Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. This prospective liquid biopsy (LB) study was based on a group of 62 metastatic castration resistant prostate cancer (mCRPC) patients and 10 healthy donors (HD) as controls. Identical blood draws were used to: (a) enumerate CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and (b) analyze CTCs and paired plasma-derived exosomes at the gene expression and DNA methylation level. CTCs were enumerated using CellSearch in 57/62 patients, with values ranging from 5 to 854 cells/7.5 mL PB. Our results revealed for the first time a significantly higher positivity of gene expression markers (CK-8, CK-18, TWIST1, PSMA, AR-FL, AR-V7, AR-567 and PD-L1 mRNA) in EpCAM-positive CTCs compared to plasma-derived exosomes. GSTP1, RASSF1A and SCHLAFEN were methylated both in CTC and exosomes. In CTCs, Kaplan–Meier analysis revealed that CK-19 (p = 0.009), PSMA (p = 0.001), TWIST1 (p = 0.001) expression and GSTP1 (p = 0.001) methylation were correlated with OS, while in exosomes GSTP1 (p = 0.007) and RASSF1A (p = 0.001) methylation was correlated with OS. Our direct comparison study of CTCs and exosomes at gene expression and DNA methylation level, revealed for the first time a significantly higher positivity in EpCAM-positive CTCs compared to plasma-derived exosomes. Future perspective of this study should be the evaluation of clinical utility of molecular biomarkers in CTCs and exosomes on independent multicentric cohorts with mCRPC patients.

Background: PD-L1, an immune checkpoint protein, is an important biomarker for monitoring cancer patients during the administration of cancer immunotherapy. Droplet digital PCR (ddPCR), is a highly sensitive and accurate tool for the quantification of cancer biomarkers in liquid biopsy. We report the development and analytical validation of a novel duplex RT-ddPCR assay for the simultaneous quantification of PD-L1 and hypoxanthine phosphoribosyltransferase (HPRT) (used as reference gene) transcripts in circulating tumor cells (CTCs).Methods: RT-ddPCR experimental conditions were first optimized and the assay was analytically validated using synthetic standards and the BB49 and SCC47 cancer cell lines. The developed assay was further applied in 71 peripheral blood (PB) samples from head and neck squamous cell carcinoma (HNSCC) patients and 20 PB samples from healthy donors. PD-L1 and HPRT transcripts were quantified in cDNAs derived from CTCs isolated by a size-dependent microfluidic device. The developed RT-ddPCR assay was directly compared to RT-qPCR using 71 identical patient cDNA samples.Results: Analytical sensitivity was 0.64 copies/μL, while estimation of intra- and interassay variation revealed a high reproducibility (within-run CV%:4.7–23%; between-run CV%:13%). Using the developed RT-ddPCR assay 33/71(46.5%) HNSCC patients’ samples were found positive for PD-L1 expression in CTCs, while by using RT-qPCR fewer samples (23/71, 32.4%) were positive (concordance: 55/71, 77.5%).Conclusions: The developed RT-ddPCR assay for PD-L1 in CTCs is highly sensitive, specific, and reproducible; additionally, it offers improved diagnostic sensitivity over RT-qPCR. The clinical utility of the assay should be prospectively evaluated for the real-time monitoring of CTCs of cancer patients under immunotherapy.

Background: AXL, a tyrosine kinase receptor, is involved in epithelial-to-mesenchymal transition (EMT), cell survival, invasion, metastasis, and resistance to EGFR-TKIs and immune checkpoint inhibitors (ICIs). Recent clinical studies have shown promising results in NSCLC patients treated with AXL inhibitors. Methods: We evaluated AXL-mRNA overexpression in CTC fractions of NSCLC patients under osimertinib treatment (Group A, n = 39), (collected in the context of a multicenter Phase II clinical study (ClinicalTrials.gov number: NCT02771314) or immunotherapy (Group B, n = 116) at different time points collected in the context of a prospective, multicenter study (ClinicalTrials.gov number: NCT04490564). Size-based CTC enrichment (Parsortix, CelLBx Health, Guildford, UK) was used, and AXL-mRNA overexpression was evaluated using RT-qPCR. Results: In Group A, AXL-mRNA overexpression in CTC fractions was detected in 5/39 (12.8%) patients’ samples at baseline, in 5/31 (16.1%) after one cycle of treatment, in 7/79 (8.9%) during treatment and in 4/32 (12.5%) at progression of disease (PD). In Group B, AXL-mRNA overexpression in CTC fractions was detected in 8/116 (6.9%) samples before immunotherapy, in 8/71 (11.3%) after three or four cycles, while no AXL transcripts were detected at PD. Conclusions: Our results indicate that AXL-mRNA overexpression in CTC fractions deserve to be further evaluated through larger clinical studies as a potential biomarker for anti-AXL targeted therapies in NSCLC.