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Rockville, MD, United States

Hembrough T.,Oncoplex Diagnostics | Hembrough T.,NantOmics LLC | Liao W.-L.,Oncoplex Diagnostics | Liao W.-L.,NantOmics LLC | And 15 more authors.
Clinical Chemistry

BACKGROUND: Crizotinib has antitumor activity in ALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non-small cell lung cancer (NSCLC). The current diagnostic test for ALK rearrangement is breakapart fluorescence in situ hybridization (FISH), but FISH has low throughput and is not always reflective of protein concentrations. The emergence of multiple clinically relevant biomarkers in NSCLC necessitates efficient testing of scarce tissue samples. We developed an anaplastic lymphoma kinase (ALK) protein assay that uses multiplexed selected reaction monitoring (SRM) to quantify absolute amounts of ALK in formalin-fixed paraffin-embedded (FFPE) tumor tissue. METHODS: After validation in formalin-fixed cell lines, the SRM assay was used to quantify concentrations of ALK in 18 FFPE NSCLC samples that had been tested for ALK by FISH and immunohistochemistry. Results were correlated with patient response to crizotinib. RESULTS: We detected ALK in 11 of 14 NSCLC samples with known ALK rearrangements by FISH. Absolute ALK concentrations correlated with clinical response in 5 of 8 patients treated with crizotinib. The SRM assay did not detect ALK in 3 FISH-positive patients who had not responded to crizotinib. In 1 of these cases, DNA sequencing revealed a point mutation that predicts a nonfunctional ALK fusion protein. The SRM assay did not detect ALK in any tumor tissue with a negative ALK status by FISH or immunohistochemistry. CONCLUSIONS: ALK concentrations measured by SRM correlate with crizotinib response in NSCLC patients. The ALK SRM proteomic assay, which may be multiplexed with other clinically relevant proteins, allows for rapid identification of patients potentially eligible for targeted therapies. © 2015 American Association for Clinical Chemistry. Source

Applebaum M.A.,University of Chicago | Thomas D.G.,University of Michigan | Hembrough T.,Oncoplex Diagnostics | Burrows J.,Oncoplex Diagnostics | And 3 more authors.
Applied Immunohistochemistry and Molecular Morphology

Targeted therapies are increasingly being evaluated for patients with Ewing sarcoma (EWS). Optimal strategies for quantifying key signaling proteins in EWS remain unclear. We sought to quantify tumor expression of signaling pathway proteins in EWS using 3 methodologies. A total of 46 blocks of formalin-fixed paraffin-embedded tissue were obtained from 40 patients with EWS. Tumor was evaluated for the expression of proteins in the insulin-like growth factor type 1 receptor (IGF- 1R), epithelial growth factor receptor (EGFR), and mTOR pathways using standard immunohistochemical analysis (IHC), automated quantitative analysis (AQUA) immunohistochemical analysis, and mass spectrometry quantification. The mean age at diagnosis was 14 years (range, 1 to 49 y). About 67.5% were male and 57.5% had localized disease. Samples displayed a wide range of expression by AQUA: mean (range) IGF-1R=10,702 (393 to 14,424); EGFR=2750 (672 to 9798); and phosphatase and tensin homolog (PTEN)=2250 (251 to 6557). Mean IGF- 1R expression by AQUA did not differ between standard IHC expression categories (low IHC=11,255; medium IHC= 11,070; high IHC=11,023; P=0.98). Mean PTEN expression by AQUA was higher in the medium and high IHC categories (low IHC=1229; medium IHC=2715; high IHC=2940; P=0.064). Only 2 samples expressed EGFR by standard IHC. Mass spectrometry trended toward correlation with standard IHC but did not yield interpretable results in the majority of samples. This study demonstrates that the relative quantification of signaling protein expression in EWS is dependent on the methodology used. Optimization and validation of these tools are necessary before clinical application for risk stratification of patients or measurement of biomarker expression. Copyright © 2014 by Lippincott Williams & Wilkins. Source

Krizman D.B.,Oncoplex Diagnostics | Burrows J.,Oncoplex Diagnostics
Methods in Molecular Biology

Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of fi ndings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin- fi xed, paraf fi nembedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease. Source

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