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Laura Pacini,* Andrew D Jenks,* Simon Vyse,* Christopher P Wilding, Amani Arthur, Paul H Huang Division of Molecular Pathology, The Institute of Cancer Research, London, UK*These authors contributed equally to this workCorrespondence: Paul H HuangDivision of Molecular Pathology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UKTel +44 207 153 5554Email paul.huang@icr.ac.ukAbstract: Insertion mutations in exon 20 (Ex20ins) of the epidermal growth factor receptor (EGFR) gene are the largest class of EGFR mutations in non-small cell lung cancer (NSCLC) for which there are currently no approved targeted therapies. NSCLC patients with these mutations do not respond to clinically approved EGFR tyrosine kinase inhibitors (TKIs) and have poor outcomes. A number of early phase clinical trials are currently underway to evaluate the efficacy of a new generation of TKIs that are capable of binding to and blocking Ex20ins. Although these agents have shown some clinical activity, patient responses have been restricted by dose-limiting toxicity or rapid acquisition of resistance after a short response. Here we review the current understanding of the mechanisms of resistance to these compounds, which include on-target EGFR secondary mutations, compensatory bypass pathway activation and acquisition of an EMT phenotype. Taking lessons from conventional EGFR inhibitor therapy in NSCLC, we also consider other potential sources of resistance including the presence of drug-tolerant persister cells. We will discuss therapeutic strategies which have the potential to overcome different forms of drug resistance. We conclude by evaluating recent technological developments in drug discovery such as PROTACs as a means to better tackle TKI resistance in NSCLC harbouring Ex20ins mutations.Keywords: EGFR, exon 20 insertions, poziotinib, lung cancer, drug resistance, PROTACs

Copyright: © 2021 The Authors. Primary cilia are microtubule-based sensory organelles surrounded by membrane. They can detect mechanical and chemical stimuli. The last few years have uncovered cilia as unique signaling hubs that host a number of receptors and effector molecules. Thus, defining how specific proteins localize and are distributed along the cilium is critical to understanding its function. Quantitative immunofluorescence can be used to accurately assess the localization of receptors and signaling molecules within the primary cilia. However, image analysis can be time consuming, and there are limited programs that can accurately determine staining intensity along the cilia. To overcome these issues, we developed a series of MATLAB scripts to accurately measure staining intensity along the length of the cilia, in both a semi-automated and automated fashion. Here, we describe the scripts and include a protocol for image analysis for each. With these scripts, the protocols can be used to analyze the distribution of any ciliary protein using immunofluorescence images. Institute of Cancer Research; Sarcoma UK grant.