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.

Immunohistochemistry assessment of tissue neutrophil-to-lymphocyte ratio predicts outcomes in melanoma patients treated with anti-programmed cell death 1 therapy.

Elevated neutrophil-to-lymphocyte ratio (NLR) is associated with diminished immunotherapy response in metastatic melanoma. Although NLR assessment in peripheral blood is established, tissue dynamics remain insufficiently explored. This study aimed to evaluate tissue NLR (tNLR)'s predictive potential through immunohistochemistry in immunotherapy-treated melanoma. Fifty melanoma patients who underwent anti-programmed cell death 1 (PD-1) therapy were assessed. Hematological, clinical and tumor features were collected from medical records. Responses were categorized using the Response Evaluation Criteria in Solid Tumors for immunotherapy (iRECIST) guidelines. Immunohistochemistry for tumor-infiltrating T cells (cluster differentiation 3) and neutrophils (myeloperoxidase) was performed on formalin-fixed paraffin-embedded tumor samples. NLR, derived NLR (dNLR) and tNLR were calculated. Overall survival (OS) and survival following immunotherapy (SFI) were calculated from diagnosis or immunotherapy start to loss of follow-up or death. Patients with high tNLR presented improved OS ( P =  0.038) and SFI with anti-PD-1 therapy ( P =  0.006). Both NLR and dNLR were associated with OS ( P =  0.038 and P =  0.046, respectively) and SFI ( P =  0.001 and P =  0.019, respectively). NLR was also associated with immunotherapy response ( P =  0.007). In conclusion, tNLR emerged as a novel potential biomarker of enhanced survival post anti-PD-1 therapy, in contrast to classical NLR and dNLR markers.

Serum Vitamin D Levels Explored in the Latvian Cohort of Patients with Basal Cell Carcinoma Linked to the Sonic Hedgehog and Vitamin D Binding Protein Cutaneous Tissue Indices.

Ultraviolet radiation is known as one of the major contributors to skin malignancies, including basal cell carcinoma (BCC), which is the most common type of skin cancer. It is a heterogeneous tumor, which presents with various types that are stratified into low- and high-risk tumors. Sunlight is important for overall health and vitamin D synthesis in the skin, whereas deviations from the optimal level of vitamin D are shown to be associated with the risk of the development of BCC. The accumulating evidence suggests the ability of vitamin D to antagonize the Sonic Hedgehog (SHH) signaling, the key tumor pathway, and play a protective role in the development of BCC. Additionally, a vitamin D binding protein (DBP) is shown to be implicated in the complex regulation of vitamin D. Here, we aimed to explore serum vitamin D in patients with different primary and recurrent BCC of the head and neck and investigate cutaneous DBP and SHH indices, confirmed immunohistochemically in these subjects. According to the results, 94.9% of the Latvian cohort of BCC patients were found to be deficient in vitamin D. No significant differences in serum vitamin D levels were found between genders, primary and recurrent tumors, and different types of BCC. Serum vitamin D was inversely associated with tumor size. Susceptible male individuals with low blood vitamin D levels were recognized at risk of developing aggressive and recurrent BCC confirmed by the use of hierarchical clustering analysis. In smaller tumors with a favorable course, such as superficial and nodular BCC, the association between high DBP and low SHH tissue expression was found, providing supportive evidence of the existence of a link between vitamin D, proteins involved in its metabolism, as exemplified by the DBP and SHH signaling pathway. The assumption of a deficiency in the protective effect of vitamin D in patients with high-risk BCCs was proposed in low DBP and high SHH tissue indices. New extensions to existing knowledge and characterization of the BCC signaling pathways and their cross-talk with vitamin D are warranted when searching for a preferential effect of vitamin D on skin cancer.

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.

Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease.

MicroRNAs have been proposed as biomarkers for equine sarcoids, the most prevalent equine skin tumors globally. This study served to validate the diagnostic and prognostic potential of whole blood microRNAs identified in a previous study for long-term equine sarcoid diagnosis and outcome prediction. Based on findings of a clinical examination at the age of 3 years and a follow-up following a further 5-12 years, 32 Franches-Montagnes and 45 Swiss Warmblood horses were assigned to four groups: horses with regression (n = 19), progression (n = 9), new occurrences of sarcoid lesions (n = 19) and tumor-free control horses (n = 30). The expression levels for eight microRNAs (eca-miR-127, eca-miR-432, eca-miR-24, eca-miR-125a-5p, eca-miR-134, eca-miR-379, eca-miR-381, eca-miR-382) were analyzed through reverse transcription quantitative polymerase chain reaction in whole blood samples collected on initial examination. Associations of sex, breed, diagnosis, and prognosis with microRNA expression levels were examined using multivariable analysis of variance. Sex and breed influenced the expression level of five and two microRNAs, respectively. Eca-miR-127 allowed discrimination between sarcoid-affected and tumor-free horses. No variation in microRNA expression was found when comparing horses with sarcoid regression and progression. Expression levels of eca-miR-125a-5p and eca-miR-432 varied in male horses that developed sarcoids throughout the study period in comparison to male control horses. While none of the investigated miRNAs was validated for predicting the prognosis of sarcoid regression / progression within young horses with this condition, two miRNAs demonstrated potential to predict if young male (though not female) tumor-free horse can develop sarcoids within the following years. Sex- and breed- biased miRNAs exist within the equine species and have an impact on biomarker discovery.

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.

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.

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.

Association of Elevated Serum Tryptase with Cutaneous Photodamage and Skin Cancers.

INTRODUCTION: Mast cells and their major protein, the serine proteinase tryptase, can be involved in cutaneous photodamage and carcinogenesis. The serum test of tryptase (S-tryptase) measures total tryptase protein (active tryptase and inactive protryptases), and S-tryptase is elevated in a variety of diseases, for example, in mastocytosis and α-tryptasemia. OBJECTIVES: The objective of this study is to study whether S-tryptase is a marker of cutaneous photodamage and carcinogenesis. METHODS: Adult subjects (n = 399, aged 21-79) evaluated to be at risk for skin cancers were recruited at the dermatological policlinic and examined for photodamage severity, mole count, actinic keratoses (AKs), skin cancers, and immunosuppression (IS). A blood sample was analyzed for S-tryptase using the ImmunoCAP® Tryptase fluoroenzymeimmunoassay. RESULTS: There was no difference in S-tryptase between non-IS (n = 321) and IS (n = 78) subjects or between genders. S-tryptase correlated slightly to photodamage and AKs in 321 non-IS subjects, and this association can be related, in part, to the age of subjects. In 34 subjects, S-tryptase was elevated (≥13.5 ng/mL), and in 20 males, but not in 14 females, the photodamage level was significantly (p = 0.031) more severe than in 179 males with normal S-tryptase. In contrast, there were more frequently subjects (n = 12) with past or present skin cancer (basal or squamous cell carcinoma or melanoma) in 14 females with elevated S-tryptase than in 186 female controls. So far, no explanation has been found for the elevated S-tryptase. CONCLUSION: There are significant associations between elevated S-tryptase and skin carcinogenesis, but the molecular mechanisms are unclear and gender differences can exist.

Low plasma fibrinogen levels are associated with poor prognosis in cutaneous angiosarcoma of the head and neck.

Angiosarcoma of the head and neck (ASHN) is one of the most aggressive malignancies of the skin, but the prognostic factors are not well known because of its rarity. Recently, high plasma fibrinogen levels were reported to predict poor prognosis in several malignancies. In the present retrospective study, we suggest that low plasma fibrinogen levels predict poor prognosis for ASHN.

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.

Predicting anti-PD-1 responders in malignant melanoma from the frequency of S100A9+ monocytes in the blood.

BACKGROUND: While programmed cell death receptor 1 (PD-1) blockade treatment has revolutionized treatment of patients with melanoma, clinical outcomes are highly variable, and only a fraction of patients show durable responses. Therefore, there is a clear need for predictive biomarkers to select patients who will benefit from the treatment. METHOD: To identify potential predictive markers for response to PD-1 checkpoint blockade immunotherapy, we conducted single-cell RNA sequencing analyses of peripheral blood mononuclear cells (PBMC) (n=8), as well as an in-depth immune monitoring study (n=20) by flow cytometry in patients with advanced melanoma undergoing treatment with nivolumab at Karolinska University Hospital. Blood samples were collected before the start of treatment and at the time of the second dose. RESULTS: Unbiased single-cell RNA sequencing of PBMC in patients with melanoma uncovered that a higher frequency of monocytes and a lower ratio of CD4+ T cells to monocyte were inversely associated with overall survival. Similarly, S100A9 expression in the monocytic subset was correlated inversely with overall survival. These results were confirmed by a flow cytometry-based analysis in an independent patient cohort. CONCLUSION: Our results suggest that monocytic cell populations can critically determine the outcome of PD-1 blockade, particularly the subset expressing S100A9, which should be further explored as a possible predictive biomarker. Detailed knowledge of the biological role of S100A9+ monocytes is of high translational relevance.

Impact of Circulating and Tissue Biomarkers in Adjuvant and Neoadjuvant Therapy for High-Risk Melanoma: Ready for Prime Time?

The prognosis of patients with metastatic melanoma has substantially improved over the last years with the advent of novel treatment strategies, mainly immune checkpoint inhibitors and BRAF and MEK inhibitors. Given the survival benefit provided in the metastatic setting and the evidence from prospective clinical trials in the early stages, these drugs have been introduced as adjuvant therapies for high-risk resected stage III disease. Several studies have also investigated immune checkpoint inhibitors, as well as BRAF and MEK inhibitors, for neoadjuvant treatment of high-risk stage III melanoma, with preliminary evidence suggesting this could be a very promising approach in this setting. However, even with new strategies, the risk of disease recurrence varies widely among stage III patients, and no available biomarkers for predicting disease recurrence have been established to date. Improved risk stratification is particularly relevant in this setting to avoid unnecessary treatment for patients who have minimum risk of disease recurrence and to reduce toxicities and costs. Research for predictive and prognostic biomarkers in this setting is ongoing to potentially shed light on the complex interplay between the tumor and the host immune system, and to further personalize treatment. This review provides an insight into available data on circulating and tissue biomarkers, including the tumor microenvironment and associated gene signatures, and their predictive and prognostic role during neoadjuvant and adjuvant treatment for cutaneous high-risk melanoma patients.

Liquid biopsy-based analysis by ddPCR and CAPP-Seq in melanoma patients.

BACKGROUND: The development of BRAF/MEK inhibitors in patients with metastatic melanoma harboring BRAF mutations has garnered attention for liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) or next-generation sequencing methods. OBJECTIVE: To investigate gene mutations in tumor DNA and cfDNA collected from 43 melanoma patients and evaluate their potential as biomarkers. METHODS: ddPCR and CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) techniques were performed to detect gene mutations in plasma cfDNA obtained from patients with metastatic melanoma. RESULTS: Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in the plasma cfDNA, and the results were partially consistent with the results of those identified in the tissues. Among the variants examined, copy numbers of MET mutations were consistent with the disease status in two melanoma patients. CONCLUSION: Liquid biopsy using CAPP-Seq and ddPCR has the potential to detect tumor presence and mutations, especially when tissue biopsies are unavailable. MET mutations in cfDNA may be a potential biomarker in patients with metastatic melanoma.