Classification of clear-cell sarcoma as a subtype of melanoma by genomic profiling.

PURPOSE: To develop a genome-based classification scheme for clear-cell sarcoma (CCS), also known as melanoma of soft parts (MSP), which would have implications for diagnosis and treatment. This tumor displays characteristic features of soft tissue sarcoma (STS), including deep soft tissue primary location and a characteristic translocation, t(12;22)(q13;q12), involving EWS and ATF1 genes. CCS/MSP also has typical melanoma features, including immunoreactivity for S100 and HMB45, pigmentation, MITF-M expression, and a propensity for regional lymph node metastases. MATERIALS AND METHODS: RNA samples from 21 cell lines and 60 pathologically confirmed cases of STS, melanoma, and CCS/MSP were examined using the U95A GeneChip (Affymetrix, Santa Clara, CA). Hierarchical cluster analysis, principal component analysis, and support vector machine (SVM) analysis exploited genomic correlations within the data to classify CCS/MSP. RESULTS: Unsupervised analyses demonstrated a clear distinction between STS and melanoma and, furthermore, showed that CCS/MSP cluster with the melanomas as a distinct group. A supervised SVM learning approach further validated this finding and provided a user-independent approach to diagnosis. Genes of interest that discriminate CCS/MSP included those encoding melanocyte differentiation antigens, MITF, SOX10, ERBB3, and FGFR1. CONCLUSION: Gene expression profiles support the classification of CCS/MSP as a distinct genomic subtype of melanoma. Analysis of these gene profiles using the SVM may be an important diagnostic tool. Genomic analysis identified potential targets for the development of therapeutic strategies in the treatment of this disease.     

Pharmacodynamic evaluation of the epidermal growth factor receptor inhibitor OSI-774 in human epidermis of cancer patients.

BACKGROUND: OSI-774 is an inhibitor of the epidermal growth factor receptor tyrosine kinase (EGFR-TK) currently in clinical development. In preclinical models, the antitumor activity of OSI-774 was directly related to its ability to inhibit the EGFR-TK. On the basis of these data, we hypothesized that inhibition of the EGFR-TK will be required for this agent to be effective in the clinic. This study evaluated the pharmacodynamic effects of OSI-774 in normal skin tissues collected from patients treated with the agent in a Phase I study. METHODS: Patients with advanced cancer who were treated in a Phase I study of OSI-774 underwent a biopsy of normal skin epidermis at baseline and after the last dose of drug in the first course of treatment. The expression and activation of the EGFR, downstream signaling extracytoplasmatic-regulated kinase (Erk), and cell cycle regulator p27 were determined in paraffin-embedded skin tissues using an immunohistochemical method (IHC). The IHC data were analyzed using both a semiquantitative scoring system and an automatic absorbance quantitative IHC method. The number of cells with nuclear staining of p27 per 500 cells was determined. Plasma samples were collected to quantitate OSI-774 plasma concentrations. RESULTS: A total of 56 skin specimens was collected from 28 patients treated with OSI-774 at doses ranging from 25 to 200 mg/day. There was a significant decrease in phospho-EGFR (Tyr 1173) expression as determined semiquantitatively with OSI-774 treatment [2.75 +/- 0.51 (mean +/- SD) pretreatment versus 2.36 +/- 0.76 after treatment, pair comparison P = 0.01]. The quantitative ratio [(phopho-EGFR/EGFR) x 100] of phospho-EGFR (Tyr1173) decreased from 64.16 +/- 36.58 pretreatment to 48.87 +/- 35.37 post-treatment (pair comparison, P = 0.02). No significant differences were observed in phospho-Erk (Thr202/Tyr204) expression. The mean number of cells with nuclear staining for p27 increased from 185 +/- 101 (mean +/- SD) pretreatment to 253 +/- 111 post-treatment (pair comparison P = 0.02). A total of 12 (42.8%), 7 (25%), and 14 (50%) patients had >25% variation in the ratio of phospho-EGFR (Tyr1173), phospho-Erk (Thr202/Tyr204), and p27 expression, respectively. Only changes in p27 expression were related to the administered dose of OSI-774. CONCLUSIONS: OSI-774 exerted pharmacodynamic effects in skin tissues of 30-50% of patients treated with the agent. Up-regulation of p27, which is a downstream effect of EGFR inhibition, was dose related. Although there was a significant decrement in phospho-EGFR (Tyr1173), it was not related to the administered dose of OSI-774. On the basis of these findings and the relatively simple and reliable method to measure p27 expression, this biomarker appears to be the most promising and is being evaluated in Phase II studies as a predictor of clinical outcome.     

Enhanced IL-4 but normal interferon-gamma production in children with isolated IgE mediated food hypersensitivity

Atopic disorders such as atopic dermatitis and asthma have been characterised by an imbalance in interferon-gamma (INF-γ) and IL-4. Whether similar imbalances are found in atopic disorders with different clinical manifestations, such as IgE mediated immediate food hypersensitivity, is not clear. We have examined the in vitro production of INF-γ and IL-4 in peripheral blood mononuclear cells (PBMC) following phytohaemagglutinin stimulation from children with isolated immediate IgE mediated food hypersensitivity (egg, milk, "nut"), children with moderate and severe atopic dermatitis, and normal children. Children with immediate food reactions were excluded if they had a history or evidence of atopic dermatitis or asthma. PBMC from children with IgE mediated food hypersensitivity produced significantly more IL-4 (p = 0.013) but equivalent INF-γ (p=0.26) compared to PBMC from control children. In contrast, PBMC from children with atopic dermatitis produced significantly less INF-γ (p < 0.001) and more IL-4 (p < 0.008) than PBMC from normal children. In addition, there was no difference in IL-4 (p = 0.74) but significantly less INF-γ (p < 0.001) produced by PBMC from the children with atopic dermatitis than food hypersensitivity. We demonstrate that children with IgE mediated food hypersensitivity and no other manifestation of atopic disease have enhanced IL-4 production without the defect in INF-γ production observed in childhood AD and asthma. We postulate that isolated IL-4 enhancement promotes the development of IgE mediated hypersensitivity disorders such as food allergy, whilst the combination of defective INF-γ and enhanced IL-4 production promotes inflammatory atopic disorders such as AD and asthma.     

Use of injury severity variables in determining disability and community integration after traumatic brain injury

BACKGROUND: Long-term outcome is important in managing traumatic brain injury (TBI), an epidemic in the United States. Many injury severity variables have been shown to predict major morbidity and mortality. Less is known about their relationship with specific long-term outcomes. METHODS: Glasgow Coma Scale, Revised Trauma Score, Injury Severity Score, and Trauma and Injury Severity Score, along with other demographic and premorbid values, were obtained for 378 consecutive patients hospitalized after TBI at a Level I trauma center between September 1997 and May 1998. Of this cohort, 120 patients were contacted for 1-year follow-up assessment with the Disability Rating Scale, Community Integration Questionnaire, and employment data. RESULTS: Univariate analyses showed these to be significant single predictors of 1-year outcome. Multivariate analyses revealed that the Revised Trauma Score and Glasgow Coma Scale had significant additive value in predicting injury variables Disability Rating Scale scores when combined with other demographic and premorbid variables studied. Predictive models of 1-year outcome were developed. CONCLUSION: Injury severity variables are significant single outcome predictors and, in combination with premorbid and demographic variables, help predict long-term disability and community integration for individuals hospitalized with TBI.     

A predictive microfluidic model of human glioblastoma to assess trafficking of blood–brain barrier-penetrant nanoparticles

The blood–brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood–brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood–brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood–brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

High-grade gliomas are the most common primary malignant brain tumors in adults (1). These include grade IV astrocytomas, commonly known as glioblastoma multiforme (GBM), which account for more than 50% of all primary brain cancers and have dismal prognoses, with a 5-y survival rate of less than 5% (2). Due to their infiltrative growth into the healthy brain tissue, surgery often fails to eradicate all tumor cells (3). While chemotherapy and radiation modestly improve median survival (4), most patients ultimately succumb to their tumors. This is primarily due to the presence of a highly selective and regulated endothelium between blood and brain parenchyma known as the blood–brain barrier (BBB) (5), which limits the entry of therapeutics into the brain tissue where tumors are located. The BBB, characterized by a unique cellular architecture of endothelial cells (ECs), pericytes (PCs), and astrocytes (ACs) (6, 7), displays up-regulated expression of junctional proteins and reduced paracellular and transcellular transports compared to other endothelia (8). While this barrier protects the brain from toxins and pathogens, it also severely restricts the transport of many therapeutics, as evidenced by the low cerebrospinal fluid (CSF)-to-plasma ratio of most chemotherapeutic agents (9). There is thus an important need to develop new delivery strategies to cross the BBB and target tumors, enabling sufficient drug exposure (10).Despite rigorous research efforts to develop effective therapies for high-grade gliomas, the majority of trialed therapeutics have failed to improve outcomes in the clinic, even though the agents in question are effective against tumor cells in preclinical models (11). This highlights the inability of current preclinical models to accurately predict the performance of therapeutics in human patients. To address these limitations, we developed an in vitro microfluidic model of vascularized GBM tumors embedded in a realistic human BBB vasculature. This BBB-GBM platform features brain microvascular networks (MVNs) in close contact with a GBM spheroid, recapitulating the infiltrative properties of gliomas observed in the clinic (12) and those of the brain tumor vasculature, with low permeability, small vessel diameter, and increased expression of relevant junctional and receptor proteins (7). This platform is well suited for quantifying vascular permeability of therapeutics and simultaneously investigating modes of transport across the BBB and into GBM tumor cells.There is strong rationale for developing therapeutic nanoparticles (NPs) for GBM and other brain tumors, as they can be used to deliver a diverse range of therapeutic agents and, with appropriate functionalization, can be designed to exploit active transport mechanisms across the BBB (13, 14). Liposomal NPs have been employed in the oncology clinic to improve drug half-life and decrease systemic toxicity (15), but, to date, no nanomedicines have been approved for therapeutic indications in brain tumors. We hypothesize that a realistic BBB-GBM model composed entirely of human cells can accelerate preclinical development of therapeutic NPs. Using our BBB-GBM model, we investigated the trafficking of layer-by-layer NPs (LbL-NPs) and ultimately designed a GBM-targeted NP. The LbL approach leverages electrostatic assembly to generate modular NP libraries with highly controlled architecture. We have used LbL-NPs to deliver a range of therapeutic cargos in preclinical tumor models (16, 17) and have recently demonstrated that liposomes functionalized with BBB-penetrating ligands improved drug delivery across the BBB to GBM tumors (18). Consistent with clinical data (19), we observed that the low-density lipoprotein receptor-related protein 1 (LRP1) was up-regulated in the vasculature near GBM spheroids in the BBB-GBM model and leveraged this information to design and iteratively test a library of NPs. We show that the incorporation of angiopep-2 (AP2) peptide moieties on the surface of LbL-NPs leads to increased BBB permeability near GBM tumors through LRP1-mediated transcytosis. With intravital imaging, we compared the vascular permeabilities of dextran and LbL-NPs in the BBB-GBM platform to those in mouse brain capillaries and validated the predictive potential of our in vitro model. Finally, we show the capability of the BBB-GBM platform to screen therapeutic NPs and predict in vivo efficacy, demonstrating improved efficacy of cisplatin (CDDP) when encapsulated in GBM-targeting LbL-NPs both in vitro and in vivo.     

Effects of an Amino Acid-Based Formula Supplemented with Two Human Milk Oligosaccharides on Growth,Tolerability, Safety,and Gut Microbiome in Infants with Cow’s Milk Protein Allergy

This open-label, non-randomized, multicenter trial (Registration: {"type":"clinical-trial","attrs":{"text":"NCT 03661736","term_id":"NCT03661736"}}NCT 03661736) aimed to assess if an amino acid-based formula (AAF) supplemented with two human milk oligosaccharides (HMO) supports normal growth and is well tolerated in infants with a cow’s milk protein allergy (CMPA). Term infants aged 1–8 months with moderate-to-severe CMPA were enrolled. The study formula was an AAF supplemented with 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT). Infants were fed the study formula for 4 months and were offered to remain on the formula until 12 months of age. Tolerance and safety were assessed throughout the trial. Out of 32 infants (mean age 18.6 weeks; 20 (62.5%) male), 29 completed the trial. During the 4-month principal study period, the mean weight-for-age Z score (WAZ) increased from –0.31 at the baseline to +0.28 at the 4-months’ follow-up. Linear and head growth also progressed along the WHO child growth reference, with a similar small upward trend. The formula was well tolerated and had an excellent safety profile. When comparing the microbiome at the baseline to the subsequent visits, there was a significant on-treatment enrichment in HMO-utilizing bifidobacteria, which was associated with a significant increase in fecal short-chain fatty acids. In addition, we observed a significant reduction in the abundance of fecal Proteobacteria, suggesting that the HMO-supplemented study formula partially corrected the gut microbial dysbiosis in infants with CMPA.