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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]

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance. [ABSTRACT FROM AUTHOR]

SCLC is an aggressive cancer type with high metastatic potential and bad prognosis. CTCs are a valuable source of tumor cells in blood circulation and are among the major contributors to metastasis. In this study we evaluated the number of CTCs that express PD-L1 in treatment-naïve ES-SCLC patients receiving ICI in a front-line setting. Moreover, we explored the percentages of different immune T-cell subsets in circulation to assess their potential role in predicting responses. A total of 43 patients were enrolled—6 of them with LS-SCLC, and 37 with ES-SCLC disease. In addition, PBMCs from 10 healthy donors were used as a control group. Different T-cell subtypes were examined through multicolor FACS analysis and patients' CTCs were detected using immunofluorescence staining. SCLC patients had higher percentages of PD-1-expressing CD3+CD4+ and CD3+CD8+ T-cells, as well as elevated PD-1 protein expression compared to healthy individuals. Additionally, in ES-SCLC patients, a positive correlation between CD3+CD8+PD-1+ T-cells and PD-L1+ CTCs was detected. Importantly, patients harboring higher numbers of CD3+CD8+PD-1+ T-cells together with PD-L1+CTCs had a survival advantage when receiving front-line immunotherapy. Thus, this study proposes, for first time possible, immune cell–CTCs interaction, as well as a potential novel clinical biomarker for ICI responses in ES-SCLC patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Studies have shown that JUNB and CXCR4 contribute to cell proliferation, migration and invasion, hence claiming an important role in tumor progression and metastasis, in various cancer types, including lung cancer. We have previously reported that JUNB and CXCR4 are overexpressed in circulating and disseminated tumor cells from breast cancer patients and are correlated with worse survival. In the present study, we investigated the expression of JUNB and CXCR4 in circulating tumor cells (CTCs) of non-small-cell lung cancer and small-cell lung cancer patients and determined their clinical significance. Our results showed that both JUNB and CXCR4 were overexpressed in CTCs from lung cancer patients. Furthermore, both proteins were correlated with poor survival in non-small-cell lung cancer patients, while only CXCR4 was associated with worse survival in small-cell lung cancer patients, suggesting that JUNB and CXCR4 are potentially important prognostic biomarkers for lung cancer. In the present study, we evaluated the expression of JUNB and CXCR4 in circulating tumor cells (CTCs) of lung cancer patients and investigated whether these proteins have prognostic clinical relevance. Peripheral blood from 30 patients with non-small-cell lung cancer (NSCLC) was filtered using ISET membranes, and cytospins from 37 patients with small-cell lung cancer (SCLC) were analyzed using confocal and VyCAP microscopy. Both JUNB and CXCR4 were expressed in the vast majority of lung cancer patients. Interestingly, the phenotypic patterns differed between NSCLC and SCLC patients; the (CK+/JUNB+/CXCR4+) phenotype was present in 50% of NSCLC vs. 71% of SCLC patients. Similarly, the (CK+/JUNB+/CXCR4–) was present in 44% vs. 71%, the (CK+/JUNB–/CXCR4+) in 6% vs. 71%, and the (CK+/JUNB–/CXCR4–) phenotype in 38% vs. 84%. In NSCLC, the presence of ≥1 CTCs with the (CK+/JUNB+/CXCR4+) phenotype was associated with worse progression-free survival (PFS) (p = 0.007, HR = 5.21) while ≥2 with poorer overall survival (OS) (p < 0.001, HR = 2.16). In extensive stage SCLC patients, the presence of ≥4 CXCR4-positive CTCs was associated with shorter OS (p = 0.041, HR = 5.01). Consequently, JUNB and CXCR4 were expressed in CTCs from lung cancer patients, and associated with patients' survival, underlying their key role in tumor progression. [ABSTRACT FROM AUTHOR]

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients. [ABSTRACT FROM AUTHOR]