PMC8107466_01
Timeline of the clinical course of the patient. Arrows indicate the link between the clinical event and the date of liquid biopsy time point.
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F1
At the post-operative CT scan performed in January 2018, a recurrence of disease was documented, involving bilateral pulmonary metastases and left pleural effusion (Figure 1). Unfortunately, following further three cycles, the patient experienced liver progression and central nervous system (CNS) progression due to the onset of multiple brain metastases (Figure 1).
CfDNA monitoring of EGFR ex19del and EGFR T790M mutations. Each dot corresponds to a different liquid biopsy time point. AF, allele frequency; CT, chemotherapy with platinum-etoposide.
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F2
The presence of EGFR activating and T790M mutations was also confirmed on liquid biopsy (Figure 2). The patient promptly started osimertinib, which led to complete metabolic response on the liver and osteoblastic reaction of pre-existing bone lesions. T790M showed up again with the known activating mutation and with two TP53 non-sense variants (Table 1). The presence of EGFR mutations on liquid biopsy were confirmed also with ddPCR (Figure 2).
Drug screening in a primary cell line following osimertinib resistance. Primary cells were treated with 250 nM osimertinib, 0.150 mug/ml etoposide, and 0.05 mug/ml cisplatin. After 72 h, cell proliferation was assessed by CellTiter 96  AQueous One Solution Cell Proliferation Assay (MTS). Data are expressed as percent of cell viability vs. control cells and are means +- SD of three measurements. *p < 0.05.
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F3
As shown in Figure 3, tumor cells were sensitive to the combination of osimertinib with chemotherapeutic agents (p < 0.05 vs. control) even if the results did not reach statistical significance vs. single drug treatments.
Immunohistochemistry analysis for p53 (upper line) and Rb (lower line). The resected tumor (lobectomy) showed a negativity for both p53 and Rb. In the first liver biopsy (LB1), p53 was overexpressed, whereas Rb was negative; the histotype of LB1 is that of a NSCLC/adenocarcinoma with morphology similar to lobectomy. In the second liver biopsy (LB2), we detected only scattered cells positive for p53 and negative for Rb; the histotype is that of a SCLC also documented by positivity for neuroendocrine markers.
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F4
In fact, p53 was negative in the surgery tissue, overexpressed in LB1, and scattered positive in LB2 (Figure 4).