Blood Samples Of Melanoma Patients Research Articles

Circulating Melanoma Cell Numbers Correlate with TIGIT-Positive Cytotoxic T Cell Counts in Advanced-Stage Melanoma Patients.

Despite the rising public awareness of the risk factors and the possible prevention of melanoma development, it remains challenging in terms of diagnosis and treatment. To improve the clinical situation of patients, it would be especially beneficial to develop prognostic methods for the effective and continuous assessment of the disease course. The solution could lie in the selection of effective biomarkers derived from the tumor microenvironment, increasing the effectiveness of melanoma prognoses and monitoring. Hence, in this study, we evaluated the number of circulating melanoma cells (CMCs) in representative blood samples of melanoma patients vs. healthy controls, as well as the proportion of particular cytotoxic T cells in the total lymphocyte and leukocyte population as a reflection of immune resistance. The results were correlated with the clinical parameters of the patients to examine the potential value of CMC quantification and lymphoid cell phenotyping in melanoma diagnostics, prognostics, and treatment outcome monitoring. The CMC numbers were significantly higher in melanoma patients than in healthy controls. However, an analysis of the correlations between the baseline CMC counts and the clinical parameters found no significant results. In turn, we found significant differences between the groups in the percentage of various profiles of CD8+ cytotoxic T lymphocytes characterized by TIGIT and TIM-3 differential expression. Importantly, the CMC number correlated with CD8+TIGIT+ and CD8+TIGIT+TIM-3- cytotoxic T cell counts in the melanoma patient group. Considering the above, the combination of CMCs and the immunological status of the patient, as defined by the prevalence of selected immune cell types, seems to be a promising approach in melanoma diagnostics and prognostics.

657 Downregulation of CTLA4 in tumor and blood samples is associated with a worse prognosis in malignant melanoma

CTLA-4 is a negative regulator of T lymphocyte activation, and the inhibition of CTLA-4 is used to treat melanoma but has limited efficacy. We measured CTLA4 mRNA expression in melanoma using TCGA tumor data and found the association of decreased CTLA4 with a worse patient prognosis. Because CTLA-4 is constitutively expressed in regulatory T (Treg) cells and upregulated in non-Treg T cells, we measured CTLA4 in blood samples. Whole blood samples from an Australian cohort (170 melanoma patients and 103 healthy controls) were measured for CTLA4 by qRT-PCR and analyzed using Kaplan-Meier (K-M), log-rank test, Cox proportional hazards model, and multivariate linear regression model. Unlike reported data of other cancers, blood CTLA4 was decreased in melanoma patients than in healthy controls. K-M survival curve demonstrated the association of low blood CTLA4 with worse patient survival. While such a trend was not evident in primary melanoma patients, the analysis of metastatic melanoma patients showed an association. This trend was confirmed using another cohort from the United States (246 melanoma patients). To understand immune cell subsets responsible for the downregulated CTLA4 in melanoma, we fractionated blood samples and found downregulated CTLA4 in T cells in melanoma patient blood. Consistent with these results, Treg cells from healthy human PBMCs showed decreased CTLA4 and CTLA-4 surface protein expression when cultured with condition media from human metastatic melanoma cell lines but not primary melanoma cell lines. In summary, we demonstrate that CTLA4 is lower in tumor and blood samples of melanoma patients, and the reduction correlates with a worse prognosis of melanoma patients, especially in metastatic melanoma. Our unpredicted and novel findings call for further investigation into the role of CTLA4 in Treg cell biology, tumor immunity, and cancer therapy.

Interleukin-37 is highly expressed in regulatory T cells of melanoma patients and enhanced by melanoma cell secretome.

Immune suppression is one of the 10 hallmarks of cancer. Interleukin-37 (IL-37), a member of the IL-1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL-37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL-37 in age- and sex-matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL-37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL-37 expression, we discovered that IL-37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (Treg ) cells in healthy individuals, and that IL-37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, Treg cells from melanoma patients exhibited the highest IL-37 gene expression levels. We provided evidence that melanoma-conditioned media induces IL-37 mRNA and protein expression in multiple lymphocyte populations, particularly in Treg cells. We further confirmed that the IL-1-mediated secretome from human melanoma cells, specifically transforming growth factor-β, induces IL-37 mRNA expression in human Treg cells. Our results suggest a potential immunosuppressive role for IL-1 and IL-37 in melanoma tumorigenesis. Highly elevated IL-37 in specific lymphocyte populations could serve as a biomarker for tumor-induced immunosuppression.

Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

Clinical significance of plasmacytoid dendritic cells and myeloid-derived suppressor cells in melanoma

BackgroundImmune markers in the peripheral blood of melanoma patients could provide prognostic information. However, there is currently no consensus on which circulating cell types have more clinical impact. We therefore evaluated myeloid-derived suppressor cells (MDSC), dendritic cells (DC), cytotoxic T-cells and regulatory T-cells (Treg) in a series of blood samples of melanoma patients in different stages of disease.MethodsFlow cytometry was performed on peripheral blood mononuclear cells of 69 stage I to IV melanoma patients with a median follow-up of 39 months after diagnosis to measure the percentage of monocytic MDSCs (mMDSCs), polymorphonuclear MDSCs (pmnMDSCs), myeloid DCs (mDCs), plasmacytoid DCs (pDCs), cytotoxic T-cells and Tregs. We also assessed the expression of PD-L1 and CTLA-4 in cytotoxic T-cells and Tregs respectively. The impact of cell frequencies on prognosis was tested with multivariate Cox regression modelling.ResultsCirculating pDC levels were decreased in patients with advanced (P = 0.001) or active (P = 0.002) disease. Low pDC levels conferred an independent negative impact on overall (P = 0.025) and progression-free survival (P = 0.036). Even before relapse, a decrease in pDC levels was observed (P = 0.002, correlation coefficient 0.898). High levels of circulating MDSCs (>4.13%) have an independent negative prognostic impact on OS (P = 0.012). MDSC levels were associated with decreased CD3+ (P < 0.001) and CD3 + CD8+ (P = 0.017) T-cell levels. Conversely, patients with high MDSC levels had more PD-L1+ T-cells (P = 0.033) and more CTLA-4 expression by Tregs (P = 0.003). pDCs and MDSCs were inversely correlated (P = 0.004). The impact of pDC levels on prognosis and prediction of the presence of systemic disease was stronger than that of MDSC levels.ConclusionWe demonstrated that circulating pDC and MDSC levels are inversely correlated but have an independent prognostic value in melanoma patients. These cell types represent a single immunologic system and should be evaluated together. Both are key players in the immunological climate in melanoma patients, as they are correlated with circulating cytotoxic and regulatory T-cells. Circulating pDC and MDSC levels should be considered in future immunoprofiling efforts as they could impact disease management.

Transcriptome Profiling of Whole Blood Cells Identifies PLEK2 and C1QB in Human Melanoma

BackgroundDeveloping analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a “nucleic acid-rich” and “inflammatory cell-rich” information reservoir and represents systemic processes altered by the presence of cancer cells.Methodology/Principal FindingsWe conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV) and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45− and CD45+ populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively.Conclusions/SignificanceThe current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45− subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients for residual disease.

High-throughput miRNA profiling of human melanoma blood samples

BackgroundMicroRNA (miRNA) signatures are not only found in cancer tissue but also in blood of cancer patients. Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool.MethodsUsing a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients. We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set.ResultsA hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls. The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81). Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis. Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis. MiRNA microarray data were validated by qRT-PCR.ConclusionsOur study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma.

Melanoma-associated markers expression in blood: MUC-18 is associated with advanced stages in melanoma patients

Multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) was originally reported to reveal melanoma-associated mRNAs (MAMs) in melanoma cells but not in the peripheral blood of healthy individuals. To evaluate the expression of MAMs in the peripheral blood of melanoma patients at different American Joint Committee on Cancer (AJCC) stages, and to correlate their presence with early and/or advanced stages of the disease. One hundred blood samples of melanoma patients (AJCC I-IV) were analysed using multimarker RT-PCR to assess the co-expression of Tyr-OH, MART-1, MAGE-3, MUC-18/MCAM and p97. Patients were stratified into two disease categories: early and advanced stages. The former includes in situ and melanoma stages AJCC I-II, the latter AJCC III-IV. chi(2) and Fisher's exact tests were used to statistically evaluate the association between each MAM and disease categories. The recognized significant associations were subsequently resubmitted to univariate logistic regression. Furthermore, sensitivity and specificity were established. At least one MAM could be detected in 24% of our series. Tyr-OH was the most common marker (14%), followed by MUC-18 (12%), MART-1 (5%), MAGE-3 (4%) and p97 (3%). No significant association among Tyr-OH, MART-1, MAGE-3, p97 and disease stages were evidenced. Only MUC-18 was statistically associated (P < 0.009) with advanced stages alone or co-expressed with other MAMs. According to logistic regression univariate analysis, MUC-18 increases the probability (odds ratio: 33) being in advanced stages and the incidence of recurrences (95% CI 2.9-374). MUC-18 RT-PCR assay could be proposed as an adjunctive molecular method in the management of melanoma patients and is useful in the monitoring of study protocols.

Facts and pitfalls in the detection of tyrosinase mRNA in the blood of melanoma patients by RT-PCR.

Reverse transcription (RT) of tyrosinase mRNA and specific cDNA amplification to facilitate the early detection of circulating tumor cells in melanoma patients have been reported. The significance and practical value of these procedures for the diagnosis of tumor dissemination in melanoma patients is, however, still unclear. We analyzed peripheral blood samples of melanoma patients of different clinical stages for the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). In addition to a nested RT-PCR-based system, we evaluated the new PCR enzyme-linked immunosorbent assay tyrosinase system for sensitivity and specificity in detecting circulating melanoma cells. Our results showed a high sensitivity and specificity for this system in detecting one melanoma cell in 1 ml of whole blood. Using different methods of detection, no tyrosinase mRNA was detectable in blood samples of patients with primary melanoma and regional lymph node metastases. In a small number of patients with visceral metastases (10-30%), we found tyrosinase mRNA-positive results. Analyses of different blood samples taken at 2-h intervals indicate that tumor cells persist only transiently in the peripheral blood. Successful establishment of melanoma cell growth from tyrosinase mRNA-positive samples indicates that viable tumor cells exist in melanoma patients' peripheral blood. Our results indicate a low amount of tyrosinase-specific transcripts in a small subset of stage IV patients and suggest that the analysis of tyrosinase mRNA in peripheral blood samples is not helpful as a prognostic marker or monitoring tool in melanoma patients.

Melanoma-inhibiting activity (MIA) mRNA is not exclusively transcribed in melanoma cells: low levels of MIA mRNA are present in various cell types and in peripheral blood.

The detection of minimal amounts of melanoma cells by tyrosinase reverse transcription polymerase chain reaction (RT-PCR) is seriously hampered by false negative reports in blood of melanoma patients with disseminated melanoma. Therefore, additional assays which make use of multiple melanoma markers are needed. It has been shown that introduction of multiple markers increases the sensitivity of detection. Melanoma inhibitory activity (MIA) is one such melanoma-specific candidate gene. To test the specificity of MIA PCR, we performed 30 and 60 cycles of PCR with two different sets of MIA specific primers on 19 melanoma and 16 non-melanoma cell lines. MIA mRNA was detected in 16 out of 19 melanoma cell lines and in seven out of 16 non-melanoma cell lines after 30 cycles of PCR. However, MIA mRNA could be detected in all cell lines after 60 cycles of PCR. Also, in 14 out of 14 blood samples of melanoma patients, five out of six blood samples of non-melanoma patients and in seven out of seven blood samples of healthy volunteers, MIA mRNA was detected after 60 cycles of PCR, whereas no MIA PCR product could be detected in any of the blood samples after 30 cycles of PCR. We conclude that low levels of MIA transcripts are present in various normal and neoplastic cell types. Therefore, MIA is not a suitable marker gene to facilitate the detection of minimal amounts of melanoma cells in blood or in target organs of the metastatic process. © 1999 Cancer Research Campaign

The Analysis of Tyrosinase-Specific mRNA in Blood Samples of Melanoma Patients by RT-PCR Is Not a Useful Test for Metastic Tumor Progression

Reverse transcription (RT) of the tyrosinase mRNA and specific cDNA amplification by nested polymerase chain reactin (PCR) have been reported to facilitate the early detection of circulating tumor cells in melanoma patients. The significance and practical value of this procedure for the diagnosis of tumor dissemination in melanoma patients are unclear. In the current study we analyzed peripheral blood samples of 65 melanoma patients of different clinical stages for the presence of tyrosinase mRNA by RT-PCR using nested oligonucleotide primers specific for tyrosinase cDNA. Furthermore, blood samples were evaluated for tumor cell growth by cell culture assays in vitro. No tyrosinase mRNA was detectable in blood samples of 26 patients with primary melanoma and 16 patients with regional lymph node metastases. In five of 13 patients with visceral metastases we found at least one blood sample positive for tyrosinase mRNA during a 2-to 4-mo interval. Analyses of different blood samples of patients with visceral metastases taken in a 2-h interval, furthermore, indicate that tumor cells only transiently persist in the peripheral blood. We obtained in vitro proliferating melanoma cells from two blood samples derived from different patients with visceral melanoma metastases. This demonstrates that viable melanoma cells indeed circulate in the peripheral blood with retained proliferative capacity in vitro. The analysis of blood samples by RT-PCR for tyrosinase mRNA, however, is not suitable for the early detection of tumor progression in melanoma patients.