Stage III-IV Melanoma Patients Research Articles

T-Cell Subtypes and Immune Signatures in Cutaneous Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy.

Immune checkpoint inhibition (ICI) improves outcomes in melanoma patients, but associated T-cell activation frequently leads to immune-related cutaneous adverse events (cutAEs). To dynamically identify T-cell subtypes and immune signatures associated with cutAEs, a pilot study was performed in stage III-IV melanoma patients using blood samples for flow cytometry and cytokine analysis. Blood samples were taken from patients before initiation of ICI (naive), at the onset of a cutAE, and after 6 months of ICI treatment. Overall, 30 patients were treated either with anti-PD1 monotherapy or with anti-PD-1/anti-CTLA-4 combination therapy. Flow cytometry analysis of PBMCs showed that ICI induced an overall shift from a Th2 towards a Th1 profile. Twelve patients (40%) developed cutAEs, which were associated with increased Th22 cells and Th17 cells, supported by a tendency to have elevated Th17/Th22-associated cytokines such as IL-17A, IL-22 and IL-23 levels in the plasma. Cytokine signatures specific for urticaria and T-cell-mediated cutAEs were identified in the plasma of patients by a bead-based assay. IL-10 was elevated in non-responders and, interestingly, during cutAEs. In conclusion, we identified distinct immune signatures based on the Th17/Th22 pathway in cutAEs, both in PBMCs and plasma. In addition, our finding of upregulated IL-10 during cutAEs supports the notion of treating these patients early and adequately to avoid implications for the overall outcome.

1090P Outcome impact of time from complete resection to start of adjuvant immunotherapy in stage III-IV melanoma patients

1090P Outcome impact of time from complete resection to start of adjuvant immunotherapy in stage III-IV melanoma patients

Association of Receipt of Systemic Treatment for Melanoma With Insurance Type in North Carolina.

Previous studies of hospital-based patients with metastatic melanoma suggest sociodemographic factors, including insurance type, may be associated with the receipt of systemic treatments. To examine whether insurance type is associated with the receipt of systemic treatment among patients with melanoma in a broad cohort of patients in North Carolina. We conducted a retrospective cohort study between 2011 and 2017 of patients with stages III-IV melanoma using data from the North Carolina Central Cancer Registry linked to Medicare, Medicaid, and private health insurance claims across the state. The primary outcome was the receipt of any systemic treatment, and the secondary outcome was the receipt of immunotherapy. A total of 372 patients met the inclusion criteria. The average age was 68 years old (interquartile range: 56-76) and 61% were male. Within the cohort 48% had Medicare only, 29% had private insurance, 12% had both Medicare and Medicaid, and 11% had Medicaid only. A total of 186 (50%) patients received systemic treatment for melanoma, 125 (67%) of whom received immunotherapy. The use of systemic therapy, including immunotherapy, increased significantly over time. Having Medicaid-only insurance was independently associated with a 45% lower likelihood of receiving any systemic treatment [0.55 (95% CI: 0.35, 0.85)] and a 43% lower likelihood of receipt of immunotherapy [0.57 (95% CI: 0.34, 0.95)] compared with private insurance. Stage III-IV melanoma patients with Medicaid-only insurance were less likely to receive systemic therapy or immunotherapy than patients with private insurance or Medicare insurance. This finding raises concerns about insurance-based disparities in treatment access.

The dynamics of circulating tumour DNA (ctDNA) during treatment reflects tumour response in advanced melanoma patients.

Despite the introduction of targeted (BRAFi/MEKi) and immune checkpoint inhibitors (ICIs) has significantly reduced the recurrence rate and improved the overall survival (OS) of patients with Stage III and IV melanoma, only a percentage will benefit of durable disease control. The aim of this study was to examine whether the levels of circulating tumour DNA (ctDNA) in plasma of advanced melanoma patients undergoing BRAFi/MEKi or ICIs vary according to the patients' survival outcomes (i.e. progression-free survival (PFS) and OS) and disease progression. Plasma samples of Stage III-IV melanoma patients were collected at baseline (treatment initiation) and thereafter every 3 months. Circulating BRAFV600E/K and NRASQ61R/K mutations were analysed through droplet digital PCR (ddPCR, Bio-Rad) in a total of 177 plasma samples from 48 melanoma patients (19 Stage III, 29 Stage IV). Baseline ctDNA concentration was significantly associated with OS (HR = 1.003, 95% CI = 1.000-1.006, p = 0.043) and PFS (HR = 1.004, 95% CI = 1.000-1.007, p = 0.029) independent of clinical-prognostic confounders. For each unit increase in the ∆ctDNA (concentration difference between the last follow-up and baseline) there was a 24% increased risk of disease progression, irrespective of treatment type and stage at diagnosis (OR = 1.24, 95% CI = 1.03-1.49, p = 0.020, AUC = 0.93). Patients with reduction of ctDNA level from baseline to the last follow-up had longer OS (HR = 0.14; 95% CI = 0.05-0.44, p = 0.001) and PFS (HR = 0.08; 95% CI = 0.03-0.27, p < 0.0001) compared to patients with increased ctDNA, including adjustment for confounding factors. Our findings suggest that variation of ctDNA over time during melanoma treatment reflects the clinical outcome and tumour response to therapy and might be helpful in clinical monitoring.

Adjuvant treatment and outcome of stage III melanoma patients: Results of a multicenter real-world German Dermatologic Cooperative Oncology Group (DeCOG) study

PurposeClinical trials demonstrated significantly improved recurrence-free survival (RFS) of melanoma patients receiving adjuvant treatment. As data from controlled trials are based on selected populations, we investigated adjuvantly treated stage III melanoma patients under real-world conditions. Patients and methodsIn a prior multicenter cohort study, stage III-IV melanoma patients were analysed for their choice of adjuvant therapy. In this follow-up study, we examined RFS, overall and melanoma-specific survival (MSS) and response to the subsequent treatment of 589 stage III patients (232 BRAF-mutated) receiving adjuvant PD-1 inhibitors (PD1; n = 479) or targeted therapy (TT; n = 110). ResultsThe median follow-up of the total cohort was 25.7 months. The main reason for premature discontinuation of adjuvant therapy was disease progression in PD1- (28.8%, n = 138/479) and adverse events in TT-treated patients (28.2%, n = 31/110).Among BRAF-mutated patients, RFS at 24 months was 49% (95% CI 40.6–59.0%) for PD1- and 67% (95% CI 58–77%) for TT-treated patients. The risk of recurrence was higher for BRAF-mutated PD1 than TT (hazard ratio 1.99; 95% CI 1.34–2.96; hazard ratio adjusted for age, sex and tumour stage, 2.21; 95% CI 1.48–3.30). Twenty-four months MSS was 87% (95% CI 81.0–94.1) for PD1 and 92% (95% CI 86.6–97.0) for TT.Response to subsequent systemic treatment for unresectable disease was 22% for all PD1- and 16% for TT-treated patients. ConclusionsPD1-treated patients had more and earlier recurrences than TT patients. In BRAF-mutated patients, adjuvant TT might prevent early recurrences more effectively than PD1 treatment. Management of recurrence despite adjuvant treatment is challenging, with low response to current therapeutic options.

S-100B in the follow-up of patients with stage III and IV melanoma: pursuit of the Holy Grail

Background: Patients with stage III-IV melanoma are at considerable risk for disease recurrence. Early detection of recurrence is important to optimize immunotherapy administration and improving progression-free and overall survival. S-100B can be used as tumor marker to evaluate whether patients are at risk for developing disease recurrence or progression. It could be a promising tool to determine whether patients need to receive additional diagnostic procedures. However, implementation in routine clinical setting is limited. Hence, this study aimed to evaluate the value of S-100B as a decision tool for the need to perform an 18F-fluorodeoxyglucose (FDG) positron emission tomography with computed tomography (PET/CT) to confirm disease recurrence or progression in stage III-IV melanoma patients.Methods: Data of 51 stage III-IV melanoma patients, presenting for follow-up after surgery with curative intent, was retrospectively extracted from a single-center electronic patient record system. 18FDG-PET/CT was performed based on clinical signs or elevated S-100B levels. S-100B measurements were treated as independent data points.Results: Fifteen out of 303 S-100B levels were elevated. Six elevated levels were causes by disease progression; 4/6 measurements were noted with concurrent clinical signs. Twenty-four events of disease progression were confirmed. The sensitivity and specificity of S-100B serum test were 25.0% [95% CI (9.8-46.7)] and 96.8% [95% CI (94.0-98.5)]. The positive predictive value (PPV) and negative predictive value (NPV) of S-100B were 40.0% [95% CI (20.6-63.2)] and 93.8% [95% CI (92.2-95.0)].Conclusions: Elevated S-100B levels did not exclude nor indicate metastatic or recurrent disease in this study. Using S-100B in routine clinical setting does not seem to be of additional value.

49P A phase II randomized trial with adjuvant autologous tumor lysate-pulsed dendritic cells (DC) in resected stage III-IV melanoma patients: Preliminary immunological results

49P A phase II randomized trial with adjuvant autologous tumor lysate-pulsed dendritic cells (DC) in resected stage III-IV melanoma patients: Preliminary immunological results

1071P A nationwide, real-life study of outcome and quality of life after the introduction of adjuvant immunotherapy for Danish melanoma patients

Clinical trials have shown promising results for adjuvant immunotherapy in resected stage III-IV melanoma patients, but real-life data are scarce. Data on efficacy and quality of life (QoL) during anti-PD-1 adjuvant therapy from a complete national cohort of melanoma patients may support treatment choices and help patient guidance.

Incidence of immune-related adverse events with pembrolizumab in advanced melanoma and correlation with treatment response: A single institution review of irAEs.

e21519 Background: Anti-PD-1 therapy has become a mainstay of advanced melanoma treatment. However, associated irAEs are common and contribute to significant delays or cessation of treatment. In this retrospective cohort study, we aim to characterize the real-world irAE profile of pembrolizumab at our institution and its prognostic implications. Methods: We evaluated unresectable stage III-IV melanoma patients treated with pembrolizumab between 2011 and 2016 at the University of Arizona Cancer Center (UACC). Information regarding demographics, treatment, irAEs, and hospitalizations were recorded. Adverse events were graded using the common terminology criteria for adverse events v.4.0 (CTCAE). Results: Seventy-three patients were identified, 4 (5.5%) with unresectable stage III and 69 (94.5%) with stage IV melanoma. The median age was 68; 48 (65.8%) patients were male and 25 (34.2%) were female. Nineteen patients (26.0%) had a preexisting autoimmune disorder. 171 irAEs were identified in 58 of the patients. 141 (82.5%) were Grade 1, 24 (14.0%) were Grade 2, and 6 (3.5%) were Grade 3. Incidence and time to onset of significant events is displayed in Table. Eleven (15%) patients were hospitalized, and 8 patients (11.0%) stopped treatment due to an irAE. Sixteen (84.2%) patients with a preexisting autoimmune condition experienced an irAE versus 78% of patients without an autoimmune condition. Twelve patients (75%) had an exacerbation of their autoimmune condition. Response could be assessed in 67 patients . 30 patients of the 67 (45%) had a CR or a PR and 37 patients (55%) had progressive disease (PD). 58 of 67 (87%) experienced an irAE. 29 (50%) of the 58 irAE patients had PD, while 8 (88.9%) of the 9 non-irAE patients had PD. The relative risk of progression in patients who experienced an irAE versus who did not was 0.56 (95% CI: 0.39-0.79, p= 0.001). Conclusions: Experience with pembrolizumab at our institution suggests a similar irAE profile compared to landmark trials, though we observed a higher percentage of patients who required discontinuation of pembrolizumab (11% versus 5% in Phase III trials). In our retrospective study, the risk for progression was significantly higher in patients without irAE versus with any irAE, suggesting that development of irAE could be a positive prognostic sign.[Table: see text]

Abstract 5083: Indoleamine 2-3 dioxygenase expression by metastatic melanoma cells correlates with increased overall survival

Abstract Though immunotherapy revolutionized the treatment of advanced melanoma, many patients fail to respond to checkpoint blockade, creating a pressing need to optimize the immune response to metastatic melanoma. Indoleamine 2-3 dioxygenase (IDO), which can be induced in a variety of cancer types and inhibits the immune response, has emerged as a potential target for future immunotherapies. Conflicting evidence exists regarding the prognostic significance of IDO expression in metastatic melanoma cells. We hypothesized that IDO expression by melanoma cells would correlate with decreased overall survival (OS). Skin or lymph node metastases from 89 patients with Stage III or IV melanoma were included in this study. Tissue microarrays (TMA) were constructed and immunohistochemical staining for IDO1 (Sigma Prestige, HPA 023072, 1:2,000 dilution) was performed. IDO staining in tumor cells was scored by an experienced dermatopathologist and was classified as positive (IDO+) when clear cytoplasmic staining was present in ≥1% of tumor cells. The main endpoint for analysis was OS as defined by the time from initial surgery to death. Multivariate Cox proportional hazards modeling was performed and Cox proportional hazards assumptions were tested using Schoenfield residual analysis. Following IHC staining, 32/89 patients were classified as IDO+ (36%). Comparison of patient demographics revealed that IDO+ patients were more likely to undergo resection with palliative intent and to have Stage IV disease. Tumor immunotype also varied by IDO status, with IDO+ patients more likely to have immunotype B or C lesions. Additionally, IDO+ tumors showed higher intratumoral density of CD20+ B cells, CD4+ and CD8+ T-cells, and FoxP3+ T-cells. Patients with IDO+ lesions had significantly better OS (median 31.6 vs 98.5 months, p-value&amp;lt;0.01). In a multivariate model incorporating IDO expression, age, sex, disease stage, BRAF mutation status, PD-L1 expression, CD8+ T-cell infiltration density, and palliative surgical intent, only IDO expression and palliative surgical intent retained statistically significant associations with overall survival (HR=0.44, p-value=0.01). Contrary to our initial hypothesis, in this cohort of immunotherapy-naïve, stage III-IV melanoma patients, IDO expression by metastatic melanoma cells was correlated with improved OS, which persisted even after controlling for sex, age, disease stage, BRAF mutation and CD8+ T cell infiltrate. It seems likely that IDO expression by melanoma cells may be triggered by a pro-inflammatory cytokine milieu within the tumor microenvironment, as has been postulated by others. Whether the positive prognostic effect of IDO expression in Stage III and IV melanoma metastases we observed is attributable entirely to increased immune infiltrate within the tumor is unclear. Nonetheless, our findings, particularly in context of the failure an IDO1 inhibitor, Epacadostat, in a recent stage 3 clinical trial highlight the need for further investigation into the function of IDO1 in melanoma metastases. Citation Format: Kevin Lynch, Sarah Gradeki, Min Kwak, Alejandro Gru, Nolan Wages, Craig Slingluff. Indoleamine 2-3 dioxygenase expression by metastatic melanoma cells correlates with increased overall survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5083.

Metabolic Biomarker-Based BRAFV600 Mutation Association and Prediction in Melanoma.

The aim of this study was to associate and predict B-rapidly accelerated fibrosarcoma valine 600 (BRAFV600) mutation status with both conventional and radiomics 18F-FDG PET/CT features, while exploring several methods of feature selection in melanoma radiomics. Methods: Seventy unresectable stage III-IV melanoma patients who underwent a baseline 18F-FDG PET/CT scan were identified. Patients were assigned to the BRAFV600 group or BRAF wild-type group according to mutational status. 18F-FDG uptake quantification was performed by semiautomatic lesion delineation. Four hundred eighty radiomics features and 4 conventional PET features (SUVmax, SUVmean, SUVpeak, and total lesion glycolysis) were extracted per lesion. Six different methods of feature selection were implemented, and 10-fold cross-validated predictive models were built for each. Model performances were evaluated with areas under the curve (AUCs) for the receiver operating characteristic curves. Results: Thirty-five BRAFV600 mutated patients (100 lesions) and 35 BRAF wild-type patients (79 lesions) were analyzed. AUCs predicting the BRAFV600 mutation varied from 0.54 to 0.62 and were susceptible to feature selection method. The best AUCs were achieved by feature selection based on literature, a penalized binary logistic regression model, and random forest model. No significant difference was found between the BRAFV600 and BRAF wild-type group in conventional PET features or predictive value. Conclusion: BRAFV600 mutation status is not associated with, nor can it be predicted with, conventional PET features, whereas radiomics features were of low predictive value (AUC = 0.62). We showed feature selection methods to influence predictive model performance, describing and evaluating 6 unique methods. Detecting BRAFV600 status in melanoma based on 18F-FDG PET/CT alone does not yet provide clinically relevant knowledge.

Anti-Melanoma immunity and local regression of cutaneous metastases in melanoma patients treated with monobenzone and imiquimod; a phase 2 a trial

ABSTRACT Vitiligo development in melanoma patients during immunotherapy is a favorable prognostic sign and indicates breakage of tolerance against melanocytic/melanoma antigens. We investigated a novel immunotherapeutic approach of the skin-depigmenting compound monobenzone synergizing with imiquimod in inducing antimelanoma immunity and melanoma regression. Stage III-IV melanoma patients with non-resectable cutaneous melanoma metastases were treated with monobenzone and imiquimod (MI) therapy applied locally to cutaneous metastases and adjacent skin during 12 weeks, or longer. Twenty-one of 25 enrolled patients were evaluable for clinical assessment at 12 weeks. MI therapy was well-tolerated. Partial regression of cutaneous metastases was observed in 8 patients and stable disease in 1 patient, reaching the statistical endpoint of treatment efficacy. Continued treatment induced clinical response in 11 patients, including complete responses in three patients. Seven patients developed vitiligo-like depigmentation on areas of skin that were not treated with MI therapy, indicating a systemic effect of MI therapy. Melanoma-specific antibody responses were induced in 7 of 17 patients tested and melanoma-specific CD8+T-cell responses in 11 of 15 patients tested. These systemic immune responses were significantly increased during therapy as compared to baseline in responding patients. This study shows that MI therapy induces local and systemic anti-melanoma immunity and local regression of cutaneous metastases in 38% of patients, or 52% during prolonged therapy. This study provides proof-of-concept of MI therapy, a low-cost, broadly applicable and well-tolerated treatment for cutaneous melanoma metastases, attractive for further clinical investigation.

Nano‐particle vaccination combined with TLR‐7 and ‐9 ligands triggers memory and effector CD8+T‐cell responses in melanoma patients

Optimal vaccine strategies must be identified for improving T-cell vaccination against infectious and malignant diseases. MelQbG10 is a virus-like nano-particle loaded with A-type CpG-oligonucleotides (CpG-ODN) and coupled to peptide16–35 derived from Melan-A/MART-1. In this phase IIa clinical study, four groups of stage III-IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan-A/MART-1-specific T-cell responses. T-cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T-cell responses in all (11/11) patients, with predominant generation of effector-memory-phenotype cells. In turn, Imiquimod induced higher proportions of central-memory-phenotype cells and increased percentages of CD127+ (IL-7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T-cell frequencies, associated with lower proportions of memory and effector-phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG-ODN induced combined memory and effector CD8+ T-cell responses.

The role of BRAF V600 mutation in melanoma

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway. About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E). BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors. The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3). The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression. Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment. Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation. In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations. Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD). Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy. For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months. A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab. In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2. Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations. Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

Humoral and cellular immune responses in stage III-IV melanoma patients: Implications for immunotherapy

Abstract Introduction: To assess humoral and cytotoxic T lymphocyte responsiveness in melanoma patients undergoing immunotherapy trials. Methods: 24 stage III (n = 5) and stage IV (n = 19) melanoma patients underwent immunization with a recombinant vaccinia virus encoding 3 tumor associated epitopes (TAA: gp100280-288, Mart-127-35, tyrosinase1-9) and CD80/CD86 costimulatory molecules (rVV). Immunization occured i.d. in a first trial (17 patients) and intranodally in a second, ongoing trial (7 patients). Frequencies of CTL precursor (CTLp) specific for TAA or influenza matrix (IM) control epitope were quantified. Humoral response against rVV was measured by ELISA. Results: Depending on specific responses to immunization patients were ranked as good responders (responsive to 3 or 2 epitopes) or poor responders (unresponsive or responsive to 1 epitope). All stage III patients showed CTL responses against all 3 epitopes. In contrast (p rVV specific humoral response was increased (OD > 50% of pre-treatment) in all stage III patients, but only in 6/13 stage IV patients (p Conclusions: Immunocompromission in stage IV melanoma patients might hamper the induction of tumor specific CTL responses. Conversely, induction of rVV specific humoral responses doesn't prevent the generation of CTL specific for TAA. These data suggest that stage III patients and immunocompetent stage IV patients are more likely to respond to immunotherapy.

Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction

Several studies have evaluated the use of polymerase chain reaction (PCR) amplification of tyrosinase mRNA to detect melanoma cells in blood. However, contradictory results have been obtained from different groups. We therefore have developed and validated a quantitative PCR method for tyrosinase and tyrosinase-related protein-2 (TRP-2) mRNA. An important methodological finding was that high concentrations of reverse transcriptase or RNA sample inhibited the following PCR. This could be abolished by dilution of the cDNA sample before the PCR. Standard curves with a linear range over at least five logs were obtained with dilutions of melanoma cell cDNA. Controls (RNA and cDNA) consisting of melanoma cells (1000/ml) added to blood were analysed repeatedly over 3 months, resulting in means between 880 and 1074 AU/ml. The RNA controls were stable, whereas the cDNA controls, as well as the calibrators, showed a tendency to change over time. The variation in the RNA controls was 25% for tyrosinase and 22% for TRP-2. Seven stage III-IV melanoma patients were tested for tyrosinase and TRP-2 transcripts in blood drawn from a peripheral vein and from a Port-a-cath. Tyrosinase mRNA was found in three patients (0.8-12.4 AU/ml). For TRP-2, the same amount was found in the patients as in healthy donors. No differences were seen between blood from a peripheral vein and from the Port-a-cath. We here present fast and sensitive methods for the quantification of tyrosinase and TRP-2 mRNA in blood.