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Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide and is largely inflicted by carcinogens contained in tobacco smoke. The generation of cell lines mimicking traits of human LADC will profoundly advance our understanding of the pathobiology of the disease, as they offer an easy and valuable tool to study the cellular and molecular aspects of carcinogenesis. Here we describe a detailed protocol for the generation of such cell lines, following the exposure of experimental mouse strains to different tobacco carcinogens and isolation of the resulting lung tumors.Patient-derived xenografts (PDXs) are created by implanting human tumor tissue or cells into immunodeficent mice, and enable the study of tumor biology, biomarkers and response to therapy in vivo. This chapter describes a method for lung adenocarcinoma (LAC) PDX generation using subcutaneous implantation of tumor tissue and cell suspensions and incorporating the humanization of PDX models by reconstitution with human immune cells.The detection of molecular alterations such as ROS1 and ALK rearrangements is performed as part of the diagnosis of advanced-stage lung adenocarcinoma. read more These alterations allow the treatments with tyrosine kinase inhibitors. Cytological samples are very useful as up to 40% patients are diagnosed with this type of sample. Here we describe the immunocytochemistry technique usable to reveal the overexpression of ALK or ROS1 tyrosine kinase receptors secondary to ALK and ROS1 rearrangements, respectively.The receptor tyrosine kinase (RTK) c-MET plays important roles in cancer, yet despite being frequently overexpressed, clinical responses to targeting this receptor have been limited in the clinical setting. A singular significant challenge has been the accurate identification of biomarkers for the selection of responsive patients. However, recently mutations which result in the loss of exon 14 (called METex14 skipping) have emerged as novel biomarkers in non-small cell lung carcinomas (NSCLC) to predict for responsiveness to targeted therapy with c-MET inhibitors. Currently, the diverse genomic alterations responsible for METex14 skipping pose a challenge for routine clinical diagnostic testing. Next generation sequencing (NGS) is the current gold standard for identifying the diverse mutations associated with METex14, but the cost for such a procedure remains to some degree prohibitive as often NGS is requested on a case-by-case basis, and many hospitals may not even have the capacity or resources to conduct NGS.However, PCR-based approaches to detect METex14 have been developed which can be conducted in most routine hospital laboratories and may therefore allow a cost-effective approach to pre-screen patients that may respond to c-MET inhibitors prior to conducting NGS, or until all patients will have NGS conducted as routine practise. In this chapter, we describe one such PCR-based approach for screening samples for the detection of METex14 in NSCLC.The profiling of EGFR mutations, the most common genetic alterations in non-small cell lung cancer (NSCLC) predictive of targeted therapy efficacy, is crucial to anticipate the patient response to EGFR tyrosine kinase inhibitors. Here, we introduce the naica® system for 6-color Crystal Digital PCRTM and describe in detail a standardized workflow for the multiplexed, single-assay detection of the 19 most prevalent sensitizing and resistance EGFR mutations in both tumor and circulating tumor DNA (ctDNA) samples. Two major advantages of the 6-color multiplexing system over current digital PCR systems are the rapid time to results, and the large quantity of mutational information obtained per patient sample, rendering the 6-color system highly cost effective. The 6-color Crystal Digital PCRTM technology enables highly sensitive and efficient therapeutic monitoring through liquid biopsy, resulting in the early detection of treatment resistance. While the assay presented here specifically addresses EGFR mutation status monitoring in NSCLC patients, 6-color Crystal Digital PCRTM assays are flexible and evolutive in design. As such, 6-color detection assays can be optimized to monitor mutations associated with a range of cancers and other genetic diseases, as well as to detect genetic changes beyond the oncology and human health domains.Driver mutations in non-small cell lung cancer (NSCLC) have a relevant significance for clinical management. EGFR mutations are the most important predictive biomarkers for NSCLC, although KRAS and BRAF mutations can also be prognostic and predictive biomarkers, respectively. PCR-based approaches followed by sequencing are useful for EGFR, KRAS, and BRAF mutational analysis. Herein, all steps for a PCR-based technique, from DNA isolation from tumor tissue sections to DNA sequencing for genetic analysis of EGFR, KRAS, and BRAF hotspot regions are described.In non-small cell lung cancer (NSCLC), mutation detection and fusion gene status are treatment predictive and, hence, key factors in clinical management. Lately, alternate splicing variants of MET have gained focus as NSCLC tumors harboring a MET exon 14 skipping event have proven sensitive toward targeted therapy. Reliable methods for detection of genetic alterations in NSCLC have proven to be of increased importance. This chapter provides with hands-on experience of the NanoString gene expression platform for detection of genetic alterations in NSCLC.The cancer phenotype is usually characterized by deregulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both preclinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines.
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