Lipophilic Toxins in Wild Bivalves from the Southern Gulf of California, Mexico.

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.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

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 (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). 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 Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Performance Characteristics of AOAC Method 2005.06 for the Determination of Paralytic Shellfish Toxins in Manila Clams, European Otter Clams, Grooved Carpet Shell Clams, Surf Clams, and Processed King Scallops.

An approach was developed for the verification of method performance of the AOAC 2005.06 LC-fluorescence detector (FLD) method for determination of paralytic shellfish poisoning (PSP) toxins in bivalve shellfish. This was developed following advice published by the Analytical Laboratory Accreditation Criteria Committee and applied to shellfish species that had not been previously subjected to a full single-laboratory validation scheme. The refined approach was developed following the need to assess performance in a number of shellfish species infrequently monitored through the UK statutory monitoring program, while reducing the impact and cost of the studies, most notably in terms of the use of valuable reference standards. The species assessed were manila clams (Ruditapes philippinarum), European otter clams (Lutraria lutraria), grooved carpet shell clams (R. decussatus), surf clams (Spisula solida), and king scallops (Pecten maximus) presented as adductor only or adductor plus roe. The method was assessed for sensitivity in terms of LOD and LOQ, toxin recovery, and method precision in each species. It incorporated the PSP toxins deemed toxic and/or prevalent in UK samples and commercially available as certified reference standards. The toxins studied included GTX1-5, dcSTX, STX, C1&2, and NEO. The toxins dcGTX2&3 were included for surf clams due to the prevalence of these toxins in this species as a result of toxin decarbamoylation. Method performance targets were met for each of the characteristics investigated. Consequently, the method was deemed fit for purpose for the screening and quantification of these clam and scallop species for PSP toxins by AOAC Method 2005.06 LC-FLD.

Development of Novel Murine BRAFV600E-Driven Papillary Thyroid Cancer Cell Lines for Modeling of Disease Progression and Preclinical Evaluation of Therapeutics.

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.

The clinical aspect of NTRK-fusions in pediatric papillary thyroid cancer.

Although adult and pediatric papillary thyroid cancer (PTC) share similar oncogenic drivers, they differ in the pathological features and outcomes of the disease. In adults with PTC, the most frequent genetic alterations are mutually exclusive point mutations in BRAFV600E or the RAS family with BRAFV600E commonly associated with invasive disease and decreased response to radioiodine therapy. In pediatric PTC, fusion oncogenes involving chromosomal translocations in tyrosine kinase (TK) receptors, most commonly RET and NTRK, are often found in patients with lateral neck and distant metastases. This brief report reviews clinical data from a single-institute's cohort of NTRK-driven pediatric PTC cases with an updated review of the literature and comparison to adult NTRK-driven PTC.

NTRK-fusions in pediatric thyroid tumors: Current state and future perspectives.

Pediatric and adult papillary thyroid cancer (PTC) share many similar oncogenic drivers, but differ in the pathological features and outcomes of the disease. The most frequent genetic alterations in adult PTCs are mutually exclusive point mutations in BRAF or the RAS family. In pediatric PTC, fusion oncogenes involving chromosomal translocations in tyrosine kinase (TK) receptors, most commonly RET and NTRK, are the most common genetic alterations observed. This review of the literature describes the current state of translational research in pediatric NTRK-driven thyroid cancer and highlights opportunities to improve our understanding and current models of pediatric PTC.

Method performance verification for the combined detection and quantitation of the marine neurotoxins cyclic imines and brevetoxin shellfish metabolites in mussels (Mytilus edulis) and oysters (Crassostrea gigas) by UHPLC-MS/MS.

A single laboratory method performance verification is reported for a rapid sensitive UHPLC-MS/MS method for the quantification of eight cyclic imine and two brevetoxin analogues in two bivalve shellfish matrices: mussel (Mytilus edulis) and Pacific oyster (Crassostrea gigas). Targeted cyclic imine analogues were from the spirolide, gymnodimine and pinnatoxin groups, namely 20-Me-SPX-C, 13-desMe-SPX-C, 13,19-didesMe-SPX-C, GYM-A, 12-Me-GYM, PnTx-E, PnTx-F and PnTx-G. Brevetoxin analogues consisted of the shellfish metabolites BTX-B5 and S-desoxy-BTX-B2. A rapid dispersive extraction was used as well as a fast six-minute UHPLC-MS/MS analysis. Mobile phase prepared using ammonium fluoride and methanol was optimised for both chromatographic separation and MS/MS response to suit all analytes. Method performance verification checks for both matrices were carried out. Matrix influence was acceptable for the majority of analogues with the MS response for all analogues being linear across an appropriate range of concentrations. In terms of limits of detection and quantitation the method was shown to be highly sensitive when compared with other methods. Acceptable recoveries were found with most analogues, with laboratory precision in terms of intra- and inter-batch precision deemed appropriate. The method was applied to environmental shellfish samples with results showing low concentrations of cyclic imines to be present. The method is fast and highly sensitive for the detection and quantification of all targeted analogues, in both mussel and oyster matrices. Consequently, the method has been shown to provide a useful tool for simultaneous monitoring for the presence or future emergence of these two toxin groups in shellfish.

Variability of Amnesic Shellfish Toxin and Pseudo-nitzschia occurrence in bivalve molluscs and water samples-Analysis of ten years of the official control monitoring programme.

As the official control laboratory for marine biotoxins within Great Britain, the Centre for Environment, Fisheries and Aquaculture Science, in conjunction with the Scottish Association for Marine Science, has amassed a decade's worth of data regarding the prevalence of the toxins associated with Amnesic Shellfish Poisoning within British waters. This monitoring involves quantitative HPLC-UV analysis of shellfish domoic acid concentration, the causative toxin for Amnesic Shellfish Poisoning, and water monitoring for Pseudo-nitzschia spp., the phytoplankton genus that produces domoic acid. The data obtained since 2008 indicate that whilst the occurrence of domoic acid in shellfish was generally below the maximum permitted limit of 20 mg/kg, there were a number of toxic episodes that breached this limit. The data showed an increase in the frequency of both domoic acid occurrence and toxic events, although there was considerable annual variability in intensity and geographical location of toxic episodes. A particularly notable increase in domoic acid occurrence in England was observed during 2014. Comparison of Scottish toxin data and Pseudo-nitzschia cell densities during this ten-year period revealed a complex relationship between the two measurements. Whilst the majority of events were associated with blooms, absolute cell densities of Pseudo-nitzschia did not correlate with domoic acid concentrations in shellfish tissue. This is believed to be partly due to the presence of a number of different Pseudo-nitzschia species in the water that can exhibit variable toxin production. These data highlight the requirement for tissue monitoring as part of an effective monitoring programme to protect the consumer, as well as the benefit of more detailed taxonomic discrimination of the Pseudo-nitzschia genus to allow greater accuracy in the prediction of shellfish toxicity.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: azaspiracids and yessotoxins.

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC-MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton. On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams. The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45-OH homo-YTX were detected throughout the monitored period. 45-OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45-OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs. This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years.

Analysis of Microcystins in Cyanobacterial Blooms from Freshwater Bodies in England.

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.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: Okadaic acid, dinophysis toxins and pectenotoxins.

Official control biotoxin testing of bivalve molluscs from Great Britain has been conducted by Cefas for over a decade. Reflecting the changes in legislation, bioassays were gradually replaced by analytical methods, firstly for analysis of Paralytic shellfish toxins, followed by introduction of liquid chromatography tandem mass spectrometric (LCMS/MS) method for lipophilic toxins (LTs) in 2011. Twelve compounds, representing three main groups of regulated lipophilic toxins, as well as two non-regulated cyclic imines were examined in over 20,500 samples collected between July 2011 and December 2016. The toxins belonging to Okadaic acid (OA) group toxins were the most prevalent and were quantified in 23% of samples, predominantly from Scotland. The temporal pattern of OA group occurrences remained similar each year, peaking in summer months and tailing off during autumn and winter, however their abundance and magnitude varied between years significantly, with concentrations reaching up to 4993 µg OA eq./kg. Three toxin profiles were identified, reflecting the relative contribution of the two main toxins, OA and dinophysis toxin-2 (DTX2). Dinophysis toxin-1 (DTX1) was less common and was never detected in samples with high proportions of DTX2. Inter-annual changes in profiles were observed within certain regions, with the most notable being an increase of DTX2 occurrences in north-west Scotland and England in the last three years of monitoring. In addition, seasonal changes of profiles were identified when OA, the dominant toxin in early summer, was replaced by higher proportions of DTX2 in late summer and autumn. The profile distribution possibly reflected the availability of individual Dinophysis species as a food source for shellfish, however persistence of DTX2 during autumn and winter in mussels might have also been attributed to their physiology. Mussels were the only species with higher average proportions of non-esterified toxins, while Pacific oysters, cockles, surf clams, razors and queen scallops contained almost exclusively ester forms. In addition, a temporal change in proportion of OA and DTX2 free form was observed in mussels. Pectenotoxin-2 (PTX2) was quantified only on rare occasions.

Phase II study of metronomic chemotherapy for recurrent malignant gliomas in adults.

Preclinical evidence suggests that continuous low-dose daily (metronomic) chemotherapy may inhibit tumor endothelial cell proliferation (angiogenesis) and prevent tumor growth. This phase II study evaluated the feasibility of this antiangiogenic chemotherapy regimen in adults with recurrent malignant gliomas. The regimen consisted of low-dose etoposide (35 mg/m2 [maximum, 100 mg/day] daily for 21 days), alternating every 21 days with cyclophosphamide (2 mg/kg [maximum, 100 mg/day] daily for 21 days), in combination with daily thalidomide and celecoxib, in adult patients with recurrent malignant gliomas. Serum and urine samples were collected for measurement of angiogenic peptides. Forty-eight patients were enrolled (15 female, 33 male). Twenty-eight patients had glioblastoma multiforme (GBMs), and 20 had anaplastic gliomas (AGs). Median age was 53 years (range, 33-74 years), and median KPS was 70 (range, 60-100). Therapy was reasonably well tolerated in this heavily pretreated population. Two percent of patients had partial response, 9% had a minor response, 59% had stable disease, and 30% had progressive disease. For GBM patients, median progression-free survival (PFS) was 11 weeks, six-month PFS (6M-PFS) was 9%, and median overall survival (OS) was 21 weeks. For AG patients, median PFS was 14 weeks, 6M-PFS was 26%, and median OS was 41.5 weeks. In a limited subset of patients, serum and urine angiogenic peptides did not correlate with response or survival (p > 0.05). Although there were some responders, this four-drug, oral metronomic regimen did not significantly improve OS in this heavily pretreated group of patients who were generally not eligible for conventional protocols. While metronomic chemotherapy may not be useful in patients with advanced disease, further studies using metronomic chemotherapy combined with more potent antiangiogenic agents in patients with less advanced disease may be warranted.

Comparison of linear and volumetric criteria in assessing tumor response in adult high-grade gliomas.

The Response Evaluation Criteria in Solid Tumors, or RECIST criteria (one-dimensional [1D] measurement), are widely used to measure response in tumors, but there are few studies evaluating these criteria in brain tumors. We compared linear and volumetric measurements in adult high-grade supratentorial enhancing gliomas to determine the agreement between measurements, in defining responses and in their subsequent relation to survival. We hypothesized that the 1D RECIST criteria maybe suitable for response assessment in adult high-grade gliomas. Tumor size on MRI scans in 104 patients with high-grade enhancing gliomas treated on clinical trial protocols was measured by using 1D (greatest length), 2D (two-dimensional: product of the two longest perpendicular diameters), 3D (three dimensional: product of the longest perpendicular diameters in one plane and the longest orthogonal diameter to that plane), enhancing volume (EV), and total volume (TV). A total of 388 T1 postgadolinium MRI scans (104 baseline and 284 follow-up scans) were evaluated. Volumetric analysis (EV and TV) was performed with commercially available software. Intraobserver and interobserver correlations (rho) were high for all modalities (rho > 0.92 and rho > 0.71, respectively). Correlation was excellent (rho > 0.9) among all modalities except for 3D (rho < 0.6). Patient response rates ranged from 12% to 26%. Median progression-free survival (mPFS) and six-month progression-free survival (6mPFS) were not significantly different among the methods (range, 5.3 months to 5.9 months and 42% to 48%, respectively). Landmark analyses of response at two months using linear methods predicted overall survival with hazard ratios of 0.19 to 0.29 (P < 0.005). These results suggest high concordance among 1D, 2D, TV, and EV, but not 3D, methods in assessing enhancing tumor progression and in estimating mPFS and 6mPFS in adult brain tumor patients. The tumor response at two months assessed by linear methods correlated better with overall survival. Thus, linear methods are comparable to volumetric methods, but simpler to implement for routine clinical use and for designing clinical trials of brain tumors.

Treatment of primary CNS lymphoma with methotrexate and deferred radiotherapy: a report of NABTT 96-07.

PURPOSE: A multicenter, phase II study of single-agent, intravenous methotrexate in newly diagnosed non-AIDS-related primary CNS lymphoma was conducted in the New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium. METHODS: Methotrexate (8 g/m(2)) was initially administered every 2 weeks. The primary end point was radiographic CR or PR, as defined by standard radiographic criteria, and secondary end points were survival and drug-related toxicity. RESULTS: Twenty-five patients were enrolled with a mean age of 60 years and median Karnofsky Performance Score of 80. Three of 14 patients who underwent lumbar puncture had malignant cells on CSF cytopathology, and five of 25 patients had ocular involvement. Two patients could not be evaluated for the primary end point because of the absence of measurable disease in one and death before radiologic imaging in another. All patients have completed the treatment program or progressed. Among 23 patients, there were 12 CR (52%), five PR (22%), one (4%) with stable disease, and five progressions (22%) while on therapy. Seven patients died of tumor progression, and two died of other causes. Median progression-free survival was 12.8 months. Median overall survival for the entire group had not been reached at 22.8+ months. The toxicity of this regimen was modest, with no grade 3 or 4 toxicity in 13 of 25 patients, grade 3 toxicity in eight of 25 patients, and grade 4 toxicity in four of 25 patients after 287 cycles of chemotherapy. CONCLUSION: These results indicate that high-dose methotrexate is associated with modest toxicity and a radiographic response proportion (74%) comparable to more toxic regimens.

Insulin-like growth factor 1 receptor as a therapeutic target in ewing sarcoma: lack of consistent upregulation or recurrent mutation and a review of the clinical trial literature.

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.

Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.

Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

High ALDH activity identifies chemotherapy-resistant Ewing's sarcoma stem cells that retain sensitivity to EWS-FLI1 inhibition.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Randomized phase II study of cilengitide, an integrin-targeting arginine-glycine-aspartic acid peptide, in recurrent glioblastoma multiforme.

PURPOSE: Cilengitide, an inhibitor of alphavbeta3 and alphavbeta5 integrin receptors, demonstrated minimal toxicity and durable activity across a wide range of doses administered to adults with recurrent glioblastoma multiforme (GBM) in a prior phase I study. The current multicenter phase II study was conducted to evaluate the activity and safety of cilengitide in GBM patients at first recurrence. PATIENTS AND METHODS: Eligible patients were randomly assigned to receive either 500 or 2,000 mg of cilengitide twice weekly on a continuous basis. Patients were assessed every 4 weeks. The primary end point was 6-month progression-free survival (PFS) rate. Secondary end points included PFS, overall survival (OS), and radiographic response, as well as quality-of-life and pharmacokinetic assessments. RESULTS: Eighty-one patients were enrolled, including 41 on the 500-mg arm and 40 on the 2,000-mg arm. The safety profile of cilengitide was excellent, with no significant reproducible toxicities observed on either arm. Antitumor activity was observed in both treatment cohorts but trended more favorably among patients treated with 2,000 mg, including a 6-month PFS of 15% and a median OS of 9.9 months. CONCLUSION: Cilengitide monotherapy is well tolerated and exhibits modest antitumor activity among recurrent GBM patients. Additional studies integrating cilengitide into combinatorial regimens for GBM are warranted.