Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC. Circulating tumor cells (CTCs) consist a unique source of information at the cellular level. Digital PCR (dPCR) is a valuable tool for accurate and valid analysis of gene mutations in liquid biopsy analysis. In the present study we detected EGFR mutations in ctDNA and paired CTCs under osimertinib therapy at two time points using crystal dPCR and the naica^® system (Stilla Technologies). We quantified mutation allele frequencies (MAF) of EGFR mutations in 91 plasma cfDNA samples of 48 EGFR mutant NSCLC patients and in 64 matched CTC-derived genomic DNA samples, and the FDA-cleared cobas^® EGFR mutation test in 80 identical plasma samples. Direct comparison between crystal dPCR and the cobas EGFR assay revealed a high concordance for all EGFR mutations. Our comparison of crystal dPCR results in ctDNA with the corresponding primary tissue has shown a strong correlation. EGFR mutations analysis in paired CTC-derived gDNA revealed a high heterogeneity. Crystal dPCR offers the unique advantages of high analytical sensitivity, precision, and accuracy for detecting and quantifying multiple EGFR mutations in plasma cfDNA and CTCs of NSCLC patients.

Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. DNA methylation has been previously implicated in NSCLC progression and often in therapy resistance, however its distinct role in osimertinib resistance is not elucidated as yet. In the present study, we directly compared DNA methylation of nine selected genes (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RARβ, SHISA3, and FOXA1) in plasma-cfDNA and paired CTCs of NSCLC patients who were longitudinally monitored during osimertinib treatment. Peripheral blood (PB) from 42 NSCLC patients was obtained at two time points: (a) baseline: before treatment with osimertinib and (b) at progression of disease (PD). DNA methylation of the selected genes was detected in plasma-cfDNA (n = 80) and in paired CTCs (n = 74). Direct comparison of DNA methylation of six genes between plasma-cfDNA and paired CTC samples (n = 70) revealed a low concordance, indicating that CTCs and cfDNA give complementary information. DNA methylation analysis of plasma-cfDNA and CTCs indicated that when at least one of these genes was methylated there was a statistically significant increase at PD compared to baseline (p = 0.031). For the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients during osimertinib therapy indicated that DNA methylation of these genes could be a possible resistance mechanism.

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.

The treatment of non-small cell lung cancer (NSCLC) has recently evolved with the introduction of targeted therapy based on the use of tyrosine kinase inhibitors (TKIs) in patients with certain gene alterations, including EGFR, ALK, ROS1, BRAF, and MET genes. Molecular targeted therapy based on TKIs has improved clinical outcomes in a large number of NSCLC patients with advanced disease, enabling significantly longer progression-free survival (PFS). Liquid biopsy is an increasingly popular diagnostic tool for treating TKI-based NSCLC. The studies presented in this article show that detection and analysis based on liquid biopsy elements such as circulating tumor cells (CTCs), cell-free DNA (cfDNA), exosomes, and/or tumor-educated platelets (TEPs) can contribute to the appropriate selection and monitoring of targeted therapy in NSCLC patients as complementary to invasive tissue biopsy. The detection of these elements, combined with their molecular analysis (using, e.g., digital PCR (dPCR), next generation sequencing (NGS), shallow whole genome sequencing (sWGS)), enables the detection of mutations, which are required for the TKI treatment. Despite such promising results obtained by many research teams, it is still necessary to carry out prospective studies on a larger group of patients in order to validate these methods before their application in clinical practice.

The PD-1/PD-L1 axis provides CTCs an escape route from the immune system. Phosphorylation of the ribosomal protein S6 is implicated in the same pathway, following mTOR activation. The aim of the study was to investigate the expression of PD-L1 and pS6 in CTCs from NSCLC patients under Osimertinib treatment at a single cell level. CTCs were isolated using ISET from NSCLC patients’ blood [37 at baseline, 25 after the 1st cycle, and 23 at the end of treatment (EOT)]. Staining was performed using immunofluorescence. Cytokeratin-positive (CK⁺) CTCs were detected in 62% of patients. CK⁺PD-L1⁺CD45⁻ and CK⁺pS6⁺ phenotypes were detected in 38% and 41% of the patients at baseline, in 28% and 32% after 1st cycle, and in 30% and 35% at EOT, respectively. Spearman’s analysis revealed statistically significant correlations between PD-L1 and pS6 phenotypes at all time points. Survival analysis revealed that CK⁺pS6⁺ (p = 0.003) and CK^lowpS6⁺ (p = 0.021) phenotypes after 1st cycle were related to significantly decreased one-year progression-free survival (PFS_12m) and PFS, respectively. CK⁺PD-L1⁺CD45⁻phenotype at baseline and after 1st cycle showed a trend for decreased PFS_12m. Increased expression of PD-L1/pS6 in CTCs of Osimertinib-treated NSCLC patients implies the activation of the corresponding pathway, which is potentially associated with poor clinical outcomes.

ESR1 mutations are now established as a key mechanism of resistance to endocrine therapy in estrogen-receptor–positive breast cancer (ER​+ ​breast cancer) and their sensitive and specific detection in plasma-cell free DNA (plasma-cfDNA) is crucial to monitor during patient treatment. In the present proof-of-principle study, we evaluated the performance of a novel multiplex assay (12plex) for the detection of ten ESR1 mutations and AKT1 E17K in plasma-cfDNA based on Crystal Digital PCR® (Stilla Technologies, France). We analyzed 35 plasma-cfDNA samples from ER+ ​breast cancer patients and 10 samples from healthy donors and further compared the results with our previously reported ESR1 NAPA assay for D538G, Y537S, Y537C and Y537 N ESR1 mutations. Using this novel 12plex ESR1-AKT 6-color Crystal Digital PCR® assay we detected both AKT1 E17K and ESR1 D538G mutations in 5/35 (14.3%) plasma-cfDNA samples. ESR1 D538G was detected in 4/35 (11.4%) of these plasma-cfDNA samples using the ESR1 NAPA assay. Direct comparison between Crystal Digital PCR™ and the ESR1 NAPA assay revealed a high concordance (97.1%, k = 0.871, p < 0.001) for the detection of D538G mutation. The Stilla 12plex ESR1-AKT 6-color Crystal Digital PCR® assay is multiplex, highly sensitive and robust and can be used in liquid biopsy.