These findings were linked to robust and sensitive DNA extraction and mutation analysis methodologies. which is available to authorized users. mutation, ctDNA, Plasma, Real-world, Liquid biopsy Introduction Non-small cell lung cancer (NSCLC) represents 80C85% of Tubacin all lung cancers and is a leading cause of cancer-related death worldwide . It is often diagnosed at advanced stages (aNSCLC). However, the management of Tubacin aNSCLC, which has been traditionally treated with systemic chemotherapy, has evolved in recent years with developments in genetic profiling and the identification of driver mutations. Investigations of the activating mutations of the epidermal growth factor receptor (mutations are present in approximately 13% of European and 47% of Japanese patients . In Spanish patients, mutations have been reported in 10C16%  of patients in some series and in 11.6% in a specific trial . The first-generation reversible EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib and the second-generation irreversible TKI afatinib elicit dramatic responses as a first-line treatment in mutation-positive aNSCLC [10C14]. However, patients will only benefit from these agents if procedures to perform mutation testing are effectively implemented in a clinical setting. A variety of methodologies are available for mutation testing [15, 16], and direct DNA sequencing is considered the gold standard . However, recently developed commercial real-time quantitative PCR kits have successfully increased sensitivity, reducing the amount of tumor DNA required to detect the mutation in a patient sample . Tubacin These molecular techniques are subject to some limitations. The primary obstacle is a lack of sufficient tumor tissue sample. Other factors include the diverse accuracy of testing techniques  and, logistically, the limited number of testing laboratories and poor reimbursement by healthcare systems . Cytology samples have proven to be a valid source of material for mutation testing [18, 20, 21]. Recently, the assessment of mutations in surrogate samples, such as serum or plasma, has gained importance . In the ASSESS study , we observed high concordance of mutation status between matched biopsy/cytology and plasma samples, suggesting that circulating free tumor-derived DNA (ctDNA) can be isolated from the plasma or serum and could serve as a feasible sample for real-world mutation analysis. These findings were linked to robust and sensitive DNA extraction and mutation analysis methodologies. ctDNA comprises ?1% of total circulating free DNA ; when the previous analysis included only Tubacin the subset of patients for whom DNA was re-extracted with an optimized method, the overall concordance rate and sensitivity further increased. The present analysis from ASSESS, which originally included European and Japanese patients, aims to increase the understanding of local practices for mutation testing and the incidence of mutations in a cohort of the Spanish population under a real-world diagnostic framework. Methods ASSESS was a large, multicenter, non-interventional, diagnostic study (ClinicalTrials.gov NCT0178588) designed to evaluate different sample types as ctDNA sources as well as to perform mutation testing in patients with aNSCLC in a real-world diagnostic setting. The study was conducted in 8 centers in Japan and 48 centers in 7 European countries, including Spain. The study protocol was approved at all participating sites. The study was conducted according to the principles of the Declaration of Helsinki and the ICH Guidelines for Good Clinical Practice and was based on the applicable regulatory requirements for non-interventional studies and AstraZenecas policy on bioethics and human biological samples. All patients provided written informed consent. The study design has been described in full elsewhere  and is summarized here. Study population Eligible patients in European countries met the following inclusion criteria: SBF age ?18?years; either histologically or cytologically confirmed local advanced NSCLC (stage IIIA/B [American Joint Committee on Cancer staging system]); treatment na?ve and unsuitable for.