Evaluation of Circulating Endothelial Cells as Direct Marker of Endothelial Damage in Allo-Transplant Recipients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome.

Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a rare but potentially fatal complication following allogenic hematopoietic cell transplantation (allo-HCT). Timely identification of SOS/VOD to allow for prompt treatment is critical, but identifying a VOD-predictive biomarker remains challenging. Given the pivotal role of endothelial dysfunction in SOS/VOD pathophysiology, the CECinVOD study prospectively evaluated levels of circulating endothelial cells (CECs) in patients undergoing allo-HCT with a myeloablative conditioning (MAC) regimen to investigate the potential of CEC level in predicting and diagnosing SOS/VOD. A total of 150 patients from 11 Italian bone marrow transplantation units were enrolled. All participants were age >18 years and received a MAC regimen, putting them at elevated risk of developing SOS/VOD. Overall, 6 cases of SOS/VOD (4%) were recorded. CECs were detected using the Food and Drug Administration-approved CellSearch system, an immunomagnetic selection-based platform incorporating ferrofluid nanoparticles and fluorescent-labeled antibodies, and were defined as CD146+, CD105+, DAPI+, or CD45-. Blood samples were collected at the following time points: before (T0) and at the end of conditioning treatment (T1), at neutrophil engraftment (T2), and at 7 to 10 days postengraftment (T3). For patients who developed VOD, additional samples were collected at any suspected or proven VOD onset (T4) and weekly during defibrotide treatment (T5 to T8). A baseline CEC count >17/mL was associated with an elevated risk of SOS/VOD (P = .04), along with bilirubin level >1.5 mg/mL and a haploidentical donor hematopoietic stem cell source. Postconditioning regimen (T1) CEC levels were elevated (P = .02), and levels were further increased at engraftment (P < .0001). Additionally, patients developing SOS/VOD after engraftment, especially those with late-onset SOS/VOD, showed a markedly higher relative increase (>150%) in CEC count. Multivariate analysis supported these findings, along with a high Endothelial Activation and Stress Index (EASIX) score at engraftment (T2). Finally, CEC kinetics corresponded with defibrotide treatment. After the start of therapy (T4), CEC levels showed an initial increase in the first week (T5), followed by a progressive decrease during VOD treatment (T6 and T7) and a return to pre-SOS/VOD onset levels at resolution of the complication. This prospective multicenter study reveals a low incidence of SOS/VOD in high-risk patients compared to historical data, in line with recent reports. The results from the CECinVOD study collectively confirm the endothelial injury in allo-HCT and its role in in the development of SOS/VOD, suggesting that CEC level can be a valuable biomarker for diagnosing SOS/VOD and identifying patients at greater risk of this complication, especially late-onset SOS/VOD. Furthermore, CEC kinetics may support treatment strategies by providing insight into the optimal timing for discontinuing defibrotide treatment.

A multiparameter liquid biopsy approach allows to track melanoma dynamics and identify early treatment resistance.

Melanoma heterogeneity is a hurdle in metastatic disease management. Although the advent of targeted therapy has significantly improved patient outcomes, the occurrence of resistance makes monitoring of the tumor genetic landscape mandatory. Liquid biopsy could represent an important biomarker for the real-time tracing of disease evolution. Thus, we aimed to correlate liquid biopsy dynamics with treatment response and progression by devising a multiplatform approach applied to longitudinal melanoma patient monitoring. We conceived an approach that exploits Next Generation Sequencing (NGS) and droplet digital PCR, as well as the FDA-cleared platform CellSearch, to analyze circulating tumor DNA (ctDNA) trend and circulating melanoma cell (CMC) count, together with their customized genetic and copy number variation analysis. The approach was applied to 17 stage IV melanoma patients treated with BRAF/MEK inhibitors, followed for up to 28 months. BRAF mutations were detected in the plasma of 82% of patients. Single nucleotide variants known or suspected to confer resistance were identified in 70% of patients. Moreover, the amount of ctDNA, both at baseline and during response, correlated with the type and duration of the response itself, and the CMC count was confirmed to be a prognostic biomarker. This work provides proof of principle of the power of this approach and paves the way for a validation study aimed at evaluating early ctDNA-guided treatment decisions in stage IV melanoma. The NGS-based molecular profile complemented the analysis of ctDNA trend and, together with CMC analysis, revealed to be useful in capturing tumor evolution.

Molecular Modeling Unveils the Effective Interaction of B-RAF Inhibitors with Rare B-RAF Insertion Variants.

The Food and Drug Administration (FDA) has approved MAPK inhibitors as a treatment for melanoma patients carrying a mutation in codon V600 of the BRAF gene exclusively. However, BRAF mutations outside the V600 codon may occur in a small percentage of melanomas. Although these rare variants may cause B-RAF activation, their predictive response to B-RAF inhibitor treatments is still poorly understood. We exploited an integrated approach for mutation detection, tumor evolution tracking, and assessment of response to treatment in a metastatic melanoma patient carrying the rare p.T599dup B-RAF mutation. He was addressed to Dabrafenib/Trametinib targeted therapy, showing an initial dramatic response. In parallel, in-silico ligand-based homology modeling was set up and performed on this and an additional B-RAF rare variant (p.A598_T599insV) to unveil and justify the success of the B-RAF inhibitory activity of Dabrafenib, showing that it could adeptly bind both these variants in a similar manner to how it binds and inhibits the V600E mutant. These findings open up the possibility of broadening the spectrum of BRAF inhibitor-sensitive mutations beyond mutations at codon V600, suggesting that B-RAF V600 WT melanomas should undergo more specific investigations before ruling out the possibility of targeted therapy.

Investigating the Retained Inhibitory Effect of Cobimetinib against p.P124L Mutated MEK1: A Combined Liquid Biopsy and in Silico Approach.

The systemic treatment of metastatic melanoma has radically changed, due to an improvement in the understanding of its genetic landscape and the advent of targeted therapy. However, the response to BRAF/MEK inhibitors is transitory, and big efforts were made to identify the mechanisms underlying the resistance. We exploited a combined approach, encompassing liquid biopsy analysis and molecular dynamics simulation, for tracking tumor evolution, and in parallel defining the best treatment option. The samples at different time points were collected from a BRAF-mutant melanoma patient who developed an early resistance to dabrafenib/trametinib. The analysis of the circulating tumor DNA (ctDNA) identified the MEK1 p.P124L mutation that confers resistance to trametinib. With an in silico modeling, we identified cobimetinib as an alternative MEK inhibitor, and consequently suggested a therapy switch to vemurafenib/cobimetinib. The patient response was followed by ctDNA tracking and circulating melanoma cell (CMC) count. The cobimetinib administration led to an important reduction in the BRAF p.V600E and MEK1 p.P124L allele fractions and in the CMC number, features suggestive of a putative response. In summary, this study emphasizes the usefulness of a liquid biopsy-based approach combined with in silico simulation, to track real-time tumor evolution while assessing the best treatment option.

Methylation Markers in Cutaneous Melanoma: Unravelling the Potential Utility of Their Tracking by Liquid Biopsy.

Malignant melanoma is the most serious, life-threatening form of all dermatologic diseases, with a poor prognosis in the presence of metastases and advanced disease. Despite recent advances in targeted therapy and immunotherapy, there is still a critical need for a better understanding of the fundamental mechanisms behind melanoma progression and resistance onset. Recent advances in genome-wide methylation methods have revealed that aberrant changes in the pattern of DNA methylation play an important role in many aspects of cancer progression, including cell proliferation and migration, evasion of cell death, invasion, and metastasization. The purpose of the current review was to gather evidence regarding the usefulness of DNA methylation tracking in liquid biopsy as a potential biomarker in melanoma. We investigated the key genes and signal transduction pathways that have been found to be altered epigenetically in melanoma. We then highlighted the circulating tumor components present in blood, including circulating melanoma cells (CMC), circulating tumor DNA (ctDNA), and tumor-derived extracellular vesicles (EVs), as a valuable source for identifying relevant aberrations in DNA methylation. Finally, we focused on DNA methylation signatures as a marker for tracking response to therapy and resistance, thus facilitating personalized medicine and decision-making in the treatment of melanoma patients.

Liquid Biopsy in Pediatric Renal Cancer: Stage I and Stage IV Cases Compared.

Pediatric renal cancer is rare, and robust evidence for treatment recommendations is lacking. In the perspective of personalized medicine, clinicians need new biomarkers to improve risk stratification and patients' follow-up. Herein, we analyzed some liquid biopsy tools, which have been never tested in pediatric renal cancer: namely, circulating tumor cells (CTCs); the expression of M30, an apoptosis marker, to test CTC metastatic potential; and c-MET expression in CTCs, because of its role in renal cancer progression and drug-resistance. Furthermore, we evaluated the Circulating Endothelial Cells (CECs), whose utility we previously demonstrated in adult metastatic renal cancer treated with anti-angiogenic therapy. We compared two renal cell carcinomas of clear-cell type, stage I and IV, which underwent surgery and surgery plus Sunitinib, respectively. Baseline CTC level and its changes during follow-up were consistent with patients' outcome. In case 2, stage IV, the analysis of CECs performed during Sunitinib revealed a late response to treatment consistent with poor outcome, as the finding of M30-negative, viable cells. Noteworthily, few CTCs were MET-positive in both cases. Our study highlights the feasibility for a change in the prognostic approach and follow-up of childhood renal cancer, with a view to guide a better treatment design.

Possible role of circulating tumor cells in early detection of lung cancer.

The prognosis of lung cancer varies highly depending on the disease stage at diagnosis, from a 5-year survival rate close to 90% in stage I, to 10% or less in stage IV disease. The enhancement of early diagnosis of this malignancy is mandatory to improve prognosis, because lung cancer patients stay long asymptomatic or few symptomatic after disease onset. Nowadays, liquid biopsy has emerged as a minimally-invasive tool to address the urgent need for real time monitoring, stratification, and personalized treatment of malignancies, including lung cancer. Liquid biopsy refers to a class of biomarkers, including circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA) and tumor-derived extracellular vesicles (tdEV). Since CTCs represent a crucial step in disease progression and metastasis, we reviewed here the scientific literature about the use of CTCs in early diagnosis of lung cancer; different techniques, and different strategies (e.g., source of analysis sample or high-risk groups of patients) were discussed showing the potential of implementing liquid biopsy in the clinical routine of non-metastatic lung cancer.

Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer.

Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3-4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306; p < 0.0001) and during therapy (139 vs. 266 days; p = 0.0009); ii) a worse OS pre-therapy (p = 0.0003, 82% survival vs. 20%) and during therapy (p = 0.0301, 67% survival vs. 38%); iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.

The Interplay between Circulating Tumor Cells and the Immune System: From Immune Escape to Cancer Immunotherapy.

Circulating tumor cells (CTCs) have aroused increasing interest not only in mechanistic studies of metastasis, but also for translational applications, such as patient monitoring, treatment choice, and treatment change due to tumor resistance. In this review, we will assess the state of the art about the study of the interactions between CTCs and the immune system. We intend to analyze the impact that the cells of the immune system have in limiting or promoting the metastatic capability of CTCs. To this purpose, we will examine studies that correlate CTCs, immune cells, and patient prognosis, and we will also discuss relevant animal models that have contributed to the understanding of the mechanisms of immune-mediated metastasis. We will then consider some studies in which CTCs seem to play a promising role in monitoring cancer patients during immunotherapy regimens. We believe that, from an accurate and profound knowledge of the interactions between CTCs and the immune system, new immunotherapeutic strategies against cancer might emerge in the future.

Critical issues in the clinical application of liquid biopsy in non-small cell lung cancer.

Current therapeutic options for non-small cell lung cancer (NSCLC) patients are chemotherapy and targeted therapy directed mainly against epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements. Targeted therapy relies on the availability of tumor biopsies for molecular profiling at diagnosis and to longitudinally monitor treatment response and resistance development. Unfortunately, tumor biopsy might be invasive, recover poor material of suboptimal quality, and cause sample bias due to tumor heterogeneity. Many studies have illustrated the potential of liquid biopsy as minimal invasive approach to respond to the urgent need for real time monitoring, stratification, and personalized optimized treatment in NSCLC patients. In principle, the liquid biopsy could provide the genetic landscape of primary and metastatic cancerous lesions, detecting "druggable" genomic alterations or associated with treatment resistance. Moreover, it would guarantee the prognostic/predictive biomarkers evaluation in patients for whom biopsies are inaccessible or difficult to repeat. At this regard, the prognostic value of circulating tumor cells (CTCs) in NSCLC patients has been largely investigated, but still their clinical utility as tumor biomarker is hampered by the lack of a consensus on the criteria necessary and sufficient to define them and on the standard operating procedures (SOPs) for their assessment. This review will summarize current developments on liquid biopsy in NSCLC, addressing the technology issues that contribute to the poor ability to track CTCs in the blood of NSCLC patients, thus limiting their extensive use in the clinical practice, and analyzing the solutions adopted to overcome such limits, on the road towards the clinical validation.

Failure mode and effects analysis of witnessing protocols for ensuring traceability during PGD/PGS cycles.

Preimplantation genetic diagnosis and aneuploidy testing (PGD/PGS) use is constantly growing in IVF, and embryo/biopsy traceability during the additional laboratory procedures needed is pivotal. An electronic witnessing system (EWS), which showed a significant value in decreasing mismatch occurrence and increasing detection possibilities during standard care IVF, still does not guarantee the same level of efficiency during PGD/PGS cycles. Specifically, EWS cannot follow single embryos throughout the procedure. This is however critical when an unambiguous diagnosis corresponds to each embryo. Failure Mode and Effects Analysis (FMEA) is a proactive method generally adopted to define tools ensuring safety along a procedure. Due to the implementation of a large quantitative PCR (qPCR)-based blastocyst stage PGD/PGS programme in our centre, and to evaluate the potential procedural risks, a FMEA was performed in September 2014. Forty-four failure modes were identified, among which six were given a moderate risk priority number (>15) (RPN; product of estimated occurrence, severity and detection). Specific corrective measures were then introduced and implemented, and a second evaluation performed six months later. The meticulous and careful application of such measures allowed the risks to be decreased along the whole protocol, by reducing their estimated occurrence and/or increasing detection possibilities.