Liquid biopsy for the management of NSCLC patients under osimertinib treatment.

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.

Pre-analytical conditions and implementation of quality control steps in liquid biopsy analysis.

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.

Liquid Biopsy Analysis as a Tool for TKI-Based Treatment in Non-Small Cell Lung Cancer.

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.

PD-L1/pS6 in Circulating Tumor Cells (CTCs) during Osimertinib Treatment in Patients with Non-Small Cell Lung Cancer (NSCLC).

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 CKlowpS6+ (p = 0.021) phenotypes after 1st cycle were related to significantly decreased one-year progression-free survival (PFS12m) and PFS, respectively. CK+PD-L1+CD45−phenotype at baseline and after 1st cycle showed a trend for decreased PFS12m. 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.

Effect of Osimertinib on CTCs and ctDNA in EGFR Mutant Non-Small Cell Lung Cancer Patients: The Prognostic Relevance of Liquid Biopsy.

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.

DNA Methylation Analysis in Plasma Cell-Free DNA and Paired CTCs of NSCLC Patients before and after Osimertinib Treatment.

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, RARß, 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.

Detection of EGFR Mutations in Plasma cfDNA and Paired CTCs of NSCLC Patients before and after Osimertinib Therapy Using Crystal Digital PCR.

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.

Androgen Receptor and PIM1 Expression in Tumor Tissue of Patients With Triple-negative Breast Cancer.

BACKGROUND/AIM: Effective targeted therapies for triple-negative breast cancer (TNBC) are limited. In a subset of TNBC, androgen receptor (AR) plays an important role, while the human proviral integration site for Moloney murine leukemia virus-1 (PIM1) overexpression is also implicated. PIM1 kinases phosphorylate AR, thus regulating its transcriptional activity, regardless of the presence or not of androgens. We evaluated the expression of AR and PIM1 and their prognostic significance in TNBC. MATERIALS AND METHODS: AR and PIM1 transcripts were quantified by quantitative reverse transcription polymerase chain reaction in formalin-fixed paraffin-embedded tumor from 141 patients with TNBC. RESULTS: AR was expressed in 38.3%, PIM1 in 10.6%, while co-expression of AR and PIM1 was detected in 7/141 cases (5.0%). No prognostic significance of AR or PIM1 was reached for overall or disease-free survival. CONCLUSION: Co-expression of AR and PIM1 exists in only in a small percentage of patients with TNBC. The implications of this finding in the therapeutic management of patients with TNBC should be investigated in larger patient cohorts.

Gene expression in circulating tumor cells reveals a dynamic role of EMT and PD-L1 during osimertinib treatment in NSCLC patients.

Liquid biopsy is a tool to unveil resistance mechanisms in NSCLC. We studied changes in gene expression in CTC-enriched fractions of EGFR-mutant NSCLC patients under osimertinib. Peripheral blood from 30 NSCLC patients before, after 1 cycle of osimertinib and at progression of disease (PD) was analyzed by size-based CTC enrichment combined with RT-qPCR for gene expression of epithelial (CK-8, CK-18, CK-19), mesenchymal/EMT (VIM, TWIST-1, AXL), stem cell (ALDH-1) markers, PD-L1 and PIM-1. CTCs were also analyzed by triple immunofluorescence for 45 identical blood samples. Epithelial and stem cell profile (p = 0.043) and mesenchymal/EMT and stem cell profile (p = 0.014) at PD were correlated. There was a strong positive correlation of VIM expression with PIM-1 expression at baseline and increased PD-L1 expression levels at PD. AXL overexpression varied among patients and high levels of PIM-1 transcripts were detected. PD-L1 expression was significantly increased at PD compared to baseline (p = 0.016). The high prevalence of VIM positive CTCs suggest a dynamic role of EMT during osimertinib treatment, while increased expression of PD-L1 at PD suggests a theoretical background for immunotherapy in EGFR-mutant NSCLC patients that develop resistance to osimertinib. This observation merits to be further evaluated in a prospective immunotherapy trial.