Significance of 5-S-Cysteinyldopa as a Marker for Melanoma.

Melanoma is one of the most lethal and malignant cancers and its incidence is increasing worldwide, and Japan is not an exception. Although there are numerous therapeutic options for melanoma, the prognosis is still poor once it has metastasized. The main concern after removal of a primary melanoma is whether it has metastasized, and early detection of metastatic melanoma would be effective in improving the prognosis of patients. Thus, it is very important to identify reliable methods to detect metastases as early as possible. Although many prognostic biomarkers (mainly for metastases) of melanoma have been reported, there are very few effective for an early diagnosis. Serum and urinary biomarkers for melanoma diagnosis have especially received great interest because of the relative ease of sample collection and handling. Several serum and urinary biomarkers appear to have significant potential both as prognostic indicators and as targets for future therapeutic methods, but still there are no efficient serum and urinary biomarkers for early detection, accurate diagnosis and prognosis, efficient monitoring of the disease and reliable prediction of survival and recurrence. Levels of 5-S-cysteinyldopa (5SCD) in the serum or urine as biomarkers of melanoma have been found to be significantly elevated earlier and to reflect melanoma progression better than physical examinations, laboratory tests and imaging techniques, such as scintigraphy and echography. With recent developments in the treatment of melanoma, studies reporting combinations of 5SCD levels and new applications for the treatment of melanoma are gradually increasing. This review summarizes the usefulness of 5SCD, the most widely used and well-known melanoma marker in the serum and urine, compares 5SCD and other useful markers, and finally its application to other fields.

Serum 5-S-cysteinyldopa behavior in the early phase of nivolumab treatment of 12 melanoma patients.

Along with the expansion of therapeutic options for metastatic melanoma, the development of useful biomarkers is urgently required to predict and monitor treatment response. Serum 5-S-cysteinyldopa (5-S-CD) has been identified as a diagnostic marker of malignant melanoma, but its utility as a biomarker for emerging therapeutic agents remains unknown. We assessed serum 5-S-CD in 12 metastatic melanoma patients (median age, 76 years; six men and six women) who had been treated with nivolumab (Nivo) at Shinshu University Hospital between 2014 and 2016. Serum 5-S-CD and lactate dehydrogenase levels before and at 3-6 weeks of Nivo treatment were obtained and their changes were compared with clinical responses as defined by the Response Evaluation Criteria in Solid Tumors criteria (version 1.1). A decrease of 10 nmol/L or more of serum 5-S-CD was observed only in partial response patients (2/3 cases, 67%), while an increase of 10 nmol/L or more of serum 5-S-CD was witnessed only in progressive disease patients (4/8 cases, 50%). Serum 5-S-CD changes were within ±10 nmol/L in the remaining six patients (partial response, one; stable disease, one; progressive disease, four). The results of the four moderately affected progressive disease patients were suspected to have been influenced by small-sized metastatic lesions, a mixed response that included diminished and enlarged metastatic lesions, prior therapy to Nivo with BRAF inhibitors or radiation, or the development of brain metastasis. Serum 5-S-CD in the early phase of Nivo treatment may be helpful to predict therapeutic response in metastatic melanoma.

Usefulness of serum 5-S-cysteinyl-dopa as a biomarker for predicting prognosis and detecting relapse in patients with advanced stage malignant melanoma.

With the recent development of novel molecular targeted drugs for advanced stage malignant melanoma (MM), including RAF and mitogen-activated protein kinase kinase inhibitors and immune checkpoint blockers, the early detection of relapse is important for managing patients with MM. In this study, we retrospectively analyzed two conventional serum biomarkers, 5-S-cysteinyl-dopa and lactate dehydrogenase, in patients with MM (n = 140) who were treated at a single Japanese institute from June 2007 to June 2015. At the initial hospital visit, serum 5-S-cysteinyl-dopa levels were significantly increased in patients with stages III (n = 38) and IV (n = 20) MM compared with patients with stages 0-II (n = 62) MM. In addition, in patients with stages III and IV MM, serum 5-S-cysteinyl-dopa levels of more than 15.0 nmol/L at initial hospital visit correlated with a poor prognosis. In 11 of 14 patients whose disease progressed during follow up (mostly from stages III-IV), serum 5-S-cysteinyl-dopa levels exceeded the normal limit of 10.0 nmol/L during the clinical detection of distant metastases. These results indicate the usefulness of measuring serum 5-S-cysteinyl-dopa levels at initial hospital visit and during follow up for early and effective therapeutic interventions using newly developed molecular targeted drugs.

Circulating melanoma cells as a potential biomarker to detect metastasis and evaluate prognosis.

TNM staging is mainly used to evaluate the prognosis of melanoma patients. Serum biomarkers such as 5-S-cysteinyldopa (5-S-CD) have occasionally been used but most do not respond until the tumour burden becomes high. Recently, circulating melanoma cells (CMC) have been reported as a possible new biomarker to detect metastasis, monitor treatment response and predict prognosis. The object of this exploratory study was to evaluate the efficacy of CMC to detect metastasis and predict prognosis by cross-sectional and prospective observational analyses, respectively. Altogether 15 patients with stages II-IV melanoma were enrolled and CMC were enumerated by CellSearch system with cut-off values of two cells/7.5 mL. Serum 5-S-CD and lactate dehydrogenase (LDH) were also measured. The sensitivity of CMC and 5-S-CD for the detection of metastasis was 33 and 50%, respectively. The combination of CMC and 5-S-CD showed a sensitivity of 67%, the best performance among CMC, 5-S-CD, LDH and any combination of two of the markers. Additionally, a 30-month prospective observation showed that CMC could segregate patients with poorer prognosis. The median survival time for the patients with <2 CMC and those with ≥2 CMC was 19.5 and 4.5 months, respectively. The limitation of this study is the small sample size. These preliminary results indicate CMC may complement the efficacy of 5-S-CD to detect metastasis and can be a prognostic marker. Although there is still room for improvement to maximise the sensitivity, the CellSearch system is reproducible, standardised and suitable for multi-centre studies.

Serum levels of leptin receptor in patients with malignant melanoma as a new tumor marker.

Leptin is known to be abnormally expressed in a variety of cancers, and leptin receptors have been reported to be expressed on human melanoma cells. In this study, we evaluated the possibility that the serum levels of leptin receptor could be a tumor marker of malignant melanoma (MM). Serum samples were obtained from 71 patients with MM, and the serum levels of leptin receptor were measured by double-determinant ELISA. Interestingly, serum levels of leptin receptor decreased gradually with the stages of MM, being highest at in situ and lowest at stage IV. There was also a trend of reverse correlation between tumor thickness and serum levels of leptin receptor. To our knowledge, this is the first report investigating the serum levels of leptin receptor in MM, and serum leptin receptor levels may be used as a useful tumor marker of MM.

Pre-study and in-study validation of a SPE-LC-MS-MS method for the determination of 5-S-cysteinyldopa, a melanoma biomarker, in human plasma.

The incidence of malignant melanoma has increased over the past decades, particularly in Caucasian population. This disease presents defavourable prognosis in terms of survey, especially when detection occurs at the metastatic phase. Reliable analytical methods for biomarker determination are thus an interesting tool in pathology detection and follow-up. In this context, a method using SPE-LC-ESI-MS-MS for the determination of 5-S-cysteinyldopa (5-SCD) in human plasma was optimized. The presence of matrix effect was investigated in details while 5-SCD stability was studied according to FDA requirements for the validation of bioanalytical methods. Pre-study and in-study validations of the entire method were then successfully performed by applying the approach based on total measurement error and accuracy profiles over a concentration ranges from 1.6 to 200 ng/ml. Good results with respect to accuracy, trueness and precision were obtained. The maximum risk of observing future measurements falling outside the acceptance limits during routine analysis was also estimated.

Increased cysteinyldopa plasma levels hint to melanocyte as stress sensor in psoriasis.

The possible role of melanocyte as a modulator of the inflammation and keratinocyte hyperproliferation in psoriasis has been hypothesised but never demonstrated on experimental basis. Aim of the present study was to assess whether plasma levels of 5-S-cysteinyldopa (CD), a metabolite reflecting melanocyte activity, undergo changes in association with psoriasis together with those of typical lipid peroxidation markers thiobarbituric acid reactive substances (TBARS). A group of 16 patients with psoriasis at different stage as indicated by the psoriasis area and severity index (PASI) were enrolled against an age and sex matched control group. Both TBARS (P<0.05) and CD (P<0.005) levels were higher than controls with statistical significance. After 1 month therapy the levels of either biomarkers decreased with respect to the starting values although with marked individual differences. CD may represent a novel and sensitive biomarker for the follow up of psoriasis and evaluation of the efficacy of therapeutic regimens beyond PASI determination.

Serum levels of 5-s-cysteinyldopa are correlated with skin colors in hemodialysis patients but not in peritoneal dialysis patients.

BACKGROUND: Diffuse hyperpigmentation is common in patients with chronic renal failure undergoing hemodialysis (HD) or peritoneal dialysis (PD). We previously reported that serum levels of 5-S-cysteinyldopa (5SCD, a pheomelanin precursor) and pheomelanin were significantly elevated in HD patients. METHODS: Skin color was assessed using a Mexameter that measures the melanin index (MI) and the erythema index (EI). The upper inner arms (non-sun-exposed site) and the foreheads (sun-exposed site) of HD and PD patients and control subjects were analyzed. RESULTS: MI values on the upper inner arms and on the foreheads of HD and PD patients were significantly higher than in controls. In HD patients, significant correlations were found for serum 5SCD levels with MI and EI on the upper inner arm, and for EI on the forehead. In PD patients, no such correlations were found. CONCLUSIONS: Hyperpigmentation in HD patients results partly from accumulation of pheomelanin in the skin.

Treatment of primary melanoma of the lung monitored by 5-S-cysteinyldopa levels.

Malignant melanoma originating outside the skin is very rare, whereas primary malignant melanoma of the lung is extremely rare. 5-S-Cysteinyldopa (5-S-CD), a melanin metabolite, has been reported to be a prognostic marker for cutaneous malignant melanoma. This is the first report in the English language literature dealing with primary malignant melanoma of the lung using serum 5-S-Cysteinyldopa levels to monitor the effects of surgery and chemotherapy.

Serum 5-S-cysteinyldopa levels in patients with psoriasis undergoing narrowband ultraviolet B phototherapy.

BACKGROUND: Ultraviolet (UV) B radiation from sunlight can result in tanning or burning of the skin. Narrowband UVB (NB-UVB), a relatively new light source that is not yet widely available, is effective for treating generalized psoriasis without the use of psoralens. AIMS: The melanin-related metabolite 5-S-cysteinyldopa (5-S-CD), which reflects pheomelanin production, has been used as a biological marker of melanoma progression, but there are no studies available on therapeutic UVB effects on serum 5-S-CD of human subjects. In the present study, we measured the time course of changes in serum levels of 5-S-CD in patients with psoriasis undergoing NB-UVB phototherapy. METHODS: In total, 11 Japanese patients with generalized psoriasis vulgaris received NB-UVB treatment five times per week, at an initial dose of 0.1 J/cm(2). The dose was increased by 10-20% per treatment for > 20 treatments. Serum samples were taken before and 3, 7, 10, 14 and 28 days after phototherapy. RESULTS: After 4 weeks of NB-UVB treatment, 9 of 11 patients were in remission, confirming the effectiveness of NB-UVB for treating Japanese patients with psoriasis. Two patients withdrew before day 28 because of other complications. Mean level of 5-S-CD in serum was significantly increased on day 7, 10 14 and 28 compared with the level before phototherapy and it peaked on day 10. CONCLUSIONS: Serum 5-S-CD levels were significantly increased by therapeutic UVB exposure. Sustained levels of 5-S-CD in serum appear to reflect the degree of skin injury during NB-UVB phototherapy.

Serum levels of pigmentation markers are elevated in patients undergoing hemodialysis.

BACKGROUND: Diffuse hyperpigmentation is common among patients with chronic renal failure undergoing hemodialysis (HD). We have examined serum levels of 5-S-cysteinyldopa (5SCD, a pheomelanin precursor), pheomelanin, eumelanin, and protein-bound (PB-) 3,4-dihydroxyphenylalanine (DOPA) and PB-5SCD in HD patients. METHODS: Pheomelanin and eumelanin were assayed by chemical degradation methods. RESULTS: Serum levels of free 5SCD in HD patients (n = 16) were 9-fold higher than in healthy controls (n = 16). Levels of pheomelanin in HD patients were 2.6-fold higher than in controls, while levels of eumelanin did not differ between HD patients and controls. Levels of PB-DOPA and PB-5SCD in HD patients were approximately 1.5-fold higher than in controls. Serum levels of free 5SCD were positively correlated to the duration of HD therapy. CONCLUSIONS: The high constitutive levels of free 5SCD, pheomelanin, and PB-DOPA in the blood may be deteriorating in HD patients through the production of reactive oxygen species.

Development of a mass spectrometry method for the determination of a melanoma biomarker, 5-S-cysteinyldopa, in human plasma using solid phase extraction for sample clean-up.

5-S-cysteinyldopa is a well-known pigment intermediate and analysis of its plasma concentration is interesting for the early diagnosis, as well as for evaluation of treatment and follow-up of malignant melanoma. A determination method of 5-SCD in human plasma was developed using solid phase extraction (SPE) on disposable cartridges and liquid chromatography electrospray mass spectrometry (LC-ESI-MS-MS). Compound's sensitivity to light and oxidation requires the addition of anti-oxidative agents (AO), to work in acidic media at 4 degrees C and to avoid light exposure of samples since blood collection. Different solid phases involving covalent binding to phenylboronic groups or dual retention mechanisms were evaluated and extraction cartridges containing both hydrophobic and strong cation exchange functionalities were finally selected. The LC separation of 5-SCD from endogenous catecholamines was achieved by gradient elution on a C18 stationary phase. 5-SCD was detected by multiple reaction monitoring (MRM) performed on ES(+) generated ions. Finally, the method was prevalidated in the lower ng/ml range. Good results with respect to accuracy, trueness and precision were obtained.

Evaluation of melanin-related metabolites as markers of solar ultraviolet-B radiation.

Ultraviolet-B (UVB) radiation due sunlight can result in sunburns and/or suntans. Sunburn occurs only several hours after solar UVB radiation, while a suntan requires several days to several weeks to develop. In the present study, we measured serum and urine levels of melanin-related metabolites, 5-S-cysteinyldopa (5-S-CD) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), in nine subjects exposed to normal sunlight over the course of 12 months. We collected samples in the middle of each month and examined the variation of the markers, the correlation between them, and their correlation with solar UVB radiation. Those markers exhibited a seasonal variation with lower values in the winter and higher values in the summer. Levels of 5-S-CD and 6H5MI2C in the serum showed 48% and 54% increases in the summer compared with those in the winter, respectively. Comparison of 5-S-CD in the serum and urine showed the highest correlation (r2 = 0.344), followed by the pair of 5-S-CD and 6H5MI2C in the serum. Levels of 5-S-CD in the serum showed the highest correlation (r2 = 0.729) with the mean solar UVB radiation during the first 10 d of the month, while 6H5MI2C in the serum was highly correlated (r2 = 0.483) with solar UVB radiation during the previous month. Levels of 5-S-CD and 6H5MI2C in the serum appear to reflect the degrees of skin injury and pigmentation in the skin, respectively.

Improved method for analysis of cysteinyldopa in human serum.

5-S-L-Cysteinyl-L-dopa is a well-known pigment intermediate and analysis of its serum concentration is well suited for evaluation of treatment and follow-up of stage III and IV malignant melanoma. A simplified analytical method is described using organic extraction followed by clean-up on a boronate gel to capture the compound containing vicinal hydroxyls. Weak acid solution elutes the 5-S-cysteinyldopa suitable for high-performance liquid chromatography (HPLC). The absolute recoveries of cysteinyldopa and its diastereomer 5-S-D-cysteinyl-L-dopa (used as an internal standard) were 81.5 +/- 2.8% and 81.3 +/- 2.7%, respectively, and use of the internal standard for the whole procedure gave an analytical recovery of 101 +/- 0.8%. The limit of quantitation was 1.5 nmol/L and the imprecision of the method was < 5.0% over the analytical range 1.5-500 nmol/L. The method is cheap and easy to perform and compares well with other described techniques. The use of the method is illustrated by results obtained during treatment of a patient with metastatic malignant melanoma.

Comparison of phaeomelanin and its precursor 5-S-cysteinyldopa in the serum of melanoma patients.

5-S-Cysteinyldopa (5-S-CD) has been used as a biochemical marker of melanoma progression. Recently we have shown that the serum level of 5-S-CD is a sensitive and specific marker in predicting distant metastases. In melanocytes and melanoma cells, cysteinyldopa isomers are oxidized to phaeomelanin, the yellow to reddish melanin pigment. In this study we have developed a new method to measure levels of phaeomelanin in serum samples and have evaluated its clinical significance. The method is based on the production of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) on reductive hydrolysis of phaeomelanin with hydriodic acid. 3-AHP is also derived from 3-nitrotyrosine-containing proteins. The isomeric 4-AHP and 3-AHP can be separated by high performance liquid chromatography. The mean +/- SD serum levels of 5-S-CD in control subjects (n = 36), in melanoma patients without recurrence (n = 92) and in melanoma patients with metastases (n = 24) were 2.7 +/- 1.2 nM (median 2.3 nM), 4.0 +/- 1.6 nM (median 3.8 nM) and 72 +/- 105 nM (median 35 nM), respectively. The serum levels of 4-AHP in these three groups were 45 +/- 21 nM (median 31 nM), 80 +/- 75 nM (median 53 nM) and 306 +/- 627 nM (median 133 nM), respectively. The serum levels of 4-AHP in patients with metastases (100 samples from 15 patients with progressive disease) correlated well (r = 0.887) with serum levels of 5-S-CD, while serum levels of 3-AHP did not (r = 0.240). The serum 5-S-CD and 4-AHP levels were serially analysed in the 15 patients with progressive disease. In two patients (13%), serum 4-AHP levels were elevated to abnormal levels before the serum 5-S-CD levels exceeded the cut-off value of 10 nM. In five patients (33%), the serum 4-AHP levels rose concurrently with the serum 5-S-CD levels. In the remaining eight patients (54%), serum 4-AHP levels were of less diagnostic value. Thus, the serum phaeomelanin level appears to be less sensitive than the serum 5-S-CD level in detecting distant metastases.

Beneficial effect of combined administration of some naturally occurring antioxidants (vitamins) and thiol chelators in the treatment of chronic lead intoxication.

Ameliorative effects of few naturally occurring antioxidants like ascorbic acid (vitamin C), alpha-tocopherol (vitamin E) either alone or in combination with meso-2,3-dimercaptosuccinic acid (DMSA) or monoisoamyl DMSA (MiADMSA), on parameters indicative of oxidative stress in the liver, kidney, brain and blood of lead-exposed rats were studied. Male Wistar rats were exposed to 0.1% lead acetate in drinking water for 3 months and treated thereafter with DMSA or its analogue MiADMSA (50 mg/kg, intraperitoneally), either individually or in combination with vitamin E (5 mg/kg, intramuscularly) or vitamin C (25 mg/kg, orally) once daily for 5 days. The effects of these treatments in influencing the lead-induced alterations in haem synthesis pathway, hepatic, renal and brain oxidative stress and lead concentration from the soft tissues were investigated. Exposure to lead produced a significant inhibition of delta-aminolevulinic acid dehydratase (ALAD) activity from 8.44+/-0.26 in control animals to 1.76+/-0.32 in lead control, reduction in glutathione (GSH) from 3.56+/-0.14 to 2.57+/-0.25 and an increase in zinc protoporphyrin level from 62.0+/-3.9 to 170+/-10.7 in blood, suggesting altered haem synthesis pathway. Both the thiol chelators and the two vitamins were able to increase blood ALAD activity towards normal, however, GSH level responded favorably only to the two thiol chelators. The most prominent effect on blood ALAD activity was, however, observed when MiADMSA was co-administered with vitamin C (7.51+/-0.17). Lead exposure produced a significant depletion of hepatic GSH from 4.59+/-0.78 in control animals to 2.27+/-0.47 in lead controls and catalase activity from 100+/-3.4 to 22.1+/-0.25, while oxidized glutathione (GSSG; 0.34+/-0.05 to 2.05+/-0.25), thiobarbituric acid reactive substance (TBARS; 1.70+/-0.45 to 5.22+/-0.50) and glutathione peroxidase (GPx) levels (3.41+/-0.09 to 6.17+/-0.65) increased significantly, pointing to hepatic oxidative stress. Altered, reduced and oxidized GSH levels showed significant recovery after MiADMSA and DMSA administration while, vitamins E and C were effective in reducing GSSG and TBARS levels and increasing catalase activity. Administration of MiADMSA alone and the combined administration of vitamin C along with DMSA and MiADMSA were most effective in increasing hepatic GSH levels to 4.88+/-0.14, 4.09+/-0.12 and 4.30+/-0.06, respectively. Hepatic catalase also reached near normal level in animals co-administered vitamin C with DMSA or MiADMSA (82.5+/-4.5 and 84.2+/-3.5, respectively). Combined treatments with vitamins and the thiol chelators were also able to effectively reduce lead-induced decrease in renal catalase activity and increase in TBARS and GPx level. Combination therapy, however, was unable to provide an effective reversal in the altered parameters indicative of oxidative stress in different brain regions, except in catalase activity. The result also suggests a beneficial role of vitamin E when administered along with the thiol chelators (particularly with MiADMSA) in reducing body lead burden. Blood lead concentration was reduced from 13.3+/-0.11 in lead control to 0.3+/-0.01 in MiADMSA plus vitamin E-treated rats. Liver and kidney lead concentration also showed a most prominent decrease in MiADMSA plus vitamin E co-administered rats (5.29+/-0.16 to 0.63+/-0.02 and 14.1+/-0.21 to 1.51+/-0.13 in liver and kidney, respectively). These results thus suggest that vitamin C administration during chelation with DMSA/MiADMSA was significantly beneficial in reducing oxidative stress however, it had little or no additive effect on the depletion of lead compared with the effect of chelators alone. Thus, the co-administration of vitamin E during chelation treatment with DMSA or MiADMSA could be recommended for achieving optimum effects of chelation therapy.

Use of serum 5-S-CD and S-100B protein levels to monitor the clinical course of malignant melanoma.

5-S-Cysteinyldopa (5-S-CD) is a precursor of pheomelanin. S-100B protein is a low molecular weight, acidic, calcium binding, cytoplasmic protein. In this study, the concentration changes of serum 5-S-CD and S-100B protein in melanomas of all stages were examined in parallel and patients were monitored during and after treatment. Serum samples were taken from 478 melanoma patients on 1924 occasions. Of these, 180 cases were regularly monitored. Concentrations of 5-S-CD were determined by high performance liquid chromatography (HPLC), S-100B protein by immunoluminometric assay. The mean/median concentrations of 5-S-CD and S-100B protein in Stage I, II and III patients and in the control group ranged around the normal level. In Stage IV patients, 58.4/50.6% sensitivity, 100% specificity and 100/86.6% positive predictive values were obtained concerning S-100B protein and 5-S-CD, respectively. Recurrence was observed in 57/180 of the regularly monitored patients in Stages I, II and III. In 10/57 (17.5%) of these patients suffering from any type of disease progression increases in both marker levels preceded the detection of metastasis by conventional methods. We can confirm that changes in both marker concentrations correlated with the stages of the patient. The markers are most sensitive in Stage IV patients and also have a high specificity in these patients. In Stage IV melanoma patients, 5-S-CD and S-100B protein levels are independent significant prognostic factors. In almost one fifth of patients both marker levels increased before the detection of metastatic disease with other appropriate, routinely scheduled investigations. This study suggests that serial serum marker measurements in the management and follow-up of melanoma patients should be examined further.