Identifying patients with cerebral infarction within the time window compatible with reperfusion therapy, diagnostic performance of glutathione S-transferase-π (GST-π) and peroxiredoxin 1 (PRDX1): exploratory prospective multicentre study FLAG-1 protocol.

INTRODUCTION: Plasma biomarkers may be useful in diagnosing acute cerebral infarction requiring urgent reperfusion, but their performance remains to be confirmed. If confirmed, these molecules could be used to develop rapid and reliable decentralised measurement methods, making it possible to initiate reperfusion therapy before hospital admission. The FLAG-1 large prospective study will constitute a plasma bank to assess the diagnostic performance of two biomarkers: glutathione S-transferase-π and peroxiredoxin 1. These molecules are involved in the oxidative stress response and could identify cerebral infarction within a therapeutic window of less than 4.5 hours following the onset of symptoms. Secondary objectives include assessing performance of these biomarkers within 3-hour and 6-hour windows; identifying additional biomarkers diagnosing cerebral infarction and significant criteria guiding therapeutic decisions: ischaemic features of stroke, presence of diffusion/fluid-attenuated inversion recovery mismatch, volume of cerebral infarction and penumbra on cerebral MRI. METHODS AND ANALYSIS: The exploratory, prospective, multicentre FLAG-1 Study will include 945 patients with acute stroke symptoms (onset ≤12 hours, National Institute of Health Stroke Scale score ≥3). Each patient's 25 mL blood sample will be associated with cerebral MRI data. Two patient groups will be defined based on the time of blood collection (before and after 4.5 hours following onset). Receiver operating characteristic analysis will determine the diagnostic performance of each biomarker, alone or in combination, for the identification of cerebral infarction <4.5 hours. ETHICS AND DISSEMINATION: The protocol has been approved by an independent ethics committee. Biological samples are retained in line with best practices and procedures, in accordance with French legislation. Anonymised data and cerebral imaging records are stored using electronic case report forms and a secure server, respectively, registered with the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés (CNIL)). Results will be disseminated through scientific meetings and publication in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT03364296).

Validation of a phosphoprotein array assay for characterization of human tyrosine kinase receptor downstream signaling in breast cancer.

BACKGROUND: Human epidermal growth factor receptor (HER) downstream signaling kinases have important effects on tumor response to anti-HER monoclonal antibodies and tyrosine kinase inhibitors. We validated an assay that uses phosphoprotein arrays for measurement of HER downstream signaling functionality in breast carcinomas. METHODS: Using the Bio-Plex(R) phosphoprotein array (BPA), we performed multiplex immunoanalysis to investigate the expression of phosphorylated epidermal growth factor receptor and phosphorylated HER downstream signaling proteins (phosphorylated protein kinase B, phosphorylated glycogen synthase kinase -3beta, phosphorylated P70 ribosomal protein S6 kinase, and phosphorylated extracellular signal regulated kinase 42/44) in 49 frozen specimens of ductal infiltrating breast carcinoma taken at diagnosis. BPA was cross-validated with Western blot analysis. Sample size, homogenicity, tumor content, protein extraction, and monoclonal antibody detection were in accordance with optimized standard operating procedures. RESULTS: Linear regression showed significant quantitative correlations between BPA and Western blot, with regression coefficient values of 0.71-0.87 (P < 0.001). BPA intra- and interassay CVs were <17% and 15%, respectively. Compared to limits of detection established by using the mean + 3SD of 10 blanks, large variations of phosphoprotein expression, up to several hundred-fold, were observed among the 49 tumor specimens. CONCLUSIONS: Our results validate the use of the multiplex phosphoprotein array assay in human clinical tumor specimens. Further prospective evaluation is warranted to investigate the use of HER downstream signaling phosphoproteins as predictive and/or surrogate markers for clinical response to anti-HER targeted therapy.

PTEN expression controls cellular response to cetuximab by mediating PI3K/AKT and RAS/RAF/MAPK downstream signaling in KRAS wild-type, hormone refractory prostate cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) and mutation of pten tumor suppressor gene in human cancer cells leads to activated EGFR downstream signaling including PI3-kinase/AKT (PI3K/AKT) and/or mitogen-activated protein kinases (RAS/RAF/MAPK) and have been linked to resistance to anti-EGFR targeted therapies. Cetuximab is a chimeric IgG1 monoclonal antibody that binds the EGFR with high specificity and have been developed as promising therapeutic anticancer treatments in several solid tumors, including colorectal and head and neck squamous cell carcinomas. Cetuximab activity is related to PI3K/AKT and RAS/RAF/MAPK signaling pathways functionality and its activity has been shown to be higher in wild-type KRAS tumors. To study the influence of PTEN expression on cell response to cetuximab, we used wild-type KRAS, PTEN-null, EGFR overexpressing PC3 prostate cancer cells. Reintroduction of PTEN significantly reduced the constitutive overexpression of phosphorylated-AKT (p-AKT) and downstream kinases (p-GSK3beta and p-P70S6 kinase) as well as phosphorylated-ERK1/2 (p-ERK1/2) and consequently significantly restored cetuximab-induced cell growth inhibition and apoptosis induction. Taken together, the results achieved in the present study show that PTEN controls the cellular response to cetuximab in KRAS wild-type prostate carcinoma PC3 cells through the regulation of AKT phosphorylation and restoration of the functionality of EGFR downstream signaling. Extrapolation of these findings to clinical situation, suggests that the assessment of EGFR downstream signaling functionality could be proposed as a diagnostic response predictive marker for anti-EGFR targeted therapies.

Sustained gene transfer and enhanced cell death following glucosylated-PEI-mediated p53 gene transfer with photochemical internalisation in p53-mutated head and neck carcinoma cells.

p53 is frequently mutated in head-and-neck squamous cell carcinoma. Wild-type p53 gene transfer induces apoptosis in vitro and tumor regression in vivo and clinical investigations of p53 gene therapy have been reported, mostly using viral vectors. Non-viral vectors are increasingly being used as an alternative to viral vectors and photochemical internalisation (PCI) of non-viral vectors has been reported to yield high gene transfer efficiency. The p53-mutated status of FaDu human pharynx carcinoma cell line was first assessed by DNA sequencing and the cells were transfected using tetraglucosylated polyethylenimine (PEI-Glu4) in conjunction with photochemical internalisation (PCI). The green fluorescent protein (GFP) was used as a reporter for determination of the transgene expression kinetics with or without PCI. p53 gene transfer was performed in these optimised conditions, and subsequent induction of apoptosis was investigated by flow cytometric determination of the phosphatidylserine externalization. Long-term cell death was assessed using colony forming assays. DNA sequencing in FaDu cells showed a G/T point mutation at codon 248 in exon 7 of p53 gene, resulting in an arginine-to-leucine substitution. As a consequence, P53 was shown to be expressed in >90% of untreated cells using immunocytochemistry. Using PEI-Glu4 as vector, PCI was found to significantly enhance GFP gene transfer whatever the formulation solution. Transfection efficiency was significantly increased with PCI. GFP expression kinetics (24-144 h) demonstrates that PCI induces sustained transgene expression with >10% of cells remaining transfected after 144 h. In such conditions, p53 gene transfer using PEI-Glu4 and PCI, resulted in spontaneous induction of apoptosis. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used, reaching up to 50% cell death. Wild-type p53 gene transfer using PEI-Glu4/DNA complexes and PCI, yields sustained transgene expression and induces cell death in p53-mutated FaDu cells.