Identification of Plasma Lipid Alterations Associated with Melanoma Metastasis.

The aim of this study was to apply a state-of-the-art quantitative lipidomic profiling platform to uncover lipid alterations predictive of melanoma progression. Our study included 151 melanoma patients; of these, 83 were without metastasis and 68 with metastases. Plasma samples were analyzed using a targeted Lipidyzer™ platform, covering 13 lipid classes and over 1100 lipid species. Following quality control filters, 802 lipid species were included in the subsequent analyses. Total plasma lipid contents were significantly reduced in patients with metastasis. Specifically, levels of two out of the thirteen lipid classes (free fatty acids (FFAs) and lactosylceramides (LCERs)) were significantly decreased in patients with metastasis. Three lipids (CE(12:0), FFA(24:1), and TAG47:2-FA16:1) were identified as more effective predictors of melanoma metastasis than the well-known markers LDH and S100B. Furthermore, the predictive value substantially improved upon combining the lipid markers. We observed an increase in the cumulative levels of five lysophosphatidylcholines (LPC(16:0); LPC(18:0); LPC(18:1); LPC(18:2); LPC(20:4)), each individually associated with an elevated risk of lymph node metastasis but not cutaneous or distant metastasis. Additionally, seventeen lipid molecules were linked to patient survival, four of which (CE(12:0), CE(14:0), CE(15:0), SM(14:0)) overlapped with the lipid panel predicting metastasis. This study represents the first comprehensive investigation of the plasma lipidome of melanoma patients to date. Our findings suggest that plasma lipid profiles may serve as important biomarkers for predicting clinical outcomes of melanoma patients, including the presence of metastasis, and may also serve as indicators of patient survival.

Prognostic Value of the Neutrophil-to-lymphocyte Ratio, Platelet-to- lymphocyte Ratio and Monocyte-to-lymphocyte Ratio in Melanoma Patients: A Cohort Study.

The prognostic value of the neutrophil-lymphocyte ratio, platelet-lymphocyte ratio and monocyte-lymphocyte ratio in patients with melanoma has yielded controversial results in the literature. A retrospective single-centre cohort study was conducted from 1998 to 2020, including patients diagnosed with invasive melanoma. A total of 2,721 patients were included in the study. The median follow-up was 8.23 years (IQR 4.41-13.25). The median baseline neutrophil- lymphocyte ratio, platelet-lymphocyte ratio and monocyte-lymphocyte ratio values increased significantly (p < 0.001) with the increasing American Joint Committee on Cancer stage. The optimal cut-off values for neutrophil-lymphocyte ratio, platelet-lymphocyte ratio and monocyte-lymphocyte ratio were determined as 2.1, 184 and 0.2, respectively. In the multivariate analysis, high levels of neutrophil-lymphocyte ratio (≥ 2.1), platelet-lymphocyte ratio (≥ 184) and monocyte-lymphocyte ratio (≥ 0.2) were independently associated with significantly shorter melanoma-specific survival (neutrophil-lymphocyte ratio: HR 1.30, 95% CI 1.06-1.60, p = 0.013; platelet-lymphocyte ratio: HR 1.37, 95% CI 1.06-1.76, p = 0.014; monocyte- lymphocyte ratio: HR 1.29, 95% CI 1.05-1.58, p = 0.015) and overall survival (neutrophil-lymphocyte ratio: HR 1.39, 95% CI 1.19-1.64, p < 0.001; platelet- lymphocyte ratio: HR 1.44, 95% CI 1.19-1.74, p < 0.001; monocyte-lymphocyte ratio: HR 1.42, 95% CI 1.21-1.66, p < 0.001). High levels of neutrophil- lymphocyte ratio and monocyte-lymphocyte ratio were also associated with poor relapse-free survival, while platelet-lymphocyte ratio was not. In conclusion, baseline neutrophil-lymphocyte ratio, platelet-lymphocyte ratio and monocyte-lymphocyte ratio were identified as independent predictors for the prognosis of melanoma.

sIL-2R- an Immuno-biomarker for Prediction of Metastases in Uveal Melanoma.

BACKGROUND/AIM: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. High serum levels of soluble IL-2 receptor (sIL-2R) have been reported in acute inflammations and metastatic cancers. This study evaluated the potential of high/increasing sIL-2R levels in predicting metastases. PATIENTS AND METHODS: The study included a total of 1,546 sera samples of subjects from three groups: 119 healthy controls (73 subjects), 566 UM 10 year (10y) disease-free (DF) (220 patients), 861 metastatic UM (268 patients). Patients were followed-up biannually with liver ultrasound and liver function tests for the presence of metastases (Mets). Blood samples to measure the levels of sIL-2R were obtained at the time of primary diagnosis, soon after initial treatment (enucleation, brachytherapy), every 6 months, 10 years from diagnosis, at Mets confirmation by CT, and after additional treatments. RESULTS: Significantly higher sIL-2R levels were detected in the Mets patients compared to healthy controls and 10y DF patients. Compared to the upper limit of the normal levels of sIL-2R, 1,000 U/ml, its levels in metastatic UM were 61%, 25% in 10y DF UM, and 6.25% in the controls. High levels of sIL-2R in metastatic patients, decreased significantly post treatments. Individual kinetics of markers, indicated similar trends of sIL-2R compared to osteopontin and S-Protein 100, predicting metastases, which were confirmed on liver imaging. CONCLUSION: Significantly higher sIL-2R levels were evident in all UM patients with Mets. Significant increases in sIL-2R levels on serial evaluations indicated and predicted UM Mets, enabling earlier treatment of Mets, to improve survival.

Therapeutic Response Evaluation in Advanced Melanoma Patients Incorporating Plasma cfDNA, LDH, VEGF, PD-L1, and IFN-γ Measurements.

BACKGROUND/AIM: Current treatment strategies for advanced melanoma require serial assessment of disease status in affected patients. In this study, we sought to examine the relationship between radiographic tumour burden and blood borne biomarkers including plasma cfDNA, serum LDH, plasma VEGF, PD-L1 and IFN-γ in advanced melanoma patients receiving immunotherapy. We hypothesized that a combination of these explanatory variables in a suitable regression analysis model may predict changes in tumour burden during patient treatment. MATERIALS AND METHODS: We extracted and quantified circulating cfDNA, LDH, VEGF, PD-L1, and IFN-γ from thirty patients with stage IV melanoma at baseline and at six months. All participating patients were evaluated with paired blood sample collection and CT scan assessments during treatment. RESULTS: Changes in radiographic tumour burden correlated with changes in levels of cfDNA (p≤0.001), LDH (p≤0.001), VEGF (p≤0.001), and PD-L1 (p<0.05) during treatment. Multiple regression analysis consisting of the follow-up to baseline assessment ratios of cfDNA, LDH, VEGF and PD-L1 explained changes in tumour burden (F (4, 23)=32.05, p<0.001); with an R2 of 0.8479 (Y=ß0+ß1*B+ß2*C+ß3*D+ß4*E). CONCLUSION: A quantitative measure of cfDNA, LDH, VEGF and PD-L1 may complement current methods of assessing tumour burden in advanced melanoma patients.

Detection of clinical progression through plasma ctDNA in metastatic melanoma patients: a comparison to radiological progression.

BACKGROUND: The validity of circulating tumour DNA (ctDNA) as an indicator of disease progression compared to medical imaging in patients with metastatic melanoma requires detailed evaluation. METHODS: Here, we carried out a retrospective ctDNA analysis of 108 plasma samples collected at the time of disease progression. We also analysed a validation cohort of 66 metastatic melanoma patients monitored prospectively after response to systemic therapy. RESULTS: ctDNA was detected in 62% of patients at the time of disease progression. For 67 patients that responded to treatment, the mean ctDNA level at progressive disease was significantly higher than at the time of response (P < 0.0001). However, only 30 of these 67 (45%) patients had a statistically significant increase in ctDNA by Poisson test. A validation cohort of 66 metastatic melanoma patients monitored prospectively indicated a 56% detection rate of ctDNA at progression, with only two cases showing increased ctDNA prior to radiological progression. Finally, a correlation between ctDNA levels and metabolic tumour burden was only observed in treatment naïve patients but not at the time of progression in a subgroup of patients failing BRAF inhibition (N = 15). CONCLUSIONS: These results highlight the low efficacy of ctDNA to detect disease progression in melanoma when compared mainly to standard positron emission tomography imaging.

Naturally Occurring Telomerase-Specific CD4 T-Cell Immunity in Melanoma.

CD4 T cells play a key role in anticancer immunity. In this study, we investigate the clinical relevance of circulating CD4 T helper type 1 (Th1) response against telomerase (anti-TERT Th1 response) in patients with melanoma. The spontaneous anti-TERT Th1 response was detected in 54.5% (85/156) of patients with melanoma before treatment. The prevalence of this systemic response was inversely related to Breslow thickness >1 mm and American Joint Committee on Cancer stage ≥II (P = 0.001 and 0.032, respectively). In contrast to patients treated with targeted therapies, the anti-TERT Th1 immunity was associated with an objective response after immune checkpoint inhibitors treatment. Hence, 86% (18/21) of responder patients exhibited pre-existing anti-TERT Th1 versus 35% (6/19) in nonresponders (P = 0.001). This response was also associated with increased progression-free survival and overall survival in patients with melanoma treated with immune checkpoint inhibitors (P = 0.0008 and 0.012, respectively). Collectively, the presence of circulating anti-TERT Th1 response is inversely related to melanoma evolution and appears to be a predictive factor of response to immunotherapy. Our results highlight the interest in telomerase-specific CD4 Th1 response as a promising blood-based biomarker of immune checkpoint inhibitors therapy in melanoma.

MCAM/MUC18/CD146 as a Multifaceted Warning Marker of Melanoma Progression in Liquid Biopsy.

Human malignant melanoma shows a high rate of mortality after metastasization, and its incidence is continuously rising worldwide. Several studies have suggested that MCAM/MUC18/CD146 plays an important role in the progression of this malignant disease. MCAM/MUC18/CD146 is a typical single-spanning transmembrane glycoprotein, existing as two membrane isoforms, long and short, and an additional soluble form, sCD146. We previously documented that molecular MCAM/MUC18/CD146 expression is strongly associated with disease progression. Recently, we showed that MCAM/MUC18/CD146 and ABCB5 can serve as melanoma-specific-targets in the selection of highly primitive circulating melanoma cells, and constitute putative proteins associated with disease spreading progression. Here, we analyzed CD146 molecular expression at onset or at disease recurrence in an enlarged melanoma case series. For some patients, we also performed the time courses of molecular monitoring. Moreover, we explored the role of soluble CD146 in different cohorts of melanoma patients at onset or disease progression, rather than in clinical remission, undergoing immune therapy or free from any clinical treatment. We showed that MCAM/MUC18/CD146 can be considered as: (1) a membrane antigen suitable for identification and enrichment in melanoma liquid biopsy; (2) a highly effective molecular "warning" marker for minimal residual disease monitoring; and (3) a soluble protein index of inflammation and putative response to therapeutic treatments.

Mortality in cutaneous malignant melanoma and its association with Neutrophil-to-Lymphocyte ratio.

INTRODUCTION: Cutaneous malignant melanoma (CMM) incidence has risen rapidly in the last 50 years. Poor progression and high mortality characterize CMM, making a thorough understanding of progression and associated factors essential for optimizing care. AIMS: We assessed the association between the Neutrophil-to-Lymphocyte Ratio (NLR) and mortality in adults with CMM from an entirely mixed-race Hispanic population during 12 consecutive years of extensive follow-up. MATERIAL & METHODS: We performed a retrospective cohort study in a tertiary hospital in Peru. NLR was categorized with a cutoff value higher or equal than 3. We collected demographic variables, laboratory results and treatments at baseline of follow-up. Cox regression analysis was performed, and we calculated crude and adjusted hazard ratios (HR) and their 95% confidence interval (95%CI). RESULTS: The analysis was from 615 CMM cases, and there were 378 deaths. Most melanomas (63.6%) were acral lentiginous. The crude analysis showed that high NLR is a risk factor for mortality, HR = 2.52; 95%CI (2.03-3.14). High NRL ratio remains statistically significant after adjusting for confounding variables, aHR = 1.61; 95%CI (1.16-2.24). Other risk factors for mortality were clinical stages III and IV, older than 60 years, females and greater Breslow thickness. CONCLUSIONS: We concluded that high NRL ratio is a risk factor for mortality and should be monitored in every patient who is diagnosed with malignant melanoma during their first blood count. It should then be carried out in follow-up controls for patients of clinical stage III and IV only, or in patients who present a relapse.

Altered Glycosylation of Human Alpha-1-Acid Glycoprotein as a Biomarker for Malignant Melanoma.

A high-resolution HILIC-MS/MS method was developed to analyze anthranilic acid derivatives of N-glycans released from human serum alpha-1-acid glycoprotein (AGP). The method was applied to samples obtained from 18 patients suffering from high-risk malignant melanoma as well as 19 healthy individuals. It enabled the identification of 102 glycan isomers separating isomers that differ only in sialic acid linkage (α-2,3, α-2,6) or in fucose positions (core, antenna). Comparative assessment of the samples revealed that upregulation of certain fucosylated glycans and downregulation of their nonfucosylated counterparts occurred in cancer patients. An increased ratio of isomers with more α-2,6-linked sialic acids was also observed. Linear discriminant analysis (LDA) combining 10 variables with the highest discriminatory power was employed to categorize the samples based on their glycosylation pattern. The performance of the method was tested by cross-validation, resulting in an overall classification success rate of 96.7%. The approach presented here is significantly superior to serological marker S100B protein in terms of sensitivity and negative predictive power in the population studied. Therefore, it may effectively support the diagnosis of malignant melanoma as a biomarker.

Upregulation of miR-29c-3p Hinders Melanoma Progression by Inhibiting CDCA4 Expression.

OBJECTIVE: To investigate the expression and regulation mechanism of miR-29c-3p and cell division cycle associated 4 (CDCA4) in melanoma (MM). Data and Methods. Fifty-nine patients with MM admitted to our hospital were enrolled as the MM group. They were followed up for 3 years to analyze the prognostic factors; meanwhile, 51 healthy subjects were allocated into a normal group. MM cell lines (M21 and C8161) were transfected with miR-29c-3p-mimics, miR-29c-3p-inhibitor, miR-NC, si-CDCA4, and sh-CDCA4. The expression of miR-29c-3p, CDCA4, Bax, Caspase3, Bcl-2, N-cadherin, vimentin, and E-cadherin was quantified, and cell proliferation, migration, invasion, and apoptosis, as well as epithelial-mesenchymal transition (EMT), were determined. RESULTS: Serum miR-29c-3p was lowly expressed and CDCA4 was highly expressed in the MM group. The area under the curve (AUC) of both for diagnosing MM was greater than 0.9. miR-29c-3p and CDCA4 were related to regional lymph node staging (N staging), distant metastasis (M staging), tumor diameter, and pathological differentiation. Low miR-29c-3p and high CDCA4 were associated with poor prognosis of MM. Overexpression of miR-29c-3p and suppression of CDCA4 hindered cell proliferation, migration, invasion, and expression of Bax, Caspase3, N-cadherin, and vimentin, but cell apoptosis and expression of Bcl-2 and E-cadherin were enhanced. Dual-luciferase reporter (DLR) assay confirmed the targeted relationship between miR-29c-3p and CDCA4. After miR-29c-3p-mimics+sh-CDCA4 was transfected into M21 and C8161 cells, the proliferation, invasion, and apoptosis were not different from those in the miR-NC group transfected with unrelated sequences. CONCLUSION: Overexpression of miR-29c-3p suppresses CDCA4 expression and decreases proliferation, migration, invasion, apoptosis, and EMT of MM cells, thus hindering MM progression.

Identification of neutralising pembrolizumab anti-drug antibodies in patients with melanoma.

Development of anti-drug antibodies (ADAs) can interfere with therapeutic monoclonal antibodies and may lead to drug neutralisation and clinical disease progression. Measurement of circulating drug levels and development of ADAs in the setting of anti-programmed cell death-1 agent pembrolizumab has not been well-studied. Enzyme-linked immunosorbent assays were used to measure pembrolizumab drug level and ADAs in 41 patients with melanoma at baseline, Time-point 1 (3 weeks) and Time-point 2 (21 weeks). Assay results were related to patient demographics and clinical outcome data at 6 months. The median pembrolizumab drug level at 3 weeks was 237 ng/µL and did not correlate with age, sex or body surface area.17/41 patients had an ADA detected at any timepoint, with the highest prevalence at Timepoint 1 (median concentration = 17 ng/µL). The presence of an ADA did not correlate with clinical progression at 6 months. 3/41 (7%) of patients displayed a falling pembrolizumab drug level and rising ADA titre between Timepoint 1 and 2 suggestive of a neutralising ADA. Pembrolizumab drug levels and ADAs can be readily measured. The rates of total and treatment-emergent ADAs may be higher in "real-word" settings than those previously reported. Larger studies are needed to determine effect of neutralising ADAs on long-term clinical outcome.

A novel prognostic biomarker for cutaneous malignant melanoma: red cell distribution width (RDW) to lymphocyte ratio.

It is well-known that inflammation plays a significant role in cancer formation and prognosis. Both lymphocyte count and red cell distribution width (RDW) has been used to predict prognosis in various cancers as an indicator of inflammation. Yet, the role of RDW-lymphocyte ratio (RLR) in determining prognosis is still unknown. We aimed to determine the prognostic role of RLR in cutaneous malignant melanoma (MM). One hundred fifteen patients with MM were included in the study retrospectively. The relationship of the clinical-pathological data with overall survival (OS) and progression-free survival (PFS) was analyzed using Kaplan-Meier curves. The cut-off values of neutrophil to lymphocyte ratio, systemic immune-inflammation index (SII), prognostic nutritional index (PNI) and RLR were determined as 2, 487, 51.5 and 6.52, respectively. OS was significantly longer in the low SII, high PNI, low RLR group, while PFS was longer in groups with high PNI and low RLR. In univariate analysis, it was determined that PFS was significantly correlated with Eastern Cooperative Oncology Group (ECOG) performance, TNM stage, PNI and RLR. Moreover, in univariate analysis, a significant correlation was determined between OS and age, ECOG performance, TNM stage, adjuvant interferon, SII, PNI and RLR. In multivariate analysis, ECOG performance, TNM stage and RLR were determined as independent prognostic factors for PFS, while TNM stage and RLR were found to be independent prognostic factors for OS. RLR could be a novel prognostic marker for both PFS and OS in patients with cutaneous MM.

Liquid Biopsy in Melanoma: Significance in Diagnostics, Prediction and Treatment Monitoring.

Liquid biopsy is a common term referring to circulating tumor cells and other biomarkers, such as circulating tumor DNA (ctDNA) or extracellular vesicles. Liquid biopsy presents a range of clinical advantages, such as the low invasiveness of the blood sample collection and continuous control of the tumor progression. In addition, this approach enables the mechanisms of drug resistance to be determined in various methods of cancer treatment, including immunotherapy. However, in the case of melanoma, the application of liquid biopsy in patient stratification and therapy needs further investigation. This review attempts to collect all of the relevant and recent information about circulating melanoma cells (CMCs) related to the context of malignant melanoma and immunotherapy. Furthermore, the biology of liquid biopsy analytes, including CMCs, ctDNA, mRNA and exosomes, as well as techniques for their detection and isolation, are also described. The available data support the notion that thoughtful selection of biomarkers and technologies for their detection can contribute to the development of precision medicine by increasing the efficacy of cancer diagnostics and treatment.

Clinical Relevance of Serum Kyn/Trp Ratio and Basal and IFNγ-Upregulated IDO1 Expression in Peripheral Monocytes in Early Stage Melanoma.

Immune escape is an early phenomenon in cancer development/progression. Indoleamine 2,3-dioxygenase 1 (IDO1) is a normal endogenous mechanism of acquired peripheral immune tolerance and may therefore be tumor-promoting. This study investigated the clinical relevance of IDO1 expression by immune cells in the lymph nodes and blood and of the serum kynurenine/tryptophan (Kyn/Trp) ratio in 65 systemic treatment naïve stage I-III melanoma patients. Blood samples were collected within the first year of diagnosis. Patients had a median follow-up of 61 months. High basal IDO1 expression in peripheral monocytes and low IFNγ-induced IDO1 upregulation correlated with worse outcome independent from disease stage. Interestingly studied factors were not interrelated. During follow-up, the risk of relapse was 9% (2/22) in the subgroup with high IFNγ-induced IDO1 upregulation in monocytes. In contrast, if IDO1 upregulation was low, relapse occurred in 30% (3/10) of patients with low basal IDO1 expression in monocytes and in 61.5% (8/13) in the subgroup with high basal IDO1 expression in monocytes (Log-Rank test, p=0.008). This study reveals some immune features in the blood of early stage melanoma that may be of relevance for disease outcome. These may offer a target for sub-stratification and early intervention.

Prognostic significance of platelet lymphocyte ratio in patients with melanoma: A meta-analysis.

BACKGROUND: The aim of this study was to systematically evaluate the prognostic role of platelet lymphocyte ratio (PLR) in patients with melanoma through performing a meta-analysis. METHODS: PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure were searched for potential studies. The basic characteristics and relevant data were extracted. Hazard ratios with 95% confidence intervals (CIs) were pooled to evaluate the prognostic role of PLR in patients with melanoma. RESULTS: Ten studies enrolling 2422 patients were included. The pooled hazard ratios of higher PLR for overall survival and progression-free survival in melanoma were 1.70 (95% CI, 1.22-2.37) and 1.65 (95% CI, 1.10-2.47), respectively. Sensitivity analysis and subgroup analyses were also performed. No significant publication bias was observed. CONCLUSION: Our results showed that higher PLR was associated with poorer overall survival and progression-free survival in patients with melanoma. These findings may help to determine the prognosis and explore future novel therapies based on modulating inflammation and immune responses in melanoma.

Clonal dynamics of circulating tumor DNA during immune checkpoint blockade therapy for melanoma.

Assessment of treatment efficacy of immune checkpoint inhibitors in melanoma patients is difficult as the response to these therapies varies among patients or lesions. The clonal evolution of cancer during immune checkpoint blockade therapy could cause treatment resistance. We investigated the potential of liquid biopsy in monitoring the mutational profiles of metastatic melanoma during immunotherapy. Plasma samples collected from 21 Japanese metastatic melanoma patients before immune checkpoint blockade therapy were subjected to whole-exome sequencing (WES). Furthermore, 14 Japanese patients with melanoma were enrolled for longitudinal analysis of circulating tumor DNA (ctDNA). Plasma samples were collected prospectively before and during therapy and sequenced. WES of the pretreatment plasma from Japanese melanoma patients showed detectable ctDNA levels with wide ranges of variant allele frequencies within a sample, suggesting clonal and subclonal mutations in ctDNA. In targeted sequencing using longitudinal samples, ctDNA levels correlated with increased tumor size, while ctDNA content immediately decreased after a surge in a patient exhibiting pseudo-progression, suggesting the potential of ctDNA analysis in discriminating between pseudo- and true progression. Mutant ctDNA levels showed different patterns within the clinical course of specific patients, suggesting that these mutations were derived from different tumor clones with distinct therapeutic responses. During further investigation, WES of plasma samples from 1 patient showed marked differences in the mutational profiles of ctDNA, including expansive tumor evolution during an acute exacerbation. Immunotherapy may induce characteristic clonal evolutions of tumors; longitudinal analysis of ctDNA has the potential of determining these tumor evolution patterns and therapeutic responses.

Plasma proteome alterations by MAPK inhibitors in BRAFV600-mutated metastatic cutaneous melanoma.

Approximately half of metastatic cutaneous melanomas (CM) harbor a mutation in the BRAF protooncogene, upregulating the mitogen-activated protein kinase (MAPK)-pathway. The development of inhibitors targeting the MAPK pathway (MAPKi), i.e., BRAF- and MEK-inhibitors (BRAFi and MEKi), have substantially improved the survival in BRAFV600E/K-mutated stage IV metastatic CM. However, most patients develop resistance to treatment and no predictive biomarkers exist in practice. This study aimed at discovering plasma proteome changes during treatment MAPKi in patients with metastatic (stage IV) CM. Matched plasma samples before (pre) and during treatment (trm) from 23 patients with stage IV CM, treated with BRAF-inhibitors (BRAFi) alone or BRAF- and MEK- inhibitors combined (BRAFi and MEKi), were collected and analyzed with targeted proteomics by proximity extension assays. Additionally, plasma from 9 patients treated with BRAFi and MEKi was analyzed with in-depth high-resolution isoelectric focusing liquid-chromatography mass-spectrometry proteomics. Alterations of plasma proteins involved in granzyme and interferon gamma pathways were detected in patients treated with BRAFi, and cell adhesion-, neutrophil degranulation-, and proteolysis pathways in patients treated with BRAFi and MEKi. Several proteins were associated with progression-free survival after MAPKi treatment. We show that the majority of the altered plasma proteins were traceable to BRAFV600E-mutant metastatic CM tissue at mRNA level in 154 patients from the TCGA, further strengthening their involvement in tumoral response to treatment. This wide screen of plasma proteins unravels proteins that may serve as predictive and/or prognostic biomarkers of MAPKi treatment, opening a window of opportunity for plasma biomarker discovery in MAPKi-treatment of BRAFV600-mutant metastatic CM.

Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.

Prospective real-world study on the pharmacokinetics of pembrolizumab in patients with solid tumors.

BACKGROUND: Dosing schemes of pembrolizumab (anti-programmed cell death protein 1 monoclonal antibody) are solely based on pharmacokinetic (PK) modelling derived from phase I-III trials. The current study aimed to determine factors affecting PK and its relationship with clinical outcome in the real-world setting. METHODS: Advanced-stage cancer patients, who were treated with pembrolizumab monotherapy (2 mg/kg Q3W or 200 mg flat Q3W), were prospectively included for serial sampling to obtain trough concentrations. A PK model was generated, covariate effects assessed and internally validated by a bootstrap procedure. PK parameters were related to overall survival (OS) and the occurrence of immune-related adverse events (irAEs). RESULTS: 588 serum samples derived from 122 patients with (non-)small-cell lung cancer ([N]SCLC), malignant pleural mesothelioma (MPM), melanoma and urothelial cell cancer (UCC) were analyzed. Median follow-up was 2.2 years. A one-compartment PK model was generated: body surface area (BSA) and serum albumin had a significant effect on drug clearance (CL; covariate estimate 1.46 and -1.43, respectively), and serum lactate dehydrogenase (LDH) on the distribution volume(Vd; 0.34). A significant inverse CL-OS relationship was determined for NSCLC (HR:1.69; 95%CI1.07-2.68; p=0.024) and MPM (HR: 3.29; 95% CI 1.08 to 10.09; p=0.037), after correction for prognostic factors, which could not confirmed for melanoma (p=0.22) or UCC (p=0.34). No relationship could be determined between CL and grade >3 irAEs (p=0.70). CONCLUSIONS: High interpatient variability of pembrolizumab PK is determined by BSA and serum albumin (on CL) and LDH (on Vd). A strong inverse CL-OS relationship was demonstrated for NSCLC and MPM, which could not be observed for melanoma and UCC. The findings suggest that personalized dosing should be prospectively explored.