Plasma Of Melanoma Patients Research Articles

Abstract 273: Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression

Abstract Melanoma is the deadliest form of skin cancer, with chronological aging as a major negative prognostic factor due to the influence of intrinsic aging on the tumor microenvironment (TME) to promote tumor initiation and progression. Ultraviolet radiation (UVR) exposure, which is a major risk factor for melanoma, also can cause a physical premature aging phenotype, known as extrinsic photoaging, from chronic UVA irradiation of the underlying dermis. Photoaging of the skin often occurs in tandem with chronological aging, and contributes to similar physical alterations in normal skin structure. However, the molecular mechanisms of how photoaging compares to chronological aging in changing the TME and on tumor progression is still lacking. Here we found that extrinsically photoaged fibroblasts have a higher level of secretion of lysosomal proteases, Cathepsins, which have been shown to be secreted via lysosomal exocytosis and play a functional role in extracellular matrix (ECM) remodeling at neutral PH levels. We found that Cathepsins B and D are secreted at higher levels in both extrinsically photoaged young fibroblasts and in intrinsically aged fibroblasts and upregulated at the endogenous protein level. Cathepsin B has been shown to be highly expressed in metastatic but not primary melanoma patient samples, and cleaves ECM proteins such as fibronectin and tenascin. Immunofluorescent staining of in-vitro fibroblast derived matrices (CDMs) confirmed that photoaging disrupts subsequent deposited extracellular matrix proteins similarly to that of intrinsically aged healthy fibroblasts and these matrix changes influences the growth pattern of melanoma lines plated on these CDMs. Mass spectrometry analysis of secreted proteomics confirmed that aged fibroblasts secrete more cathepsins than intrinsically young fibroblasts, and Cathepsins B and D were also detected in extracellular vesicle (EV) cargo proteomics. Single-Particle Interferometric Reflectance Imaging (SP-IRIS) of EVs showed that UVA irradiation induced a significant increase in the total number of secreted of exosomes and in expression of tetraspanin protein CD63, which plays a role in cargo sorting and has been detected in high levels in the plasma of melanoma patients. We hypothesize that extrinsic photoaging of fibroblasts induces the secretion of exosomes packaged with Cathepsins that play an integral role in ECM remodeling and matrix degradation in a beneficial manner for melanoma invasion. Further studies will assess the influence on secreted cathepsins on melanomagenesis and melanoma invasion through both mechanical and biochemical changes in the TME with in-vitro and in-vivo studies. Citation Format: Vania Wang, Yash Chhabra, Laura Hueser, Ashani Weeraratna. Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 273.

Alterations in Plasma Lipid Profiles Associated with Melanoma and Therapy Resistance.

Dysfunctions of lipid metabolism are associated with tumor progression and treatment resistance of cutaneous melanoma. BRAF/MEK inhibitor resistance is linked to alterations of melanoma lipid pathways. We evaluated whether a specific lipid pattern characterizes plasma from melanoma patients and their response to therapy. Plasma samples from patients and controls were analyzed for FASN and DHCR24 levels and lipidomic profiles. FASN and DHCR24 expression resulted in association with disease condition and related to plasma cholesterol and triglycerides in patients at different disease stages (n = 144) as compared to controls (n = 115). Untargeted lipidomics in plasma (n = 40) from advanced disease patients and controls revealed altered levels of different lipids, including fatty acid derivatives and sphingolipids. Targeted lipidomics identified higher levels of dihydroceramides, ceramides, sphingomyelins, ganglioside GM3, sphingosine, sphingosine-1-phosphate, and dihydrosphingosine, saturated and unsaturated fatty acids. When melanoma patients were stratified based on a long/short-term clinical response to kinase inhibitors, differences in plasma levels were shown for saturated fatty acids (FA 16:0, FA18:0) and oleic acid (FA18:1). Our results associated altered levels of selected lipid species in plasma of melanoma patients with a more favorable prognosis. Although obtained in a small cohort, these results pave the way to lipidomic profiling for melanoma patient stratification.

Immunosuppressive functions of melanoma cell-derived exosomes in plasma of melanoma patients.

Tumor-derived exosomes (TEX) are a subset of small extracellular vesicles (sEV) present in all body fluids of patients with cancer. In plasma of patients with metastatic melanoma, numbers of exosomes produced by melanoma cells called MTEX are elevated. To study the role of MTEX in melanoma progression, immunoaffinity-based separation of MTEX from total plasma exosomes was performed. The surface of MTEX was decorated by various checkpoint inhibitory proteins, and upon coincubation with immune recipient cells, MTEX suppressed anti-tumor functions of these cells. MTEX emerge as a major mechanism of immune suppression in melanoma and thus might play a role in promoting melanoma progression.

The plasma exosomal miR-1180-3p serves as a novel potential diagnostic marker for cutaneous melanoma

BackgroundExosomes are a promising tool in disease detection because they are noninvasive, cost-effective, sensitive and stable in body fluids. MicroRNAs (miRNAs) are the main exosomal component and participate in tumor development. However, the exosomal miRNA profile among Asian melanoma patients remains unclear.MethodsExosomal miRNAs from the plasma of melanoma patients (n = 20) and healthy individuals (n = 20) were isolated and subjected to small RNA sequencing. Real-time PCR was performed to identify the differential miRNAs and to determine the diagnostic efficiency. Proliferation, scratch and Transwell assays were performed to detect the biological behavior of melanoma cells.ResultsExosomal miRNA profiling revealed decreased miR-1180-3p expression as a potential diagnostic marker of melanoma. The validation group of melanoma patients (n = 28) and controls (n = 28) confirmed the diagnostic efficiency of miR-1180-3p. The level of miR-1180-3p in melanoma cells was lower than that in melanocytes. Accordingly, the level of miR-1180-3p was negatively associated with the proliferation, migration and invasion of melanoma cells. Functional analysis and target gene prediction found that ST3GAL4 was a potential target and highly expressed in melanoma tissues and was negatively regulated by miR-1180-3p. Knockdown of ST3GAL4 hindered the malignant phenotype of melanoma cells.ConclusionsThis study indicates that reduced exosomal miR-1180-3p in melanoma patient plasma is a promising diagnostic marker and provides novel insight into melanoma development.

Abstract 2903: The exosome marker CD63 and Caveolin-1 in breast cancer patients

Abstract The tetraspanin CD63, a proposed exosome marker, has been reported to be expressed in solid tumors. For instance, CD63 status in carcinoma cells was reported to be a significant independent prognostic factor in gastric cancer patients, with its overexpression causing adversity in patients with gastrointestinal stromal tumors. However, the expression of CD63 has remained virtually unknown in breast cancer pathological specimens. In addition, the detection of high levels of exosomes expressing CD63 and Caveoloin-1 in the plasma of melanoma patients was reported as a novel tool for the clinical management of cancer patients. Caveolin-1 has also been reported to negatively regulate the endocytosis of exosomes. Tetraspanin proteins are shown to form complicated interaction networks in cells to exert their functions. In this study, we immunolocalized CD63 and Caveolin-1 in the surgical pathology specimens of 88 breast cancer patients. CD63 was immunolocalized in the cytoplasm of both the cancer and stromal cells. The number of CD63-positive cancer cells was significantly higher in HER2-positive patients and the levels of CD63-positive stromal cells were also significantly higher in those with high pathologic T factor (pT) and proliferation marker protein Ki-67 labeling index and low pathologic N factor (pN). The number of CD63-double-positive cancer cells and stromal cells was associated with high histological grade, high pT, and high Ki67 expressions. CD63-positive cancer cells and Caveolin-1-positive cancer cells were both significantly higher in Estrogen receptor-positive breast cancer patients with high histological grade and pT. To the best of our knowledge, this is the first study demonstrating the significance of CD63 in breast cancer. Our results indicated that CD63-double-positive cancer cells and stromal cells could be involved in clinically and biologically aggressive subtypes of breast cancer. Further studies are required to clarify the clinical significance of the associations between CD63 and Caveolin-1 in patients with breast cancer. Citation Format: Erina Iwabuchi, Yasuhiro Miki, Ayako Kanai, Takanori Ishida, Hironobu Sasano. The exosome marker CD63 and Caveolin-1 in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2903.

Immunoaffinity-Based Isolation of Melanoma Cell-Derived and T Cell-Derived Exosomes from Plasma of Melanoma Patients.

Tumor-derived exosomes (TEX), a subset of small extracellular vesicles (EVs) which originate from the endocytic compartment of tumor cells, are emerging as key players in cancer progression. TEX circulate freely in patients' body fluids and transfer bioactive cargos from tumor to various recipient cells. The molecular cargo of melanoma cell-derived exosomes (MTEX) mimics that of the tumor, and MTEX serve as a liquid biopsy that provides potentially useful information for cancer diagnosis, prognosis, or responses to therapy. Plasma of melanoma patients contains a mix of MTEX and exosomes produced by nonmalignant cells (NMTEX). Isolation of these exosome subtypes from the bulk of plasma exosomes is necessary to evaluate contributions of each as potential biomarkers of melanoma progression and outcome. Here, methods for separation of MTEX from T cell-derived exosomes from a single small volume of plasma and their subsequent molecular and functional characterization are described. Following size exclusion chromatography (SEC) to isolate total plasma exosomes, immune affinity-based capture of MTEX with anti-CSPG4 antibody and then of exosomes produced by T cells with anti-CD3 antibody is used to sequentially isolate the two subsets. This immune capture method enables the recovery of MTEX and CD3+ exosomes in quantities sufficient both for molecular profiling by flow cytometry or western blotting and for functional analyses.

Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma.

Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and –5p transfection in cutaneous melanoma cell lines are investigated. Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.

Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors.

Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity in vitro and in vivo. Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101+CD56+ nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56+ NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101+CD56+ exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.

439 Melanoma cells export functional proteasomes by way of microvesiculation: a possible mechanism for generation of plasmatic proteasomes

Plasmatic proteasomes (p-proteasomes) are a reliable marker for metastatic dissemination in melanoma patients. Recent works have suggested that p-proteasome partially originated from tumour cell secretion via exosomes, which are also considered as a new biomarker for metastatic melanoma. Our objectives were to investigate the origin, mechanism of secretion and functionality of the melanoma p-proteasomes. We show by proteasome purification either from culture supernatant of melanoma cell lines (A-375, SK-MEL-28) or from plasma of melanoma patients, that the extracellular proteasome is released essentially as an assembled 20S core particle, contrarily to proteasome analyzed from cell lysate, which presents additional 26S capped proteasome. Using specific fluorogenic substrates we demonstrate that these extracellular proteasomes are functional and possess all the peptidase activities associated with b1, b2 and b5 subunits. There is no correlation between exosome and proteasome concentrations determined by ELISA in the plasma of melanoma patients. Immunoisolation of secreted proteasome using a7 antibody adsorbed on magnetic beads and pull-down experiments show that secreted proteasomes are not enwrapped in exosomes. To our point of view, previous reports of 20S proteasome subunits in exosomes are rather due to a co-purification along the exosome preparation. Extracellular proteasome concentration is increased in the secretome of melanoma cell lines after stimulation of membrane blebbing with the ionophore A23187. Moreover, proteasome is associated with microvesicles as shown by gel filtration experiments and immobilization of microvesicles on lectin-sepharose. Altogether our results show that melanoma cells secrete functional 20S proteasomes at least in part by membrane blebbing and microvesiculation and not through exosomal pathway.

Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma

ABSTRACTExtracellular vesicles (EVs), including exosomes and microvesicles, are secreted from all cells, and convey messages between cells in health and disease. However, the diversity of EV subpopulations is only beginning to be explored. Since EVs have been implicated in tumour microenvironmental communication, we started to determine the diversity of EVs specifically in this tissue. To do this, we isolated EVs directly from patient melanoma metastatic tissues. Using EV membrane isolation and mass spectrometry analysis, we discovered enrichment of mitochondrial membrane proteins in the melanoma tissue-derived EVs, compared to non-melanoma-derived EVs. Interestingly, two mitochondrial inner membrane proteins MT-CO2 (encoded by the mitochondrial genome) and COX6c (encoded by the nuclear genome) were highly prevalent in the plasma of melanoma patients, as well as in ovarian and breast cancer patients. Furthermore, this subpopulation of EVs contains active mitochondrial enzymes. In summary, tumour tissues are enriched in EVs with mitochondrial membrane proteins and these mitochondrial membrane proteins can be detected in plasma and are increased in melanoma, ovarian cancer as well as breast cancer.

Abstract 687: Implementation of inter-patient variability in semi-mechanistic model of melanoma treatment with vemurafenib

Abstract Introduction: The number of parameters in quantitative systems pharmacology (QSP), mechanistic and semi-mechanistic models is greater than in conventional PK/PD models. That is why it is not possible to implement inter-patient variability using only clinical data. The objective of this study was to describe inter-patient variability in the semi-mechanistic model of melanoma treatment with vemurafenib, a BRAF inhibitor, on the basis of published human in vitro, in vivo and clinical data, and to predict overall response from phase 2 clinical trial. Methods: Model described melanoma progression and its treatment with vemurafenib. It includes (i) proliferation, apoptosis, and transition between states (c-MET negative/positive and sensitive/resistant to BRAF inhibitor) of cancer cells; (ii) HGF effect on proliferation of melanoma cells; (iii) PK/PD of vemurafenib. Various types of data were used to extract inter-patient variability, for example: (a) in vitro data on effect of vemurafenib on melanoma cell lines [1]; (b) in vivo data on tumor growth in melanoma patients without treatment [2]; (c) in vivo data on HGF level in blood plasma of melanoma patients [3]. Results: Different virtual patients developed by the model reproduce all types of tumor response to vemurafenib monotherapy according to RECIST criteria: complete response, partial response, stable disease and progressive disease. To evaluate the predictive ability of the model in a quantitative manner we compared results of simulation of virtual patients population with clinical outcomes. An overall response rate was 70% in the model versus 53% (95% CI, 44 to 62) observed in phase 2 clinical trial [4]. Overestimation can be explained by intermittent administration and dose reduction in case of adverse events in clinical trials which were not taken into account in the model. Conclusions: Our work establishes the benefit of using of in vitro and in vivo data in addition to clinical data to predict the variability in treatment outcomes of cancer patients. [1] Stones et al. Front Genet. 2013 May 8;4:66 [2] Hartung et al. PLoS One. 2017 May 4;12(5):e0176080 [3] Hügel et al. Melanoma Res. 2016 Aug;26(4):354-60 [4] Sosman et al. N Engl J Med. 2012 Feb 23;366(8):707-14 Citation Format: Dmitry Shchelokov, Oleg Demin Jr, Oleg Demin. Implementation of inter-patient variability in semi-mechanistic model of melanoma treatment with vemurafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 687.

Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy

Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ Tcell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ Tcells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

PO-471 Circular RNA detection in melanoma patients’ plasma

IntroductionMelanoma is an aggressive disease, curable by surgical resection if caught at an early stage (>95% 5 year survival) but with a poor prognostic outcome when diagnosed at advanced metastatic stage (<5% 5 year survival). Therefore, there is an urgent need for biomarkers of early diagnosis. In this respect, liquid biopsies hold great promise as they are fast, non-invasive and allow repeated sampling and monitorisation of patient response.Circular RNAs (circRNAs) are covalently closed RNA molecules that much more stable than linear RNAs to the high level of RNase activity in blood, and therefore have great potential as circulating biomarkers for cancer patient management. We therefore set out to identify and validate potential biomarker circRNAs from the plasma of melanoma patients’ representing differing stages of the disease, along with healthy controls.Material and methodsNext generation sequencing (RNAseq) was performed on pools of plasma from melanoma patients with stages 0, I/II, III or IV disease, as well as healthy individuals. Back-spliced junction reads were mapped using a combination of TopHat and TopHat-Fusion, and differential expression analysis carried out with the DEseq algorithm. Three circRNA (CR-3320,–2465 and −1445) were selected for validation in 106 plasma samples from 84 melanoma patients and 22 healthy controls by qRT-PCR.Results and discussionsWe obtained 20–30 million reads by RNAseq and identified between 120–200 circRNA species (after QC) in each sample. Of these 37 were differentially (p<0.05) expressed. Of the three circRNAs measured in an independent validation cohort, all were expressed at higher levels in melanoma patient samples than in controls. The highest levels were observed in stage 0 and I patients, decreasing in stage II and stage III/IV patients, probably due to treatment differences in the cohorts. The AUC values of CR-3320 and CR-2465 were 0.72 and 0.66 with a sensitivity of 53.33% and 30.77% and a specificity of 82.53% and 100%, respectively.ConclusionThis study suggests that circRNAs could represent a novel source of biomarkers for liquid biopsies for melanoma, and in all probability other cancers.

Immunoaffinity-based isolation of melanoma cell-derived exosomes from plasma of patients with melanoma

ABSTRACTTumour-derived exosomes (TEX) are a subset of extracellular vesicles (EVs) present in body fluids of patients with cancer. The role of this exosome subset in melanoma progression has been of interest ever since ex vivo studies of exosomes produced by melanoma cell lines were shown to suppress anti-melanoma immune responses. To study the impact of melanoma-derived exosomes (MTEX) present in patients’ plasma on melanoma progression, isolation of MTEX from total plasma exosomes is necessary. We have developed an immunoaffinity-based method for MTEX capture from plasma of melanoma patients. Using mAb 763.74 specific for the CSPG4 epitope uniquely expressed on melanoma cells, we separated MTEX from non-tumour cell-derived exosomes and evaluated the protein cargo of both fractions by quantitative flow cytometry. Melanoma-associated antigens were carried by MTEX but were not detectable in exosomes produced by normal cells. Separation of plasma-derived MTEX from non-MTEX provides an opportunity for future evaluation of MTEX as potential biomarkers of melanoma progression and as surrogates of melanoma in tumour liquid biopsy studies.

Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of EVs reflects the type and status of the originating cell and EVs in melanoma patient’s plasma could be indicative for the tumor. Likewise, EVs might influence tumor progression by regulating immune responses. We performed a broad protein characterization of EVs from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer (NK) cells, monocytes, monocyte-derived dendritic cells (moDCs), and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on EVs. Hierarchical clustering of protein intensity patterns grouped EVs according to their originating cell type. The analysis of EVs from stimulated B cells and moDCs revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of EVs from platelets, antigen-presenting cells and NK cells as shown by platelet markers, co-stimulatory proteins, and a NK cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers, indicating a changed vesicle secretion or protein loading of EVs by platelets and a lower CD8 signal that might be associated with a diminished activity of NK cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of EVs in melanoma plasma, but rather argue for an indirect influence of melanoma cells on the vesicle secretion or vesicle protein loading by blood cells.

Quantitative analysis of the BRAF V600E mutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR.

BRAF V600E is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA. The level of BRAF V600E ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients. The castPCR assay was performed using a mixture of BRAF V600E DNA and BRAF wild DNA and found to be able to detect BRAF V600E at a fractional abundance of ≥0.5% in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAF V600E mutation. BRAF V600E ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0% of total BRAF cell-free DNA. The abundance of BRAF V600E ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAF V600E ctDNA preceded exacerbation of clinical symptoms. The castPCR assay can detect and quantitate small amounts of BRAF V600E ctDNA in samples containing large amounts of BRAF wild cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients.

Detection of Exosomal miRNAs in the Plasma of Melanoma Patients

MicroRNAs (miRNAs) are a class of 22–25 nucleotide RNAs that control gene expression at the post-transcriptional level. MiRNAs have potential as cancer biomarkers. Melanoma is a highly aggressive form of skin cancer accounting for almost 4% of cancers among men and women, and ~80% of skin cancer-related deaths in the US. In the present study we analyzed plasma-derived exosomal miRNAs from clinically affected and unaffected familial melanoma patients (CDKN2A/p16 gene carriers) and compared them with affected (nonfamilial melanoma) and unaffected control subjects in order to identify novel risk biomarkers for melanoma. Intact miRNAs can be isolated from the circulation because of their presence in exosomes. A number of differentially regulated miRNAs identified by NanoString human V2 miRNA array were validated by quantitative PCR. Significantly, miR-17, miR-19a, miR-21, miR-126, and miR-149 were expressed at higher levels in patients with metastatic sporadic melanoma as compared with familial melanoma patients or unaffected control subjects. Surprisingly, no substantial differences in miRNA expression were detected between familial melanoma patients (all inclusive) and unaffected control subjects. The miRNAs differentially expressed in the different patient cohorts, especially in patients with metastatic melanoma, may play important roles in tumor progression and metastasis, and may be used as predictive biomarkers to monitor remission as well as relapse following therapeutic intervention.

Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations.

We assessed the utility of droplet digital PCR (ddPCR) to evaluate the potential of using circulating tumour DNA (ctDNA) as a post therapy monitoring tool in melanoma by comparing it to serum LDH levels and RECIST scores. ddPCR was shown to be reliable in distinguishing mutant from wild type alleles with no false positives. Subsequently, we quantified ctDNA (V600EBRAF,V600KBRAF or Q61HNRAS) in 6 stage IV melanoma patients across several time points during their treatment course. All tested patients had detectable ctDNA, which exhibited dynamic changes corresponding to the changes in their disease status. The ctDNA levels fell upon treatment response and rose with detectable disease progression. In our group of patients, ctDNA was more consistent and informative than LDH as a blood-based biomarker. In addition, BRAF mutant ctDNA as detected by ddPCR could be used diagnostically where the tumour block was unavailable. In conclusion, this study demonstrates the applicability of using ddPCR to detect and quantify ctDNA in the plasma of melanoma patients.

Detection of BRAF mutations in the plasma of melanoma patients as an early marker of treatment efficiency.

9069 Background: Mutant BRAF DNA can be detected in plasma of melanoma patients bearing a mutation in their tumor. Detection of these genetic alterations in circulating DNA has potential clinical a...

Osteopontin expression in plasma of melanoma patients and in melanocytic tumours

While the serological tumour marker S100 is well established for the detection of metastatic melanoma, the extracellular matrix protein osteopontin (OPN) seems to be a promising novel marker for invasive melanoma. We analysed the potential of OPN as a serological tumour marker for metastatic melanoma and evaluated its combination with S100 and lactate dehydrogenase (LDH) levels to increase the reliability of these biomarkers for the detection of metastatic disease. We examined OPN in the peripheral blood of 110 melanoma patients using enzyme-linked immunosorbent assay and combined it with S100 and LDH levels. In addition, the protein expression of OPN was analysed in tissue sections of melanocytic nevi and melanomas of different progression stages by immunohistochemistry. The independent comparison of S100 and OPN levels in metastatic vs. non-metastatic patients revealed a P-value <0.001 respectively. The predictiveness of OPN, S100 and LDH was 0.85, 0.89 and 0.69 as measured by the area under the receiver operating curve (AUC) respectively, while the combination of the two biomarkers OPN and S100 showed an AUC of 0.97. The optimal cut-off of the combination of OPN and S100 yielded a specificity of 85.9% and a sensitivity of 95.5%. By immunohistochemistry, OPN protein expression was detected in 29% (7/24) of melanocytic nevi, 67% (30/45) of primary melanomas and 39% (7/18) of metastatic melanomas. Together, OPN seems to be a promising novel biomarker for the detection of metastatic disease in melanoma patients, showing elevated plasma levels in metastatic disease and increased protein expression in melanocytic lesions. The combination of OPN with the well-established tumour marker S100 might increase the prediction of metastases.