Raw data related to article "A multiparameter liquid biopsy-based approach allows longitudinal tracking of cutaneous melanoma dynamics and early resistance to treatment"

<p><strong>Background:</strong> Melanoma heterogeneity is an obstacle in metastatic disease management. Although the advent of targeted therapy has significantly improved patient outcome, the occurrence of resistance makes monitoring of the tumor genetic landscape mandatory. Liquid biopsy could represent an important biomarker to track the evolution of the disease in real time. Thus, we aimed to correlate liquid biopsy dynamics with treatment response/progression by devising a multiplatform approach applied to longitudinal monitoring. We exploited Next Generation Sequencing and droplet digital PCR to analyze circulating tumor DNA genetic profile. The approach was applied to 17 stage IV melanoma patients treated with BRAF/MEK inhibitors, longitudinally tracked for up to 28 months.</p><p><strong>Description</strong>: Data of hybridization capture-based target enrichment sequencing (SureSelect XT HS Target Enrichment System, Agilent Technologies) for 17 patients longitudinally followed for up to 28 months (median follow-up of 480 days, range: 77-853) for a total of 48 samples. Patients had a median age of 64 and there was equal representation of males and females (47% females). The following genes were sequenced on a NextSeq 550 instrument (Illumina) with a 300-cycle NextSeq 500/550 Mid Output v2 kit: ABL1, ADAMTS18, ARID2, ATM, BAP1, BRAF, CDK4, CDKN2A, CTNNB1, CYSLTR2, EGFR, EIF1AX, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, GNA11, GNAQ, GRIN2A, HOXD8, HRAS, IDH1, KDR, KIT, KRAS, MAP2K1, MAP2K2, MC1R, MET, MITF, MLH1, NF1, NRAS, PDGFRA, PIK3CA, PLCB4, PPP6C, PREX2, PTEN, PTPN11, RAC1, RB1, SF3B1, SRC, SRSF2, STK11, STK19, TERT, TP53, U2AF1 for SNP/INDEL analysis, and BRAF, CDC42, CDK4, ERBB2, FGFR1, FGFR3, NEDD9, NFAT5, PIK3CA, PPARG, PTK2, TUSC3 for CNV assessment.</p&gt

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

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

Simple Summary Malignant melanoma is the most lethal form of skin cancer. While new therapeutic approaches have improved survival in patients with metastatic melanoma, responses are rarely sustained due to the high degree of heterogeneity at the inter- and intra-metastatic levels. The development of reliable biomarkers to monitor therapeutic response and disease progression is critical. While attention has been focused on dissecting the molecular basis responsible for treatment resistance, it is clear that epigenetic changes warrant further in-depth investigation. Indeed, many aberrantly methylated genes play a role in cell cycle control, apoptosis, and cell invasion, as well as in melanoma progression. Longitudinal monitoring of DNA methylation via liquid biopsy can provide real-time information on the behavior and stage of melanoma. 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

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

Simple Summary This work focuses on the peculiar contribution made by molecular dynamics simulation and in silico tools, in the choice of an effective second line therapy for a BRAF-mutated melanoma patient who developed resistance to the undergoing targeted therapy with BRAF/MEK inhibitors. Among the MEK inhibitors, we identified a drug alternative to trametinib, able to block the target even in the presence of a damaging mutation, and supported these findings, gathered by an in silico approach, with a liquid biopsy tracking of the response to treatment. The evolution of the disease, before and after the therapy change, was followed by analysis of the circulating tumor DNA and circulating melanoma cells. 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

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

Evaluation of Circulating Endothelial Cells (CECs) As Marker of Endothelial Damage in Allo-Transplanted Patients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS): The Cecinvod Study

Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a potentially fatal complication after allogeneic stem cell transplantation (alloSCT). Identifying a predictive biomarker for VOD has been challenging. Since endothelial injury is considered one of the main pathogenic factors for VOD onset, with the CECinVOD prospective study we aimed to evaluate Circulating Endothelial Cells (CEC) in allo-transplanted patients (pts) at higher risk to develop VOD. From October 2020 to November 2022, 150 pts have been enrolled in the CECinVOD study from 11 Italian Bone Marrow Transplantation Units. All pts must be older than 18 years and undergoing myeloablative alloSCT. CECs were detected using the CellSearch system, the FDA-approved immunomagnetic selection approach incorporating ferrofluid nanoparticles (anti CD146) and fluorophore-labelled antibodies (anti CD105, CD45 and DAPI). CEC were defined as CD146+, CD105+, DAPI+ and CD45-. CEC were collected at the following timepoints: before conditioning regimen (T0), at the end of conditioning regimen and before alloSCT (T1), at the time of neutrophils engraftment (T2), and 7-10 days after engraftment (T3). In pts who developed VOD, additional timepoints were collected as follows: at any time of suspected or proven VOD onset (T4), and then weekly during Defibrotide treatment (T5-T8). SOS/VOD was defined according to the 2016 European Group for Blood and Marrow Transplantation criteria. Pts' main characteristics are summarized in Table 1. Six out of 150 pts (4%) developed VOD during the follow up (4 “severe”, and 2 “very severe”). All pts were treated with Defibrotide, obtaining a complete remission in 5 of them, while 1 pt died due to VOD complications. Pts receiving TBI-based regimen were more likely to develop VOD compared to those receiving Treosulfan (10 to 14 g/m2) or Busulfan ev (9.6 to 12.8 mg/kg) (p 0.08). Similarly, higher baseline levels of bilirubin were associated with a higher incidence of VOD (p 0.08). Considering the CECs analysis, 615 samples were evaluated. At the enrollment, CECs levels were not related with any of clinical characteristics analyzed, except for the number of previous treatments. Indeed, those pts with 2 or more previous lines of treatments had higher levels of CECs (OR=0.53; p<0.001). Considering the different timepoints, conditioning regimen and alloSCT result in higher levels of CECs. Thus, CEC were higher at T1 than at T0 (p 0.02), as well as they were even higher at T2 than at T1 (p<0.0001) (Fig.1). Conversely, no significant differences have been observed between T3 and T2. No other clinical characteristic was correlated with CEC counts at the different timepoints. Pts who developed VOD had higher median levels of CEC at all the timepoints analyzed, but this difference has not achieved statistical significance, probably due to the low number of pts in the VOD group. At VOD onset, the pts always had an increase in CEC levels compared with the previous timepoint. After defibrotide treatment, the CEC levels increased in the first week, while they progressively decreased during the VOD treatment (T6 and T7, -50,7% and -71,5%, respectively). Recently, another endothelial activation marker has been considered: the Easix score. We didn't find any relationship between the Easix score at transplant and the VOD onset, even if a trend may be observed. We also investigated whether the CEC levels may be related with Easix score, but we didn't find any relationship at each timepoint. Interestingly, in a subgroup of pts CECs have been observed as a cluster of multiple cells. This data is firstly described in allo-SCT. Its functional significate remains unclear, but we found a relationship between CECs cluster and the number of CECs (p < 0.001). The incidence of VOD in our prospective study was low with respect to the one reported in the literature. This can be explained by the changes in alloSCT from the past (better selection of the pts, lower use of TBI, higher use of reduced intensity regimen) and the retrospective nature of most of the previous reports. We show that CECs can be considered reliable marker of endothelial damage in alloSCT pts, highlighting the impact of previous treatments, the conditioning regimen, and allo-SCT itself. Increased CEC level may be helpful to confirm VOD diagnosis, as well as their monitoring may be useful to evaluate the response to the treatment for VOD