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Background: The heterogeneous and complex genetic landscape of NSCLC impacts the clinical outcomes of patients who will eventually develop resistance to osimertinib. Liquid biopsy (LB) analysis as a minimally invasive approach is a key step to efficiently identify resistance mechanisms and adjust to proper subsequent treatments. Materials and methods: In the present study, we combined plasma-cfDNA and CTC analysis from 30 NSCLC patients in samples collected before treatment and at the progression of disease (PD). We detected molecular alterations at the DNA mutation (EGFR, PIK3CA, KRAS G12C, BRAF V600E), DNA methylation (RASSF1A, BRMS1, FOXA1, SLFN1, SHISA3, RARβ,, WIF-1, RASSF10 and APC), gene expression (CK-19, CK-18, CK-8, AXL, TWIST-1, PD-L1, PIM-1, Vimentin, ALDH-1 , and B2M) and chromosomal level (HER2 and MET amplification) as possible resistance mechanisms and druggable targets. We also studied the expression of PD-L1 in single CTCs using immunofluorescence. Results: In some cases, T790M resistance EGFR mutation was detected at baseline in CTCs but not in the corresponding plasma cfDNA. PIK3CA mutations were detected only in plasma-cfDNA but not in corresponding CTCs. KRAS G12C and BRAF V600E mutations were not detected in the samples analyzed. MET amplification was detected in the CTCs of two patients before treatment whereas HER2 amplification was detected in the CTCs of three patients at baseline and in one patient at PD. DNA methylation analysis revealed low concordance between CTCs and cfDNA, indicating the complementary information obtained through parallel LB analysis. Results from gene expression analysis indicated high rates of vimentin-positive CTCs detected at all time points during osimertinib. Moreover, there was an increased number of NSCLC patients at PD harboring CTCs positive in PD-L1. AXL and PIM-1 expression detected in CTCs during treatment suggesting new possible therapeutic strategies. Discussion: Our results reveal that comprehensive liquid biopsy analysis can efficiently represent the heterogeneous molecular landscape and provide prominent information on subsequent treatments for NSCLC patients at PD since druggable molecular alterations were detected in CTCs. [ABSTRACT FROM AUTHOR]

Therapeutic management of NSCLC patients is quite challenging as they are mainly diagnosed at a late stage of disease, and they present a high heterogeneous molecular profile. Osimertinib changed the paradigm shift in treatment of EGFR mutant NSCLC patients achieving significantly better clinical outcomes. To date, osimertinib is successfully administered not only as first- or second-line treatment, but also as adjuvant treatment while its efficacy is currently investigated during neoadjuvant treatment or in stage III, unresectable EGFR mutant NSCLC patients. However, resistance to osimertinib may occur due to clonal evolution, under the pressure of the targeted therapy. The utilization of liquid biopsy as a minimally invasive tool provides insight into molecular heterogeneity of tumor clonal evolution and potent resistance mechanisms which may help to develop more suitable therapeutic approaches. Longitudinal monitoring of NSCLC patients through ctDNA or CTC analysis could reveal valuable information about clinical outcomes during osimertinib treatment. Therefore, several guidelines suggest that liquid biopsy in addition to tissue biopsy should be considered as a standard of care in the advanced NSCLC setting. This practice could significantly increase the number of NSCLC patients that will eventually benefit from targeted therapies, such as EGFR TKIs. [ABSTRACT FROM AUTHOR]

Over the last decade, great advancements have been made in the field of liquid biopsy through extensive research and the development of new technologies that facilitate the use of liquid biopsy for cancer patients. This is shown by the numerous liquid biopsy tests that gained clearance by the US Food and Drug Administration (FDA) in recent years. Liquid biopsy has significantly altered cancer treatment by providing clinicians with powerful and immediate information about therapeutic decisions. However, the clinical integration of liquid biopsy is still challenging and there are many critical factors to consider prior to its implementation into routine clinical practice. Lack of standardization due to technical challenges and the definition of the clinical utility of specific assays further complicates the establishment of Standard Operating Procedures (SOPs) in liquid biopsy. Harmonization of laboratories to established guidelines is of major importance to overcome inter-lab variabilities observed. Quality control assessment in diagnostic laboratories that offer liquid biopsy testing will ensure that clinicians can base their therapeutic decisions on robust results. The regular participation of laboratories in external quality assessment schemes for liquid biopsy testing aims to promptly pinpoint deficiencies and efficiently educate laboratories to improve their quality of services. Accreditation of liquid biopsy diagnostic laboratories based on the ISO15189 standard in Europe or by CLIA/CAP accreditation procedures in the US is the best way to achieve the adaptation of liquid biopsy into the clinical setting by assuring reliable results for the clinicians and their cancer patients. Nowadays, various organizations from academia, industry, and regulatory agencies collaborate to set a framework that will include all procedures from the pre-analytical phase and the analytical process to the final interpretation of results. In this review, we underline several challenges in the analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) concerning standardization of protocols, quality control assessment, harmonization of laboratories, and compliance to specific guidelines that need to be thoroughly considered before liquid biopsy enters the clinic. [ABSTRACT FROM AUTHOR]

Simple Summary: Osimertinib has become the standard of care for the first-line treatment of EGFR-mutant NSCLC patients. The aim of this current translational research study was to assess the clinical relevance of liquid biopsy in 47 patients receiving osimertinib. Effects on circulating tumor cells (CTCs) and plasma-DNA (ctDNA) were investigated before, after one treatment cycle, and at the end of treatment. ctDNA and CTCs decreased after one treatment cycle, but increased at the end of treatment. The detection of ctDNA before and after one treatment cycle was associated with shorter progression-free and overall survivals (PFS and OS), whereas ctDNA clearance after one treatment cycle resulted in a significantly longer PFS and OS. ctDNA at baseline emerged as an independent predictor of shorter PFS. Thus, changes in liquid biopsy status (CTCs, ctDNA) during osimertinib treatment can be used as a tool for treatment efficacy. Introduction: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. Methods: Forty-seven EGFR-mutant NSCLC patients who had progressed on prior first- or second-generation EGFR inhibitors were enrolled in the study and treated with osimertinib, irrespective of the presence of the T790M mutation in the primary tumor or the plasma. Peripheral blood was collected at baseline (n = 47), post-Cycle 1 (n = 47), and at the end of treatment (EOT; n = 39). CTCs were evaluated in 32 patients at the same time points (n = 32, n = 27, and n = 21, respectively) and phenotypic characterization was performed using triple immunofluorescence staining (CK/VIM/CD45). Results: Osimertinib resulted in an ORR of 34% (2 CR) and a DCR of 76.6%. The median PFS and OS values were 7.5 (range, 0.8–52.8) and 15.1 (range, 2.1–52.8) months, respectively. ctDNA was detected in 61.7%, 27.7%, and 61.5% of patients at baseline, post-Cycle 1, and EOT, respectively. CTCs (CK+/CD45-) were detected in 68.8%, 48.1%, and 61.9% of patients at the three time points, respectively. CTCs expressing both epithelial and mesenchymal markers (CK+/VIM+/CD45-) were detected in 56.3% and 29.6% of patients at baseline and post-Cycle 1, respectively. The detection of ctDNA at baseline and post-Cycle 1 was associated with shorter PFS and OS, whereas the ctDNA clearance post-Cycle 1 resulted in a significantly longer PFS and OS. Multivariate analysis revealed that male sex and the detection of ctDNA at baseline were independent predictors of shorter PFS (HR: 2.6, 95% C.I.: 1.2–5.5, p = 0.015 and HR: 3.0, 95% C.I.: 1.3–6.9; p = 0.009, respectively). Conclusions: The decrease in both CTCs and ctDNA occurring early during osimertinib treatment is predictive of better outcome, implying that liquid biopsy monitoring may be a valuable tool for the assessment of treatment efficacy. [ABSTRACT FROM AUTHOR]