A Novel Diagnostic Approach to Screen for Chemotherapy Drug Response in Non-Small Cell Lung Cancer Using Sortase Technology
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CitationMerluzzo, Angela M. 2016. A Novel Diagnostic Approach to Screen for Chemotherapy Drug Response in Non-Small Cell Lung Cancer Using Sortase Technology. Master's thesis, Harvard Extension School.
AbstractDrug resistance is the most significant barrier to improving long-term outcomes for patients with non-small cell lung cancer (NSCLC). Recent advances in the understanding of cell biology and molecular pathways have enabled researchers to find a link between the altered expression of many molecular biomarkers in tumor cells and patient outcomes, making it possible to identify markers with prognostic and predictive value that could help guide treatment.
This research used a new Multicomponent Detection System for the duel detection of co-expressed targets and protein fusions with potential diagnostic value for predicting drug response in NSCLC. The Multicomponent Detection System takes advantage of the protein ligation activity of Sortase A (SrtA), a transpeptidase enzyme from Staphylococcus aureus. In this system two modified antibody species are used, each recognizing a unique molecule on the surface of a biological sample. Attached to one antibody species is the Sortase A enzyme (Ab-SrtA), and to the second antibody species the LPXTG recognition sequence for SrtA-mediated molecular ligation (Ab-LPXTG). Binding of both antibody species to their respective targets in close proximity will result in SrtA-mediated ligation of an antigenic peptide to the LPXTG containing antibody. Subsequent detection of the antigenic peptide indicates the concurrent presence of two targets (i.e. biomarkers) within a given sample.
In this study we investigated the use of this system to develop a diagnostic assay for NSCLC. We tested the following NSCLC associated protein pairs and protein fusion: B7 homolog 3 (B7-H3) paired with Napsin A, mutant epidermal growth factor receptor [EGFR (L858R)] paired with mesenchymal epidermal transition (MET) receptor, and EFGR (L858R) with Programed death-1 ligand (PD-L1), and the EML4-ALK oncogenic fusion. We found that while all the protein pairs tested showed positive results in our cell-free ELISA based assay, In-Cell ELISA based assays yielded weak or negative signals. However, testing for protein fusions of Anaplastic lymphoma kinase (ALK) with echinoderm microtubule-associated protein-like 4 (EML4) showed promising results. We demonstrated that this platform could successfully detect EML4-ALK fusions in a cytological immunofluorescence assay. The work in this study may pave the way for the development of the first protein-based diagnostic assay for EML4-ALK fusion positive NSCLC.
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