*2.1. EGFR and TP53 Mutation Analysis*

*EGFR* mutation analyses were performed on both cytologic and histologic samples, accurately selected by a dedicated expert pathologist from each center at the time of diagnosis. The same DNA specimens were used for the determination of *TP53* mutation status, blindly to the clinical outcomes. Quality controls were periodically performed during the course of the study to ensure concordance of molecular results.

DNA was extracted by macro-dissection of an area comprising at least 50% of tumor cells. Cells were lysed in a digestion buffer of 50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, and Tween-20 0.45%; proteinase K at 1.25 mg/mL were added to each specimen, with an overnight incubation at 56 ◦C. After proteinase K inactivation at 95 ◦C for 10 min, samples were centrifuged twice to eliminate debris and supernatant DNA quantity and quality was assessed by Nanodrop (Celbio) before molecular analyses.

Mutation status for exons 5–8 of *TP53* gene was performed by PCR amplification and Direct Sequencing using 3130 Genetic Analyzer (Applied Biosystems,Monza, Italy), orNext-Generation Sequencing by Ion S5 platform (Thermofisher, Monza, Italy), or MySeq platform (Illumina, San Diego, CA, USA).
