,2,7 1Department of Pharmacy, the Hospital for Sick Children, Toronto, Ontario, Canada, 2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada, 3Department of Communication Disorders, 4Department of Nephrology, 5Department of Paediatric Laboratory Medicine, 6Division of Infectious Diseases, Department of Paediatrics, 7Child Health Evaluation Services, the Hospital for Sick Children, Toronto, Ontario, Canada A Pharmacokinetic Validation Study of Once Daily Dosing of Intravenous Gentamicin and Tobramycin in Paediatric Patients

A fully automated, high-throughput method was developed to profile the fatty acids of phospholipids from human plasma samples for application to a large epidemiological sample set (n > 25,000). We report here on the data obtained for the quality-control materials used with the first 860 batches, and the validation process used. The method consists of two robotic systems combined with gas chromatography, performing lipid extraction, phospholipid isolation, hydrolysis and derivatization to fatty-acid methyl esters, and on-line analysis. This is the first report showing that fatty-acid profiling is an achievable strategy for metabolic phenotyping in very large epidemiological and genetic studies.