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Background ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib. Methods Twenty-four patients with advanced NSCLC harboring ALK fusion were administered crizotinib in a phase IV trial which included blood sampling prior to treatment. Targeted proteomics of 327 proteins using MRM-MS was used to measure plasma levels at baseline (including pre-treatment and early treatment blood samples) and assess potential clinical association. Results Patients were categorized by duration of response: long-term responders [PFS ≥ 24 months (n = 7)], normal responders [3 Conclusion In conjunction with ALK mutation, the expression of this proteomic signature may represent a liquid biopsy-based marker of long-term response to crizotinib in NSCLC. Expanding the utility of prognostic biomarkers of response duration could influence choice of therapy, therapeutic sequencing, and potentially the need for alternative or combination therapy. Trial registration ClinicalTrials.gov, NCT02041468. Registered 22 January 2014, https://clinicaltrials.gov/ct2/show/NCT02041468?term=NCT02041468&rank=1

Additional file 1: Table S1. Patient Groups based on PFS.

Additional file 3: Table S2. Top 15 proteins differentially detected between long-term and normal responders.

Additional file 5: Table S3. Candidate proteins signature for long-term response.

The epidermal growth factor receptor (EGFR) pathway is one of the most deregulated molecular pathways in human epithelial cancers. Many approved drugs were optimized to directly target EGFR but yielded only modest clinical improvement in cancer patients due to low efficacy and drug resistance. Transactivation of EGFR by other cell surface receptors such as G-protein-coupled receptors (GPCRs) was proposed to explain this lack of efficacy. Even if direct EGFR activation and transactivation by GPCR contribute to the activation of the same signaling pathways, they are often studied as independent events resulting in partial investigation of a drug's mechanism of action. We present a novel high-throughput approach that integrates interrogation of direct activation of EGFR and its transactivation via GPCR activation. Using distinct technology platforms, three readouts were used to measure (1) direct activation of GPCR via cyclic adenosine monophosphate (cAMP) detection, (2) direct activation of EGFR through the release of intracellular Ca(2+), and (3) EGFR transactivation by GPCR using the detection of p-extracellular-signal-regulated kinases 1/2 (p-ERK1/2). In addition to being simple, quick, and homogenous, our methods were shown to be more sensitive than those in current use. These enabling tools should improve the knowledge pertaining to GPCRs and receptor tyrosine kinases trans-regulation and facilitate the design of more potent and better targeted new therapeutic strategies.

Objective: Cerebral accumulation of amyloid beta (A beta) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by alpha- or beta-secretase results in two soluble metabolites, sAPP alpha and sAPP beta, respectively. However, previous data have shown that both alpha- and beta-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPP alpha and sAPP beta in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. Methods: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPP alpha and sAPP beta from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPP alpha. Results: Four different C-terminal forms of sAPP were identified, sAPP beta-M671, sAPP beta-Y681, sAPP alpha-Q686, and 5APP alpha-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R-2) between the two immunoassays was 0.41 for sAPP alpha and 0.45 for sAPP beta. Conclusion: Using high resolution MS, we show here for the first time that sAPP alpha in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPP alpha and sAPP beta levels are unaltered in AD. (C) 2013 Elsevier B.V. All rights reserved. (Less)

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reflect brain biochemistry. Using combined immunoprecipitation and mass spectrometry, we have shown that amyloid beta 1-15 (Aβ1-15) is produced by concerted β- and α-secretase cleavage of amyloid precursor protein (APP) and that the relative levels of Aβ1-16 in AD compared to controls are increased. Furthermore, drug-induced γ-secretase inhibition enhances the relative levels of Aβ1-15 and Aβ1-16. Here, we investigate a novel immunoassay for Aβ1-15/16 in a broad range of neurodegenerative conditions. The CSF level of Aβ1-15/16 was measured by the bead-based amplified luminescent proximity homogeneous assay (Alpha technology). Concentrations of Aβ1-15/16 were analyzed in subjects with Parkinson disease (PD; n = 90), PD with dementia (PDD) (n = 32), dementia with Lewy bodies (DLB) (n = 68), AD (n = 48), progressive supranuclear palsy (PSP) (n = 45), multiple system atrophy (MSA) (n = 46), and corticobasal degeneration (CBD) (n = 12). The detecting antibody is specific to the C-terminal epitope of Aβ15. We found that a carboxypeptidase (CPB) present in fetal bovine serum (FBS), a component of the buffers used, degrades Aβ1-16 to Aβ1-15, which is then detected by the Aβ1-15/16 assay. Significantly, lower levels of Aβ1-15/16 were detected in PD, PDD, PSP, and MSA compared to other neurodegenerative diseases and controls. Using the specific Aβ1-15/16 assay, a reliable quantification of Aβ1-15 or Aβ1-15/16 in CSF samples is obtained. We found reduced levels of Aβ1-15 in parkinsonian disease groups. The molecular mechanism behind this reduction is at present unknown.

Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the authors investigated and characterized the biological properties of a novel target, IRE1alpha, a bifunctional kinase/RNase stress sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology AlphaScreen to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1alpha. They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using AlphaScreen were biologically relevant. Preliminary characterization of assay robustness indicates that both AlphaScreen assays should be useful in HTS for the identification of IRE1 activity modulators.

Magdalena Nutu, Philippe Bourgeois, Henrik Zetterberg, Erik Portelius, Francesco Lipari, St ephane Parent, Oskar Hansson, Kaj Blennow, The Sahlgrenska Academy, Gothenburg, Sweden; PerkinElmer Biosignal, Inc, Montreal, Quebec, Canada; University of Gothenburg, M€olndal, Sweden; Institute of Neuroscience and Physiology, M€olndal, Sweden; PerkinElmer Inc, Montreal, Quebec, Canada; Depatment of Neurology, Sk ane University Hospital, Malm€o, Sweden; Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, M€olndal, Sweden.

The aggregation and accumulation of amyloid beta peptides is one of the pathological hallmarks of Alzheimer’s disease (AD). Amyloid beta is produced by sequential proteolytic cleavage of Amyloid Pr...

PerkinElmer's bead-based AlphaLISA® immunoassays are designed for the detection of analytes in biological samples. These chemiluminescent, no-wash assays are ideally suited for miniaturization and automation. They exhibit remarkable sensitivity, wide dynamic range and robust performance that compares advantageously with conventional enzyme-linked immunosorbent assay (ELISA).