PMC8226241_01
Timeline and histological transformation. H&E: Hematossil and Eosin staining (magnification 20X). Immunohistochemistry analyses were performed using mouse monoclonal antibodies anti TTF-1 and p40, clone 8G7G3/1 and clone BC28 respectively, on the Ventana Medical System (Roche). Immonuhistochemistry images have been reported with a 20X magnification. Gene mutational analysis was performed by Sequenom MassArray at baseline, EGFR mutational status was determined by digital droplet PCR at progression times. ALK and PD-L1 were evaluated by immunohistochemistry and gene fusions and amplifications by fluorescent in situ hybridization. LUAD, lung adenocarcinoma; LUSC, lung squamous-cell carcinoma.
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The tumor was characterized by immunohistochemical (IHC) stain on cell-block from pleural effusion with anti-thyroid transcription factor (TTF-1) antibody (mouse monoclonal antibody; clone 8G7G3/1; Ventana Medical System - Roche, Monza, Italy), which showed a strong positive nuclear staining ( Figure 1 ). EGFR mutational status in circulating tumor DNA (ctDNA) was evaluated every 3 months ( Figure 1 ). ctDNA was purified from 3 ml of plasma by the QIAamp Circulating kit (Qiagen) and EGFR was analyzed by the digital droplet PCR (Bio-Rad, Hercules, California, USA) with the PrimePCR  ddPCR  Mutation Assay for human EGFR. Terminology Criteria for Adverse Events). The CT scan confirmed the PR and the ctDNA analysis highlighted a decrease of L858R amount ( Figures 1  and  2 ). Tumor cells were not detected in the effusion, but ctDNA analysis revealed an increased L858R amount ( Figure 1 ). An ultrasound-guided thoracentesis was performed with a diagnosis of adenocarcinoma metastasis, TTF1 positive ( Figure 1 ).
Clinical Timeline. The immunohistochemistry evaluation of PD-L1 was performed using a monoclonal primary antibody SP263 clone on the Ventana Medical System (Roche). MET amplification was evaluated by fluorescence in situ hybridization using the probes: LSI MET spectrum red and CEP7 spectrum green (Vysis - Abbott). PR, partial response; SD, stable disease; PD, progression disease; NGS, Next Generation Sequencing; LUAD, lung adenocarcinoma; LUSC, lung squamous-cell carcinoma.
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The sensitive EGFR mutation L858R, within exon 21, was detected and 10% of cancer cells expressed PD-L1 ( Figure 2 ). Terminology Criteria for Adverse Events). The CT scan confirmed the PR and the ctDNA analysis highlighted a decrease of L858R amount ( Figures 1  and  2 ). TTF1, with PD-L1 expression observed in 60% of cancer cells ( Figure 2 ). MET amplification (MET/CEP7: 5,8 - cut-off<2; MET copy number: 16,6 - cut-off<5) and EGFR L858R mutation were detected on the squamous lesion ( Figure 2 ). The MRI showed the disappearance of brain metastases, and T790M mutation was no longer detectable in ctDNA, but the L858R amount increased.