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In the identification of subjects with lung cancer, increased DNA methylationof the SHOX2 gene locus in bronchial aspirates has previously been proven to bea clinically valuable biomarker. This is particularly true in cases where thecytological and histological results following bronchoscopy are undetermined.This previous case control study was conducted using research assay componentsand a complex work flow. To facilitate the use in a diagnostic setting, a CE markedin vitro diagnostic test kit to quantify SHOX2 DNA methylation in bronchial aspirateswas developed and characterized. The presented assay for measuring SHOX2 DNA methylationin bronchial aspirates is based on two major steps: generation of bisulfite convertedtemplate DNA from patient samples followed by subsequent determination of SHOX2biomarker methylation by real-time PCR. Individual kits for DNA preparation, real-timePCR analysis and work flow control were developed. This study describes the analyticalperformance (reproducibility, accuracy, interfering substances, cross-reactivity)of the in vitro diagnostic (IVD) test kit 'Epi proLung BL Reflex Assay'. In addition,the intended use of the test was validated in a clinical performance evaluation(case control) study comprised of 250 patients (125 cases, 125 controls). Theresults describe the test as a robust and reliable diagnostic tool for identifyingpatients with lung cancer using Saccomanno-fixed bronchial lavage specimens (AUC[95% confidence intervals] = 0.94 [0.91-0.98], sensitivity 78% [69-86]/specificity96% [90-99]). This test may be used as a diagnostic adjunct to existing clinicaland pathological investigations in lung cancer.

Performance evaluation of the DNA methylation biomarker SHOX2 for the aid in diagnosis of lung cancer based on the analysis of bronchial aspirates