A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood.

INTRODUCTION: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. METHODS: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. RESULTS: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. CONCLUSIONS: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment.

NK cells are central to anti-tumor immunity and recently showed efficacy for treating hematologic malignancies. However, their dysfunction in the hostile tumor microenvironment remains a pivotal barrier for cancer immunotherapies against solid tumors. Using cancer patient samples and proteomics, we found that human NK cell dysfunction in the tumor microenvironment is due to suppression of glucose metabolism via lipid peroxidation-associated oxidative stress. Activation of the Nrf2 antioxidant pathway restored NK cell metabolism and function and resulted in greater anti-tumor activity in vivo. Strikingly, expanded NK cells reprogrammed with complete metabolic substrate flexibility not only sustained metabolic fitness but paradoxically augmented their tumor killing in the tumor microenvironment and in response to nutrient deprivation. Our results uncover that metabolic flexibility enables a cytotoxic immune cell to exploit the metabolic hostility of tumors for their advantage, addressing a critical hurdle for cancer immunotherapy.

Co-creating local socioeconomic pathways for achieving the sustainable development goals.

The Sustainable Development Goals (SDGs) recognise the importance of action across all scales to achieve a sustainable future. To contribute to overall national- and global-scale SDG achievement, local communities need to focus on a locally-relevant subset of goals and understand potential future pathways for key drivers which influence local sustainability. We developed a participatory method to co-create local socioeconomic pathways by downscaling the SDGs and driving forces of the shared socioeconomic pathways (SSPs) via a local case study in southern Australia through contextual analysis and community engagement. We linked the SSPs and SDGs by identifying driving forces and describing how they affect the achievement of local SDGs. We co-created six local socioeconomic pathways with the local community which track towards futures with different levels of fulfilment of the SDGs and each encompasses a narrative storyline incorporating locally-specific ideas from the community. We tested and validated the local pathways with the community. This method extends the SSPs in two dimensions-into the broader field of sustainability via the SDGs, and by recontextualizing them at the local scale. The local socioeconomic pathways can contribute to achieving local sustainability goals from the bottom up in alignment with global initiatives.

The Frequency and Context of Snacking among Children: An Objective Analysis Using Wearable Cameras.

Snacking is a common eating behaviour, but there is little objective data about children's snacking. We aimed to determine the frequency and context of children's snacking (n = 158; mean age = 12.6 years) by ethnicity, gender, socioeconomic deprivation and body mass index (BMI) children. Participants wore wearable cameras that passively captured images of their surroundings every seven seconds. Images (n = 739,162) were coded for snacking episodes, defined as eating occasions in between main meals. Contextual factors analysed included: snacking location, food source, timing, social contact and screen use. Rates of total, discretionary (not recommended for consumption) and healthful (recommended for consumption) snacking were calculated using negative binomial regression. On average, children consumed 8.2 (95%CI 7.4, 9.1) snacks per day, of which 5.2 (95%CI 4.6, 5.9) were discretionary foods/beverages. Children consumed more discretionary snacks than healthful snacks in each setting and at all times, including 15.0× more discretionary snacks in public spaces and 2.4× more discretionary snacks in schools. Most snacks (68.9%) were sourced from home. Girls consumed more total, discretionary and healthful snacks than boys, and Maori and Pacific consumed fewer healthful snacks than New Zealand (NZ) Europeans. Results show that children snack frequently, and that most snacking involves discretionary food items. Our findings suggest targeting home buying behaviour and environmental changes to support healthy snacking choices.

Global trends in ultraprocessed food and drink product sales and their association with adult body mass index trajectories.

This study evaluated global trends in ultraprocessed food and drink (UPFD) volume sales/capita and associations with adult body mass index (BMI) trajectories. Total food/drink volume sales/capita from Euromonitor for 80 countries (2002-2016) were matched to mean adult BMI from the NCD Risk Factor Collaboration (2002-2014). Products were classified as UPFD/non-UPFD according to the NOVA classification system. Mixed models for repeated measures were used to analyse associations between UPFD volume sales/capita and adult BMI trajectories, controlling for confounding factors. The increase in UPF volume sales was highest for South and Southeast Asia (67.3%) and North Africa and the Middle East (57.6%), while for UPD, the increase was highest for South and Southeast Asia (120.0%) and Africa (70.7%). In 2016, baked goods were the biggest contributor to UPF volume sales (13.1%-44.5%), while carbonated drinks were the biggest contributor to UPD volume sales (40.2%-86.0%). For every standard deviation increase (51 kg/capita, 2002) in UPD volume sales, mean BMI increased by 0.195 kg/m2 for men (P < .001) and 0.072 kg/m2 for women (P = .003). For every standard deviation (40 kg/capita, 2002) increase in UPF volume sales, mean BMI increased by 0.316 kg/m2 for men (P < .001), while the association was not significant for women. Increases in UPFD volume sales/capita were positively associated with population-level BMI trajectories.

Expanded CD56superbrightCD16+ NK Cells from Ovarian Cancer Patients Are Cytotoxic against Autologous Tumor in a Patient-Derived Xenograft Murine Model.

Natural killer (NK) cells are useful for cancer immunotherapy and have proven clinically effective against hematologic malignancies. However, immunotherapies for poor prognosis solid malignancies, including ovarian cancer, have not been as successful due to immunosuppression by solid tumors. Although rearming patients' own NK cells to treat cancer is an attractive option, success of that strategy is limited by the impaired function of NK cells from cancer patients and by inhibition by self-MHC. In this study, we show that expansion converts healthy donor and immunosuppressed ovarian cancer patient NK cells to a cytotoxic CD56superbrightCD16+ subset with activation state and antitumor functions that increase with CD56 brightness. We investigated whether these expanded NK cells may overcome the limitations of autologous NK cell therapy against solid tumors. Peripheral blood- and ascites-derived NK cells from ovarian cancer patients were expanded and then adoptively transferred into cell-line and autologous patient-derived xenograft models of human ovarian cancer. Expanded ovarian cancer patient NK cells reduced the burden of established tumors and prolonged survival. These results suggest that CD56bright NK cells harbor superior antitumor function compared with CD56dim cells. Thus, NK cell expansion may overcome limitations on autologous NK cell therapy by converting the patient's NK cells to a cytotoxic subset that exerts a therapeutic effect against autologous tumor. These findings suggest that the value of expanded autologous NK cell therapy for ovarian cancer and other solid malignancies should be clinically assessed. Cancer Immunol Res; 6(10); 1174-85. ©2018 AACR.

Ex vivo-expanded NK cells from blood and ascites of ovarian cancer patients are cytotoxic against autologous primary ovarian cancer cells.

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.

Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3ß.

In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 µg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3ß on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of ß-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3ß (Ad-GSK-3ß S9A) resulted in a >2-fold increase in GSK-3ß activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3ß activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

Oxidized low-density lipoprotein promotes osteoblast differentiation in primary cultures of vascular smooth muscle cells by up-regulating Osterix expression in an Msx2-dependent manner.

We have previously shown that oxidized low-density lipoproteins (oxLDLs) act synergistically with ß-glycerophosphate to induce the osteogenic differentiation of primary bovine aortic smooth muscle cells (BASMCs). In the present study, we attempt to resolve the mechanism responsible for this effect by examining the expression of several osteoblast-specific transcription factors. Thus, by culturing BASMCs in the absence or presence of ß-glycerophosphate and/or oxLDL, we demonstrate that ß-glycerophosphate induces both Runx2 and Osterix (Osx) expression. In contrast, oxLDL has no effect on Runx2 expression but rather it enhances ß-glycerophosphate-induced osteoblast differentiation by further up-regulating Osx expression. In an attempt to elucidate the mechanism responsible for this latter effect, we examined the ability of oxLDL to affect Msh homeobox 2 (Msx2) expression. Similar to its effect on Osx expression, oxLDL was found to synergistically enhance ß-glycerophosphate-induced Msx2 expression in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2)-dependent manner. Furthermore, oxLDL's ability to enhance both ß-glycerophosphate-induced Osx expression and alkaline phosphatase activity was prevented when the BASMCs were first transfected with Msx2-specific siRNA. Taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteoblast differentiation and vascular calcification in vivo.

Effect of leptin on vascular calcification in apolipoprotein E-deficient mice.

OBJECTIVE: The adipocytokine leptin has been proposed to increase cardiovascular risk in both obese and diabetic individuals. In the current study, therefore, we used apoE-deficient mice to examine the effects of leptin on both lesion size and calcification. METHODS AND RESULTS: Mice were treated with once daily intraperitoneal injections of leptin (125 microg/mouse/d) for 2 months. The mice were then euthanized, and sections of the aortic root and thoracic aorta analyzed histomorphometrically. Measurements of lesion size and surface area occupied by atherosclerotic lesions did not reveal any differences between nontreated and leptin-treated animals. However, von Kossa staining of the aortic root demonstrated an 8.3+/-2.0-fold increase in lesion calcification as well as a 2.5+/-0.6-fold increase in valvular calcification in those animals treated with leptin. In addition, the percent total lesion area demonstrating ALP-positive staining was 5.4+/-2.1-fold greater in leptin-treated mice when compared to nontreated control mice. This increase in ALP staining was also accompanied by an increase in the expression of the osteoblast-specific markers, osteocalcin, and osteopontin. CONCLUSIONS: Based on these observations, we conclude that leptin may increase cardiovascular risk by promoting osteogenic differentiation and thus vascular calcification.

Inhibition of osteolytic bone metastasis by unfractionated heparin.

In the current study, we examine heparin's anti-metastatic properties by using a well-defined mouse model of osteolytic bone metastasis. C57BL/6 mice were treated with increasing doses of unfractionated heparin (15, 20, or 25 units/mouse) 30 min prior to the left ventricular injection of GFP-transfected B16F10 melanoma cells. Heparin's effect on tumour burden and bone strength was then quantified 14 days later by bone histomorphometry and biomechanical testing, respectively. Based on histomorphometric analysis of the femurs, injection of GFP-transfected melanoma cells resulted in a 37% decrease in cancellous bone volume and a 68% increase in osteoclast surface. This was associated with a 13% reduction in bone strength as measured by biomechanical testing. However, when the mice were first pre-treated with 25 units of heparin, tumour burden was decreased by 73% and tumour cell-dependent decreases in both cancellous bone volume and bone strength were prevented. Based on these observations, we conclude that heparin inhibits the ability of tumour cells to metastasize to bone and that as such, prevents tumour cell-induced decreases in bone strength.

Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells.

Previous studies have localized osteoblast specific markers to sites of calcified atherosclerotic lesions. We therefore decided to use an established in vitro model of vascular calcification in order to confirm earlier reports of oxidized low-density lipoprotein (oxLDL) promoting the osteogenic differentiation of vascular smooth muscle cells. Treatment of primary bovine aortic smooth muscle cells (BASMCs) with beta-glycerophosphate was found to induce a time-dependent increase in osteoblast differentiation. In contrast, no effect was seen when BASMCs were cultured in the presence of oxLDL alone. However, when the BASMCs were cultured in the presence of both beta-glycerophosphate and oxLDL, beta-glycerophosphate's ability to induce osteoblast differentiation was significantly enhanced. In an attempt to resolve the mechanism by which this effect was occurring, we examined the effect of beta-glycerophosphate and oxLDL on several pathways known to be critical to the differentiation of osteoblasts. Surprisingly, beta-glycerophosphate alone was found to enhance Osterix (Osx) expression by inducing both Smad 1/5/8 activation and Runx2 expression. In contrast, oxLDL did not affect either Smad 1/5/8 activation or Runx2 activation but rather, it enhanced both beta-glycerophosphate-induced Osx expression and osteoblast differentiation in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2) -dependent manner. When taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteogenic differentiation and vascular calcification in vivo. J. Cell. Biochem. 105: 185-193, 2008. (c) 2008 Wiley-Liss, Inc.

The effects of heparin and low molecular weight heparins on bone.

Recent clinical trials have shown that the risk of developing osteoporosis is substantially lower when low molecular weight heparins (LMWHs) are used in place of unfractionated heparin. While the reason(s) for this difference has not been fully elucidated, studies with animals have suggested that heparin causes bone loss by both decreasing bone formation and increasing bone resorption. In contrast, LMWHs appear to cause less bone loss because they only decrease bone formation. Whether all LMWHs decrease bone formation and therefore cause bone loss is unknown. For example, preliminary in vitro studies with the synthetic pentasaccaride, Fondaparinux, have suggested that it may not decrease bone formation and thus, may have no deleterious effects on bone. Further studies are required in order to determine if all LMWHs cause bone loss equally.

Heparin synergistically enhances interleukin-11 signaling through up-regulation of the MAPK pathway.

Using an animal model of heparin-induced osteoporosis we previously demonstrated that heparin causes bone loss, in part, by increasing osteoclast number and activity. Furthermore, we found that, although heparin alone has no effect, it is able to synergistically enhance Interleukin-11 (IL-11)-induced signal transducer and activator of transcription 3 (STAT3) activation and thus increase osteoclast formation in vitro. In the present study, we examine the effect of various serine kinase inhibitors on the ability of heparin to act synergistically with IL-11. Inhibition of the c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or the phosphatidylinositol 3-kinase pathways had no effect on the ability of heparin to promote either IL-11-induced STAT3.DNA complex formation or osteoclast formation in vitro. In contrast, PD098059, a MAPK kinase inhibitor, completely abolished the synergy between heparin and IL-11. In an attempt to resolve the mechanism by which this was occurring, we examined the effect of heparin on STAT3 Ser-727 phosphorylation and extracellular signal-regulated kinases 1 and 2 (Erk1/2) activation, either in the presence or absence of IL-11. Heparin alone was found to have no effect on Ser-727 phosphorylation, nor did heparin alter the phosphorylation status of Ser-727 in the presence of IL-11. Heparin was, however, found to increase Erk1/2 activation in both a time- and dose-dependent manner. When taken together, these findings suggest that heparin enhances IL-11-induced STAT3 activation and thus osteoclast formation, by a mechanism that is independent of STAT3 Ser-727 phosphorylation but that involves up-regulation of the MAPK pathway.

The effect of heparin on osteoblast differentiation and activity in primary cultures of bovine aortic smooth muscle cells.

Recent studies have suggested that aortic smooth muscle cells undergo a phenotypic transition into osteoblast-like cells and mineralize when cultured in the presence of beta-glycerophosphate. Since we had previously demonstrated that heparin could inhibit osteoblast differentiation and mineralization in primary cultures of murine calvaria cells, we were interested in determining if heparin would have a similar effect when primary aortic smooth muscle cells were cultured in the presence of beta-glycerophosphate. The effect of heparin and low molecular weight heparin (LMWH) on osteoblast differentiation and activity was therefore examined in primary cultures of bovine aortic smooth muscle cells (BASMC) over a 14-day period. Here, we report that BASMC differentiate into osteoblast-like cells when cultured in the presence of beta-glycerophosphate. Moreover, we report that heparin not only inhibits this process but that it also inhibits the ability of BASMC to mineralize as well. Importantly, these effects were found not to be dependent upon heparins' anticoagulant activity since unfractionated heparin and heparins with low anti-thrombin III affinities inhibited the mineralization process equally well. Sulfation, however, was found to be a major determinant of heparins ability to inhibit BASMC mineralization since neither dermatan sulfate nor N-desulfated heparin were able to demonstrate an effect. We conclude that BASMC cultures can undergo a phenotypic transition into mature osteoblasts and that both the differentiation process and their ability to mineralize are inhibited by heparin.

Differential effects of heparin and low molecular weight heparin on osteoblastogenesis and adipogenesis in vitro.

We have previously demonstrated that heparin produces cancellous bone loss in rats due in part to a decrease in the number of osteoblasts lining the trabecular bone surface. In the present study, we use a stromal-derived cell culture system together with measurements of alkaline phosphatase (ALP) activity, to compare the effects of heparin and the low molecular weight heparin (LMWH), Fragmin, on osteoblast differentiation in vitro. In addition, we examined the possibility that both heparin and LMWH can induce adipogenesis in our stromal cell culture system. Both heparin and LMWH were found to produce a statistically significant (P < 0.01) and concentration-dependent decrease in the number of osteoblasts while increasing the number of adipocytes. When the effects of gravimetrically equivalent amounts of heparin and LMWH were compared, heparin had a 4-fold greater effect than LMWH. In contrast to heparin, N-desulfated heparin was found to have minimal effects on both osteoblast and adipocyte differentiation indicating that the heparin effect is not only chain-length dependent but also charge-dependent. The observation that LMWH has less of an effect on bone formation than heparin is compatible with the results of clinical trials indicating that LMWH produces less bone loss after long-term administration.

Neutralization of interleukin-11 activity decreases osteoclast formation and increases cancellous bone volume in ovariectomized mice.

The issue of whether interleukin-11 (IL-11) contributes to bone loss during states of estrogen deficiency has not been previously determined. We therefore randomized ovariectomized (OVX) mice to once daily interperitoneal injections of either sheep anti-murine IL-11 Ab or normal sheep IgG (NSIgG) for 21 days, and then determined the effects on bone using bone histomorphometry. Here we report that treatment of OVX mice with anti-IL-11 Ab significantly increases both trabecular width and cancellous bone volume. Osteoblast activity, as measured by the percentage of trabecular surface covered by osteoid and rates of bone formation, were also significantly increased following treatment with anti-IL-11 Ab. In contrast, treatment of OVX mice with anti-IL-11 Ab significantly decreased both osteoclast number and activity. Ex-vivo assays of osteoclast formation and activity confirmed the histomorphometric data. Thus, bone marrow cells isolated from anti-IL-11 Ab treated OVX mice formed fewer osteoclasts and resorbed less bone in culture than did marrow cells isolated from either untreated or NSIgG-treated OVX mice. Based on these results we conclude that IL-11 contributes to the bone loss which is observed during states of estrogen deficiency.