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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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

Liquid biopsy, a minimally invasive approach, is a highly powerful clinical tool for the real‐time follow‐up of cancer and overcomes many limitations of tissue biopsies. Epigenetic alterations have a high potential to provide a valuable source of innovative biomarkers for cancer, owing to their stability, frequency, and noninvasive accessibility in bodily fluids. Numerous DNA methylation markers are now tested in circulating tumor DNA (ctDNA) as potential biomarkers, in various types of cancer. DNA methylation in combination with liquid biopsy is very powerful in identifying circulating epigenetic biomarkers of clinical importance. Blood‐based epigenetic biomarkers have a high potential for early detection of cancer since DNA methylation in plasma can be detected early during cancer pathogenesis. In this review, we summarize the latest findings on DNA methylation markers in ctDNA for early detection, prognosis, minimal residual disease, risk of relapse, treatment selection, and resistance, for breast, prostate, lung, and colorectal cancer. [ABSTRACT FROM AUTHOR]

Liquid biopsy analysis, mainly based on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK3CA hotspot mutations (E545K, H1047R) in EpCAM‐positive CTCs and paired plasma‐ctDNA in breast cancer (BrCa). PIK3CA hotspot mutations in CTCs and ctDNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma‐ctDNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK3CA hotspot mutations in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma‐ctDNA, in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma‐ctDNA,PIK3CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors' plasma‐ctDNA samples were positive. Our direct comparison study in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma‐ctDNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by ddPCR methodology, and the concordance between our assay and ddPCR for PIK3CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK3CA hotspot mutations were detected in samples found to be negative for CTCs in CellSearch®. Our data demonstrated for the first time that (a) PIK3CA hotspot mutations are present at high frequencies in CTCs isolated from CellSearch® cartridges and paired plasma‐ctDNA both in early and metastatic BrCa, (b) the detection and concordance of PIK3CA hotspot mutations between plasma‐ctDNA and CTCs are higher in the metastatic setting, (c) PIK3CA mutational status significantly changes after therapeutic intervention, and (d) PIK3CA mutation detection in CTCs and plasma‐ctDNA provides complementary information. [ABSTRACT FROM AUTHOR]

Ovarian cancer remains the most lethal disease among gynecological malignancies despite the plethora of research studies during the last decades. The majority of patients are diagnosed in an advanced stage and exhibit resistance to standard chemotherapy. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) represent the main liquid biopsy approaches that offer a minimally invasive sample collection. Both have shown a diagnostic, prognostic and predictive value in many types of solid malignancies and recent studies attempted to shed light on their role in ovarian cancer. This review is mainly focused on the clinical value of both CTCs and ctDNA in ovarian cancer and, more specifically, on their potential as diagnostic, prognostic and predictive tumor biomarkers. [ABSTRACT FROM AUTHOR]

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients. [ABSTRACT FROM AUTHOR]

A variety of molecular assays have been developed for CTCs detection and molecular characterization. Molecular assays are based on the nucleic acid analysis in CTCs and are based on total RNA isolation and subsequent mRNA quantification of specific genes, or isolation of genomic DNA that can be for DNA methylation studies and mutation analysis. This review is mainly focused on gene expression and methylation studies in CTCs in various types of cancer. [ABSTRACT FROM AUTHOR]

Lung cancer is the most common cause of cancer-related deaths that frequently metastasizes prior to disease diagnosis. Circulating tumor cells (CTCs) are found in many different types of epithelial tumors and are of great clinical interest in terms of prognosis and therapy intervention. Here, we present and discuss epithelial cell adhesion moleculedependent and -independent capture of CTCs in non-small cell lung cancer (NSCLC) and the clinical relevance of CTC detection and characterization. Taking blood samples and analyzing CTCs as "liquid biopsy" might be a far less invasive diagnostic strategy than biopsies of lung tumors or metastases. Moreover, sequential blood sampling allows to study the dynamic changes of tumor cells during therapy, in particular the development of resistant tumor cell clones. [ABSTRACT FROM AUTHOR]

The presence of circulating tumor cells (CTCs) in peripheral blood can serve as a 'liquid biopsy' approach and has thus emerged lately as one of the hottest fields in cancer research. CTCs can be isolated from blood in a non-invasive approach, and can be used to follow patients over time since these cells can provide significant information for a better understanding of tumor biology and tumor cell dissemination. CTC molecular characterization offers the unique potential to better understand the biology of metastasis and resistance to established therapies, and analysis of these cells presents a promising field for both advanced and early-stage patients. CTC detection, enumeration, and molecular characterization are very challenging since CTCs are rare, and the amount of available sample is very limited. Since detection of CTCs has been shown to be of considerable utility in the clinical management of patients with solid cancers, various analytical systems for their isolation and detection have been developed. New areas of research are directed towards developing novel assays for single-CTC isolation and molecular characterization. The clinical significance of CTCs has been evaluated in many types of solid cancers, and the CTC enumeration test in metastatic breast, colorectal, and prostate cancer was cleared by the FDA almost a decade ago. This review is mainly focused on the clinical potential of CTCs as novel biomarkers in 10 different types of solid cancers: breast, ovarian, prostate, lung, colorectal, hepatocellular carcinoma, pancreatic, head and neck, bladder cancer and melanoma. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) provide unique information for the management of breast cancer patients, since their detection and monitoring is useful for prognosis, prediction of response to therapy, or monitoring clinical course in patients with localized or metastatic disease. Currently, the most practical application of CTCs is monitoring of patients with metastatic disease. Elevated CTC levels prior to initiation of a new systemic therapy are associated with a worse prognosis while persistently elevated CTC levels strongly suggest that the therapeutic regimen with which the patient is being treated is not working. New areas of research are directed toward developing novel sensitive assays for CTC molecular characterization. Molecular characterization of CTCs is very important for the future use of CTCs as targets of novel therapies. This review has focused on the presentation of recent data showing that CTCs are emerging as novel tumor biomarkers for prognostic and predictive purposes in breast cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid biopsy is a highly useful tool for the management of NSCLC patients and could provide valuable information on early detection of resistance to osimertinib. Epigenetic biomarkers are very promising for the early diagnosis, prognosis, and prediction of drug response in many types of cancer. We performed a DNA methylation analysis in plasma cell-free DNA and paired CTCs of NSCLC patients before osimertinib treatment and at progression of disease (PD). Our results revealed a significant increase in DNA methylation at PD. Epigenetic alterations should be further evaluated as a possible resistance mechanism to osimertinib and their detection in liquid biopsy samples can be valuable for the follow-up of patients in real time. 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. [ABSTRACT FROM AUTHOR]

In metastatic breast cancer (MBC) the molecular characterization of circulating tumor cells (CTCs) provides a unique tool to understand metastasis-biology and therapy-resistance. We evaluated the prognostic significance of gene expression in EpCAM(+) CTCs in 46 MBC patients based on a long follow-up. We selected a panel consisting of stem cell markers (CD24, CD44, ALDH1), the mesenchymal marker TWIST1, receptors (ESR1, PGR, HER2, EGFR) and the epithelial marker CK-19. Singleplex RT-qPCR was used for TWIST1 and CK-19 and multiplex RT-qPCR for stem cell markers and receptors. A group of 19 healthy donors (HD) was used as control. Univariate (p = 0.001) and multivariate analysis (p = 0.002) revealed the prognostic value of combined gene expression of CK-19(+), CD44high/CD24low, ALDH1high/CD24low and HER2 over-expression for overall survival (OS). The Kaplan–Meier estimates of OS were significantly different in patients positive for CK-19 (p = 0.028), CD44high/CD24low (p = 0.002), ALDH1high/CD24low (p = 0.007) and HER2-positive (p = 0.022). Our results indicate that combined gene expression analysis in EpCAM(+) CTCs provides prognostic information in MBC. [ABSTRACT FROM AUTHOR]

(1) Background: The aim of the study was to evaluate the prognostic significance of EMT-associated (TWIST1) and stem-cell (SC) transcript (CD24, CD44, ALDH1) quantification in EpCAM+ circulating tumor cells (CTCs) of early breast cancer patients. (2) Methods: 100 early stage breast cancer patients and 19 healthy donors were enrolled in the study. CD24, CD44, and ALDH1 transcripts of EpCAM+ cells were quantified using a novel highly sensitive and specific quadraplex RT-qPCR, while TWIST1 transcripts were quantified by single RT-qPCR. All patients were followed up for more than 5 years. (3) Results: A significant positive correlation between overexpression of TWIST1 and CD24−/low/CD44high profile was found. Kaplan–Meier analysis revealed that the ER/PR-negative (HR-) patients and those patients with more than 3 positive lymph nodes that overexpressed TWIST1 in EpCAM+ cells had a significant lower DFI (log rank test; p < 0.001, p < 0.001) and OS (log rank test; p = 0.006, p < 0.001). Univariate and multivariate analysis also revealed the prognostic value of TWIST1 overexpression and CD24−/low/CD44high and CD24−/low/ALDH1high profile for both DFI and OS. (4) Conclusions: Detection of TWIST1 overexpression and stem-cell (CD24, CD44, ALDH1) transcripts in EpCAM+ CTCs provides prognostic information in early stage breast cancer patients. [ABSTRACT FROM AUTHOR]

Objectives: Chemoradiation can induce immunogenic (ICD) or tolerogenic cell death. ICD relies on the generation of damage-associated molecular patterns which can stimulate toll-like receptors (TLRs). We sought to determine whether we can predict responses to chemoradiation by measuring surrogate biomarkers of ICD in a cohort of patients with locally advanced (LA) head and neck squamous cell carcinoma (HNSCC).Materials and Methods: In a cohort of 113 LA HNSCC pts we evaluated expression of TLR4, TLR7 and TLR9 in the EpCAM + circulating tumor cell (CTC) fraction at baseline and after cisplatin chemoradiation. We also quantified changes in chemokines CXCL10, CXCL16 and IL-2R in the serum.Results: Seventy three patients had evaluable specimens. Among cases with biomarker assessment at baseline and post treatment, 36.8% had an increase in CXCL10 levels (p = 0.022), 73.7% had an increase in CXCL16 levels (p = 0.002) and 63.8% had an increase in IL2Ra levels (p = 0.032) with treatment. 52.0% of evaluable cases at baseline and post-treatment had an increase in TLR4 levels (p = 0.996), 42.9% had an increase in TLR7 levels (p = 0.042) and 27.7% had increase in TLR9 levels (p = 0.011) with treatment. CXCL10 levels at baseline were significantly associated with PFS and OS (p = 0.010 and p = 0.032, respectively).Conclusions: Our results suggest that chemoradiation leads to quantifiable effects in surrogate markers of ICD. These effects may inform trials combining chemoradiation with immune checkpoint inhibitors. In addition, CXCL10 has prognostic effect in pts treated with chemoradiation. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: : The development and validation of a nested RT-PCR methodology for the detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer and evaluation of its prognostic significance. Design and methods: : Different combinations of specific primers were in silico designed and selected, so that false positive results due to genomic DNA contamination were avoided. The specificity of the primers used was evaluated in 30 healthy individuals, 20 patients with colorectal cancer and 20 patients with non-small cell lung cancer. The method was applied in 101 patients with operable breast cancer before the administration of adjuvant chemotherapy and 39 patients with metastatic breast cancer. Results: : Mammaglobin A-mRNA-positive cells were detected in 14/101 (13.9%) of early breast cancer patients but not in the control population studied (0%); 9 of them (64.3%) relapsed during the follow-up period. Mammaglobin A was detected in 7/39 (17.9%) of patients with verified metastasis. Multivariate analysis revealed the detection of Mammaglobin A-mRNA-positive cells, as an independent risk factor for reduced DFI. Conclusions: : Mammaglobin A is a highly specific molecular marker for the detection of circulating tumor cells in operable breast cancer, with important prognostic applications. [Copyright &y& Elsevier]