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Cyanobacterial blooms in freshwater bodies in England are currently monitored reactively, with samples containing more than 20,000 cells/mL of potentially toxin-producing species by light microscopy resulting in action by the water body owner. Whilst significantly reducing the risk of microcystin exposure, there is little data describing the levels of these toxins present in cyanobacterial blooms. This study focused on the quantitative LC-MS/MS analysis of microcystins in freshwater samples, collected across England during 2016 and found to contain potentially toxin-producing cyanobacteria. More than 50% of samples contained quantifiable concentrations of microcystins, with approximately 13% exceeding the WHO medium health threshold of 20 μg/L. Toxic samples were confirmed over a nine-month period, with a clear increase in toxins during late summer, but with no apparent geographical patterns. No statistical relationships were found between total toxin concentrations and environmental parameters. Complex toxin profiles were determined and profile clusters were unrelated to cyanobacterial species, although a dominance of MC-RR was determined in water samples from sites associated with lower rainfall. 100% of samples with toxins above the 20 μg/L limit contained cell densities above 20,000 cells/mL or cyanobacterial scum, showing the current regime is suitable for public health. Conversely, with only 18% of cell density threshold samples having total microcystins above 20 μg/L, there is the potential for reactive water closures to unnecessarily impact upon the socio-economics of the local population. In the future, routine analysis of bloom samples by LC-MS/MS would provide a beneficial confirmatory approach to the current microscopic assessment, aiding both public health and the needs of water users and industry.

Harmful algal biotoxins in the marine environment are a threat to human food safety due to their bioaccumulation in bivalve shellfish. Whilst official control monitoring provides ongoing risk management for regulated toxins in live bivalve molluscs, no routine monitoring system is currently in operation in the UK for other non-regulated toxins. To assess the potential presence of such compounds, a systematic screen of bivalve shellfish was conducted throughout Great Britain. A rapid dispersive methanolic extraction was used with UHPLC-MS/MS analysis to test for fifteen cyclic imines and seven brevetoxins in 2671 shellfish samples taken from designated shellfish harvesting areas around Great Britain during 2018. Out of the 22 toxins incorporated into the method, only pinnatoxin G, 13-desmethyl spirolide C and 20-methyl spirolide G were detected, with maximum concentrations of 85.4 µg/kg, 13.4 µg/kg and 51.4 µg/kg, respectively. A follow up study of pinnatoxin G-positive samples examined its potential esterification to fatty acids and concluded that following hydrolysis, pinnatoxin G concentration increased by an average of 8.6%, with the tentative identification of these esters determined by LC-HRMS. This study highlights the requirement for ongoing monitoring of emerging threats and the requirement for toxicological and risk assessment studies.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis) common mussels (Mytilus edulis) and hard clams (Mercenaria mercenaria), but not found in cockles (Cerastoderma edule), razors (Ensis species) or scallops (Pecten maximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrum cordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Laboratory experimental models are essential for advancements in thyroid cancer translational research. In this study, we discuss the development and characterization of six cell-based models of BRAF[sup.V600E] -driven papillary thyroid cancer that closely mimic the heterogeneous pathological progression of the disease seen in patients despite sharing a common driver mutation. We anticipate that these newly developed models will allow for the discovery of novel mechanisms that determine differences in disease progression among BRAF[sup.V600E] -driven thyroid cancers and facilitate the testing of therapeutic interventions. The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAF[sup.V600E] -mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAF[sup.V600E] -driven papillary thyroid cancer (PTC) cell lines established from a BrafV600E[sup.+/−] /Pten[sup.+/−] /TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAF[sup.V600E] -driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAF[sup.V600E] -driven PTC.

Most of the shellfish fisheries of Mexico occur in the Gulf of California. In this region, known for its high primary productivity, blooms of diatoms and dinoflagellates are common, occurring mainly during upwelling events. Dinoflagellates that produce lipophilic toxins are present, where some outbreaks related to okadaic acid and dinophisystoxins have been recorded. From January 2015 to November 2017 samples of three species of wild bivalve mollusks were collected monthly in five sites in the southern region of Bahía de La Paz. Pooled tissue extracts were analyzed using LC-MS/MS to detect lipophilic toxins. Eighteen analogs of seven toxin groups, including cyclic imines were identified, fortunately individual toxins did not exceed regulatory levels and also the total toxin concentration for each bivalve species was lower than the maximum permitted level for human consumption. Interspecific differences in toxin number and concentration were observed in three species of bivalves even when the samples were collected at the same site. Okadaic acid was detected in low concentrations, while yessotoxins and gymnodimines had the highest concentrations in bivalve tissues. Although in low quantities, the presence of cyclic imines and other lipophilic toxins in bivalves from the southern Gulf of California was constant.

The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a BrafV600E+/−/Pten+/−/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal. We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy.

Thyroid cancer is the most prevalent endocrine malignancy and is estimated to have affected 52,890 individuals in the United States in the year 2020. Papillary thyroid cancer (PTC) accounts for up to 80% of all thyroid cancer diagnoses. Within PTC, BRAFV600E is the most common mutation in adults and the second most common mutation in pediatrics. While there are several validated human thyroid cancer cell lines harboring BRAF mutations, no pediatric derived cell line has been established to date, limiting generalizations in pediatric disease. Patient-derived pre-clinical models are valuable tools but are limited due to their need to be studied in vitro or in an immunocompromised host. However, in vivo mouse models recapitulate the complex interactions between tumor cells, the immune system, and components of the microenvironment, allowing more comprehensive investigation of thyroid oncogenesis and response to therapy. In this study we describe a new subcutaneous mouse model that can be used to understand differences between pediatric onset and adult onset PTC. We have developed multiple independent, congenic murine cell lines from different stages of thyroid cancer progression. Two independent BRAFV600E-driven cell lines harboring the same genetic mutations were utilized in this study, one representing a well-differentiated tumor (WD) and one representing a poorly-differentiated tumor (PD). Despite coming from different stages of disease, both cell lines showed similar expression of the thyroid specific genes: Pax8, Ttf1, Tg, and Slc5a5, but expression of Pax8, Tg, and Slc5a5 was lower compared to WT thyrocytes. Activation of the Pi3Kinase pathway and the Akt Pathway were assessed via western blot analysis. The WD cell line had increased pERK activation compared to the PD cell line, and the PD cell line had increased pAKT compared to WD cell line. Both cell lines were injected subcutaneously into the hind flank of Wt SJV129 mice of pediatric age (4-5 weeks) and adult age (20-22 weeks). These animals were monitored for 10 weeks post injection. While the PD cell line developed tumors at approximately the same rate and penetrance in both age groups, tumors reached end point more rapidly in the adult cohort. Adult hosts injected with the WD cell line developed tumors more rapidly than in pediatric hosts, however, tumors receded in both cohorts. While adult and pediatric thyroid cancer share common driving mutations, there are distinct differences in disease pathogenesis between these patient populations. There are known differences in the immune system, metabolism, and other variables between pediatrics and adults. However, it is unknown what causes the difference in PTC pathogenesis between pediatric versus adult patients. We believe these new models provide a great opportunity to evaluate the role of age in PTC development and progression. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Introduction: Fibrolamellar carcinoma (FLC) displays upregulation of several oncogenes, including HER2, and multiple immune-suppressive mechanisms. We investigated the efficacy and safety of the pan-HER tyrosine kinase inhibitor neratinib as monotherapy (SUMMIT phase 2 basket study) or with immune checkpoint and/or mammalian target of rapamycin (mTOR) inhibitors (compassionate-use program) in patients with FLC. Methods: Patients received neratinib 240 mg/day orally in SUMMIT, or as doublet or triplet combinations with pembrolizumab 2 mg/kg intravenously every 3 weeks, nivolumab 240 mg intravenously every 2 weeks, everolimus 7.5 mg/day orally, or sunitinib 37.5 mg/day orally under compassionate use. The primary endpoint in SUMMIT was objective response rate; safety was a secondary endpoint. Results: Fifteen patients with FLC received neratinib monotherapy in SUMMIT. The objective response rate was 5% (95% confidence interval [CI]: 0–21.8) and the disease control rate was 13.3% (95% CI: 1.7–40.5). Upon progression, five had added immune checkpoint inhibitors with or without everolimus or sunitinib. Two additional patients received neratinib-based combinations outside of SUMMIT, for a total of 17 neratinib-treated patients. One patient who received neratinib plus pembrolizumab had a confirmed partial response, one treated with neratinib plus everolimus had stable disease lasting 6 months, and one who received neratinib plus pembrolizumab plus sunitinib had stable disease lasting 16 months. Grade 3/4 adverse events with neratinib monotherapy occurred in 10 (66.7%)/2 (13.3%) patients, respectively. Grade 3 adverse events with neratinib-based combinations were hyperglycemia (n = 1; neratinib plus pembrolizumab), hepatic failure, and anaphylaxis (n = 1 each, neratinib plus pembrolizumab plus everolimus). There were no grade 4 adverse events with combination therapy. Conclusion: In patients with FLC, single-agent neratinib had limited efficacy, but clinical benefit was observed with neratinib in combination with immunotherapy and/or mTOR-targeted agents.

The insulin-like growth factor 1 receptor (IGF-1R) has been considered an important therapeutic target in Ewing sarcoma (ES), generating a need to identify the subset of patients most likely to respond to IGF-1R inhibitors. We assessed IGF-1R expression in ES cell lines and patient tumors to understand the variable clinical responses to anti-IGF-1R therapy. Using ligand-binding displacement, we measured between 13,000 and 40,000 receptors per cell in ES cell lines. We used ELISA to quantify IGF-1R in patient tumors, which expressed 4.8% ± 3.7 to 20.0% ± 0.2 of the levels in a positive control cell line overexpressing IGF-1R. Flow cytometry showed markedly reduced IGF-1R expression in ES cell lines compared to a standard positive control cell line. The IGF1R gene was sequenced in 47 ES tumor samples and 8 ES cell lines; only one tumor sample showed a nonsynonymous mutation, R1353H, in a region with low functional impact. Finally, we assessed IGF-1R pathway activity in the ES stem cell (ESSC) population, to characterize its potential for resistance to anti-IGF-1R therapy, using Luminex technology. We found no significant differences in IGF-1R pathway activity between ESSCs and the total cell population. Overall, our findings suggest that IGF-1R as a therapeutic target in this sarcoma may require reevaluation.