Comparative study of serum 6-hydroxy-5-methoxyindole-2-carboxylic acid, 5-S-cysteinyl-dopa, and lactate dehydrogenase levels as markers for malignant melanoma

Comparative study of serum 6-hydroxy-5-methoxyindole-2-carboxylic acid, 5-S-cysteinyl-dopa, and lactate dehydrogenase levels as markers for malignant melanoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Comparative study of serum 6-hydroxy-5-methoxyindole-2-carboxylic acid, 5-S-cysteinyl-dopa, and lactate dehydrogenase levels as markers for malignant melanoma Hiroshi Umemura, Masaki Takiwaki, Yoshikuni Kikutani, Seketsu Fukuzawa, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4470628/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background With the development of novel therapies for advanced malignant melanoma (MM), biomarkers that can accurately reflect the progression of MM are needed. Recently, we have developed a method to measure the serum levels of 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), a melanin-related metabolite, using mass spectrometry and reported that it may be a potential marker for MM. Objective In the present study, we compared the usefulness of serum levels of 6H5MI2C, 5-S-cysteinyl-dopa (5-S-CD), and lactate dehydrogenase (LD) as tumor markers for MM (n = 63). Results Among the three markers, only 6H5MI2C was significantly elevated in the metastatic group (stages III and IV) compared to the non-metastatic group (stages 0 to II). Additionally, 6H5MI2C with a cut-off level of 1.00 ng/mL showed higher sensitivity for metastatic MM than 5-S-CD and LD. Conclusions Thus, serum 6H5MI2C level has the potential to be a good marker for MM in comparison to 5-S-CD and LD. 5-S-cysteinyl-dopa 6-hydroxy-5-methoxyindole-2-carboxylic acid biomarker lactate dehydrogenase melanoma Figures Figure 1 Introduction Malignant melanoma (MM) is one of the most aggressive skin cancers, and it has a high mortality rate in the metastatic phase [ 1 ]. The recently developed serine-threonine protein kinase B-Raf and mitogen-activated protein kinase kinase inhibitors, and immune checkpoint inhibitors have improved the prognosis of metastatic MM [ 2 , 3 ]. However, the development of cancer biomarkers that can accurately reflect the progression of advanced MM is still needed for the efficient use of these novel therapies. Lactate dehydrogenase (LD) is widely used as a serum biomarker for MM, and it is also used for staging [ 1 ]. In addition, 5-S-cysteinyl-dopa (5-S-CD), a eumelanin-related metabolite, has been widely studied as a serum biomarker for MM [ 4 – 6 ]. Recently, we developed a mass spectrometry-based assay for measuring the serum levels of the pheomelanin-related metabolite 5-hydroxy-6-methoxyindole-2-carboxylic acid and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), and reported that the serum 6H5MI2C levels may be a potential marker for MM [ 7 ]. In the present study, we compared the usefulness of 6H5MI2C, 5-S-CD, and LD as tumor markers for MM. Methods In our previous report, the serum 6H5MI2C levels were evaluated in samples obtained from 81 patients with MM between December 2011 and August 2021 at the Department of Dermatology, Okayama University Hospital; the 6H5MI2C levels were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) [ 7 ]. In 63 of these 81 patients, the 5-S-CD and LD levels were evaluated at the same time when serum samples were collected. In the present study, we used the data for these 63 MM patients. The serum 5-S-CD levels were measured using high-performance liquid chromatography (HPLC) by SRL (Tokyo, Japan) according to a previously published method with minor modifications [ 8 ]. As previously described, 10.0 nmol/L was considered to be the cut-off value for the serum 5-S-CD level [ 4 , 5 ]. The serum LD levels were determined using conventional methods in the Department of Laboratory Medicine, Okayama University Hospital. The reference range was 124–222 IU/L. The median age of the 63 patients was 70 years (range, 28 to 90 years), and there were 36 males and 27 females. None had amelanotic melanoma. Clinical staging was performed according to the eighth edition of the American Joint Committee on Cancer Melanoma Staging System 2018 [ 1 ]. We performed statistical analyses using EZR version 1.61 statistical software [ 9 ]. The serum 6H5MI2C, 5-S-CD, and LD levels were compared among patients with different stages of MM, i.e. , the non-metastatic (stages 0 to II) group, stage III group, stage IV group, and a combined stages III and IV group, using the Mann–Whitney U-test. The statistical significance level was set at P < 0.05. Results Figure 1 shows the comparisons of the serum levels of each marker between the non-metastatic (stages 0 to II) group and each of the stage III, stage IV, and metastatic (stages III and IV) groups. No significant elevation was seen in any of the three markers in the stage III group when compared to the non-metastatic group. However, the levels of the three markers were significantly elevated in the stage IV group when compared to the non-metastatic group. Only 6H5MI2C was significantly elevated in the combined stages III and IV group when compared to the non-metastatic group. In addition, we calculated the sensitivity of the three markers for each disease stage. Since there is no fixed cut-off value for 6H5MI2C, sensitivity was calculated for each stage using three cut-off values of 0.80, 0.90, and 1.00 ng/mL. As shown in Table 1, the sensitivity of serum 6H5MI2C levels with 1.00 ng/mL cut-off value was higher than that of serum 5-S-CD and LD levels for the metastatic group, whereas the sensitivity of the three markers was similar for the non-metastatic group. Discussion In our previous study, we compared the serum 5-S-CD and LD levels as markers of MM, and reported that 5-S-CD was more efficient for detecting advanced-stage MM. In that study, the 5-S-CD levels were significantly higher in patients with stage III or IV disease than in patients with non-metastatic disease (stages 0 to II) [ 5 ]. However, in the present study, the 6H5MI2C and 5-S-CD levels were not significantly higher in the stage III group when compared to the non-metastatic group. One possible reason for this discrepancy is the differences between the MM populations analyzed in the two studies. The method for measuring serum 6H5MI2C levels has just been established and cut-off values have not been determined [ 7 ]. In the present study, we examined the sensitivity of 6H5MI2C to each stage of MM at three different cut-off values (0.80, 0,90, and 1.00 ng/mL). With reference to the sensitivity of 5-S-CD and LD to MM in the non-metastatic group, a cut-off value of 1.00 ng/mL was considered appropriate for serum 6H5MI2C levels. Among the three markers examined in the present study, the serum 6H5MI2C level appeared to best reflect MM progression. Similarly, Hara et al. previously reported that the plasma 6H5MI2C levels determined by HPLC reflected MM progression better and more reliably than the plasma 5-S-CD levels [ 10 ]. However, there have been papers showing that the serum and urinary levels of 5-S-CD reflect MM progression better than those of 6H5MI2C [ 11 – 13 ]. Although it remains to be determined whether 6H5MI2C or 5-S-CD is a better marker for MM, in the future, comparisons should be made with a larger number of cases. Although various tumor markers for MM have been developed, only LD has been widely used worldwide as a serum biomarker. Serum LD is also included in the staging criteria for MM, and increased baseline LD levels reflect the prognosis of patients receiving nivolumab or pembrolizumab therapy [ 14 ]. LD can be measured with commonly available apparatuses for laboratory testing, and is thus relatively inexpensive and does not require specialized knowledge and skills to measure. In contrast, the HPLC for measuring 5-S-CD and the LC-MS/MS for measuring 6H5MI2C have the disadvantages of being expensive and requiring specialized knowledge and skills to perform, limiting their application. We have previously reported that the serum 5-S-CD levels may be a better predictive marker of nivolumab treatment efficacy than LD in patients with advanced MM [ 15 ]. Taken together with the results of the present study, serum melanin metabolites, especially 6H5MI2C measured by LC-MS/MS, appear to have the potential to be good markers for MM. Therefore, further experiments to examine their usefulness, optimize the measurement technology, and decrease costs are essential for the practical clinical application of melanin metabolites, including 6H5MI2C, as markers of MM. Limitations Samples from healthy volunteers are necessary to obtain precise cut-off values, sensitivity, and specificity of tumor markers. In the present study, however, the cut-off values and sensitivity of 6H5MI2C could only be determined using samples from MM patients only, because serum samples from healthy volunteers were not available. Additionally, the present study compared the serum 6H5MI2C levels measured by LC-MS/MS to the serum 5-S-CD levels measured by HPLC, and it is possible that the difference in measurement methods affected the results; thus, the development of a technique for measuring the serum 5-S-CD levels by LC-MS/MS is also desirable. Abbreviations 6H5MI2C 6-hydroxy-5-methoxyindole-2-carboxylic acid 5-S-CD 5-S-cysteinyl-dopa HPLC High-performance liquid chromatography LC-MS/MS Liquid chromatography tandem mass spectrometry LD Lactate dehydrogenase MM Malignant melanoma Declarations Acknowledgements We are grateful to Dr. Mamoru Satoh and Prof. Fumio Nomura (Division of Clinical Mass Spectrometry, Chiba University, Chiba, Japan) for providing insightful comments and suggestions for this work. Author contributions Hiroshi Umemura, Kentaro Abe, Kiyotaka Fujino and Osamu Yamasaki designed the study concept. Masaki Takiwaki, Yoshikuni Kikutani and Seketsu Fukuzawa performed the experiments. Hiroshi Umemura and Masaki Takiwaki drafted data analysis and manuscript. Satoru Sugihara, Kota Tachibana, Shin Morizane and Osamu Yamasaki collected serum samples and data from patients. All authors researched and collated this paper as a group effort. All authors have read and approved the final manuscript. Funding This work was supported in part by JSPS KAKENHI Grant Number 23K07796, and the 28 th Research Grant of the Kurozumi Medical Foundation. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate This study was approved by the ethics committees of the Nihon University Itabashi Hospital (no. RK-210910-1), Nihon University School of Medicine (no. 30-9-0), and Okayama University Hospital (no. 1902-011). Written informed consent was obtained from each participant. Participation was entirely voluntary. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Gershenwald JE, Scolyer RA, Hess KR, Sondak VK, Long GV, Ross MI, et al. Melanoma staging: Evidence-based changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA Cancer J Clin. 2017;67:472–92. https://doi.org/10.3322/caac.21409 . Queirolo P, Spagnolo F. BRAF plus MEK-targeted drugs: a new standard of treatment for BRAF-mutant advanced melanoma. Cancer Metastasis Rev. 2017;36:35–42. https://doi.org/10.1007/s10555-017-9660-6 . Carlino MS, Larkin J, Long GV. Immune checkpoint inhibitors in melanoma. Lancet. 2021;398:1002–14. https://doi.org/10.1016/s0140-6736(21)01206-x . Wakamatsu K, Kageshita T, Furue M, Hatta H, Kiyohara Y, Nakayama J, et al. Evaluation of 5-S-cysteinyldopa as a marker of melanoma progression: 10 years' experience. Melanoma Res. 2002;12:245–53. https://doi.org/10.1097/00008390-200206000-00008 . Umemura H, Yamasaki O, Kaji T, Otsuka M, Asagoe K, Takata M, et al. Usefulness of serum 5-S-cysteinyl-dopa as a biomarker for predicting prognosis and detecting relapse in patients with advanced stage malignant melanoma. J Dermatol. 2017;44:449–54. https://doi.org/10.1111/1346-8138.13651 . Wakamatsu K, Fukushima S, Minagawa A, Omodaka T, Hida T, Hatta N, et al. Significance of 5-S-Cysteinyldopa as a Marker for Melanoma. Int J Mol Sci. 2020;21:432. https://doi.org/10.3390/ijms21020432 . Takiwaki M, Umemura H, Kikutani Y, Fukuzawa S, Abe K, Fujino K et al. A method for measuring serum levels of melanin-associated indole metabolites using LC-MS/MS and its application to malignant melanoma. Clin Chim Acta 2024 Mar 16;557:117873. https://doi.org/10.1016/j.cca.2024.117873 . Wakamatsu K, Ito S. Improved HPLC determination of 5-S-cysteinyldopa in serum. Clin Chem. 1994;40:495–96. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transpl. 2013;48:452–58. https://doi.org/10.1038/bmt.2012.244 . Hara H, Walsh N, Yamada K, Jimbow K. High plasma level of a eumelanin precursor, 6-hydroxy-5-methoxyindole-2-carboxylic acid as a prognostic marker for malignant melanoma. J Invest Dermatol. 1994;102:501–5. https://doi.org/10.1111/1523-1747.ep12373153 . Horikoshi T, Ito S. Serum 5-S-cysteinyldopa (5-S-CD) as a marker of melanoma progression. J Dermatol. 1992;19. https://doi.org/10.1111/j.1346-8138.1992.tb03787.x . :809 – 13. Horikoshi T, Ito S, Wakamatsu K, Onodera H, Eguchi H. Evaluation of melanin-related metabolites as markers of melanoma progression. Cancer. 1994;73:629–36. https://doi.org/10.1002/1097-0142(19940201)73:3%3C629 . ::aid-cncr2820730321%3E3.0.co;2-w. Kärnell R, Kågedal B, Lindholm C, Nilsson B, Arstrand K, Ringborg U. The value of cysteinyldopa in the follow-up of disseminated malignant melanoma Melanoma. Res. 2000;10:363–9. https://doi.org/10.1097/00008390-200008000-00008 . Diem S, Kasenda B, Spain L, Martin-Liberal J, Marconcini R, Gore M, et al. Serum lactate dehydrogenase as an early marker for outcome in patients treated with anti-PD-1 therapy in metastatic melanoma. Br J Cancer. 2016;114:256–61. https://doi.org/10.1038/bjc.2015.467 . Umemura H, Kaji T, Tachibana K, Morizane S, Yamasaki O. Serum 5-S-cysteinyl-dopa levels as a predictive marker for the efficacy of nivolumab in advanced malignant melanoma. Int J Biol Markers. 2019;34:414–20. https://doi.org/10.1177/1724600819883658 . Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files 04Table1.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4470628","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":311475503,"identity":"908f113f-eed9-4498-9159-44e16cd200e3","order_by":0,"name":"Hiroshi Umemura","email":"data:image/png;base64,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","orcid":"","institution":"Nihon University","correspondingAuthor":true,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Umemura","suffix":""},{"id":311475505,"identity":"8133d442-2713-4c2a-8b2d-431e4fb09363","order_by":1,"name":"Masaki Takiwaki","email":"","orcid":"","institution":"JEOL (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Takiwaki","suffix":""},{"id":311475507,"identity":"5cf6bde4-74ce-4781-a92e-f3c1c9c2757c","order_by":2,"name":"Yoshikuni Kikutani","email":"","orcid":"","institution":"JEOL (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Yoshikuni","middleName":"","lastName":"Kikutani","suffix":""},{"id":311475510,"identity":"b40f5948-6d24-4559-a79f-bed4aa106009","order_by":3,"name":"Seketsu Fukuzawa","email":"","orcid":"","institution":"JEOL (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Seketsu","middleName":"","lastName":"Fukuzawa","suffix":""},{"id":311475513,"identity":"e4f52e5d-fc3b-4510-b4c6-726ee8be91cb","order_by":4,"name":"Kentaro Abe","email":"","orcid":"","institution":"JEOL (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Kentaro","middleName":"","lastName":"Abe","suffix":""},{"id":311475517,"identity":"cad9a2a8-ff49-4027-8ce1-638abd41e339","order_by":5,"name":"Kiyotaka Fujino","email":"","orcid":"","institution":"JEOL (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Kiyotaka","middleName":"","lastName":"Fujino","suffix":""},{"id":311475522,"identity":"f7d189cb-ce51-44a7-a86c-15e92ed02642","order_by":6,"name":"Satoru Sugihara","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Sugihara","suffix":""},{"id":311475525,"identity":"2791c555-936c-4a89-9117-6d30222e78d8","order_by":7,"name":"Kota Tachibana","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Kota","middleName":"","lastName":"Tachibana","suffix":""},{"id":311475526,"identity":"4283dcf4-c7c7-4824-80d9-00b39971430f","order_by":8,"name":"Shin Morizane","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Shin","middleName":"","lastName":"Morizane","suffix":""},{"id":311475529,"identity":"5b01daad-a102-499e-9ab0-6a28c2f0997a","order_by":9,"name":"Osamu Yamasaki","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Osamu","middleName":"","lastName":"Yamasaki","suffix":""}],"badges":[],"createdAt":"2024-05-24 07:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4470628/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4470628/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58047536,"identity":"5a3d9c08-8fc7-437e-bf89-2876bd84d316","added_by":"auto","created_at":"2024-06-10 11:55:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77950,"visible":true,"origin":"","legend":"\u003cp\u003eThe serum 6H5MI2C, 5-S-CD, and LD levels were evaluated. Asterisks indicate a significant difference at a level of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 in the Mann–Whitney U-test. (a) The 6H5MI2C level was significantly elevated in the stage IV group and in the metastatic (stages III and IV) group when compared to the non-metastatic (stages 0 to II) group. (b) The 5-S-CD level was significantly elevated only in the stage IV group when compared to the non-metastatic (stages 0 to II) group. (c) The LD level was also significantly elevated only in the stage IV group when compared to the non-metastatic (stages 0 to II) group.\u003c/p\u003e","description":"","filename":"03Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4470628/v1/e2c1b592eb08d2877fcd3573.png"},{"id":66073955,"identity":"644775a0-2211-4f29-acf9-9e67723692c8","added_by":"auto","created_at":"2024-10-07 12:47:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":375524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4470628/v1/1330c34d-2460-4ee3-9b1a-92050e1f54c7.pdf"},{"id":58047537,"identity":"57de27f3-dda6-437f-9e28-6b95a1096cb8","added_by":"auto","created_at":"2024-06-10 11:55:55","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11240,"visible":true,"origin":"","legend":"","description":"","filename":"04Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4470628/v1/933da902f568836b3a942e51.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative study of serum 6-hydroxy-5-methoxyindole-2-carboxylic acid, 5-S-cysteinyl-dopa, and lactate dehydrogenase levels as markers for malignant melanoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMalignant melanoma (MM) is one of the most aggressive skin cancers, and it has a high mortality rate in the metastatic phase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The recently developed serine-threonine protein kinase B-Raf and mitogen-activated protein kinase kinase inhibitors, and immune checkpoint inhibitors have improved the prognosis of metastatic MM [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the development of cancer biomarkers that can accurately reflect the progression of advanced MM is still needed for the efficient use of these novel therapies. Lactate dehydrogenase (LD) is widely used as a serum biomarker for MM, and it is also used for staging [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition, 5-S-cysteinyl-dopa (5-S-CD), a eumelanin-related metabolite, has been widely studied as a serum biomarker for MM [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recently, we developed a mass spectrometry-based assay for measuring the serum levels of the pheomelanin-related metabolite 5-hydroxy-6-methoxyindole-2-carboxylic acid and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), and reported that the serum 6H5MI2C levels may be a potential marker for MM [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the present study, we compared the usefulness of 6H5MI2C, 5-S-CD, and LD as tumor markers for MM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eIn our previous report, the serum 6H5MI2C levels were evaluated in samples obtained from 81 patients with MM between December 2011 and August 2021 at the Department of Dermatology, Okayama University Hospital; the 6H5MI2C levels were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In 63 of these 81 patients, the 5-S-CD and LD levels were evaluated at the same time when serum samples were collected. In the present study, we used the data for these 63 MM patients. The serum 5-S-CD levels were measured using high-performance liquid chromatography (HPLC) by SRL (Tokyo, Japan) according to a previously published method with minor modifications [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As previously described, 10.0 nmol/L was considered to be the cut-off value for the serum 5-S-CD level [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The serum LD levels were determined using conventional methods in the Department of Laboratory Medicine, Okayama University Hospital. The reference range was 124\u0026ndash;222 IU/L. The median age of the 63 patients was 70 years (range, 28 to 90 years), and there were 36 males and 27 females. None had amelanotic melanoma. Clinical staging was performed according to the eighth edition of the American Joint Committee on Cancer Melanoma Staging System 2018 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. We performed statistical analyses using EZR version 1.61 statistical software [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The serum 6H5MI2C, 5-S-CD, and LD levels were compared among patients with different stages of MM, \u003cem\u003ei.e.\u003c/em\u003e, the non-metastatic (stages 0 to II) group, stage III group, stage IV group, and a combined stages III and IV group, using the Mann\u0026ndash;Whitney U-test. The statistical significance level was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the comparisons of the serum levels of each marker between the non-metastatic (stages 0 to II) group and each of the stage III, stage IV, and metastatic (stages III and IV) groups. No significant elevation was seen in any of the three markers in the stage III group when compared to the non-metastatic group. However, the levels of the three markers were significantly elevated in the stage IV group when compared to the non-metastatic group. Only 6H5MI2C was significantly elevated in the combined stages III and IV group when compared to the non-metastatic group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we calculated the sensitivity of the three markers for each disease stage. Since there is no fixed cut-off value for 6H5MI2C, sensitivity was calculated for each stage using three cut-off values of 0.80, 0.90, and 1.00 ng/mL. As shown in Table\u0026nbsp;1, the sensitivity of serum 6H5MI2C levels with 1.00 ng/mL cut-off value was higher than that of serum 5-S-CD and LD levels for the metastatic group, whereas the sensitivity of the three markers was similar for the non-metastatic group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our previous study, we compared the serum 5-S-CD and LD levels as markers of MM, and reported that 5-S-CD was more efficient for detecting advanced-stage MM. In that study, the 5-S-CD levels were significantly higher in patients with stage III or IV disease than in patients with non-metastatic disease (stages 0 to II) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, in the present study, the 6H5MI2C and 5-S-CD levels were not significantly higher in the stage III group when compared to the non-metastatic group. One possible reason for this discrepancy is the differences between the MM populations analyzed in the two studies.\u003c/p\u003e \u003cp\u003eThe method for measuring serum 6H5MI2C levels has just been established and cut-off values have not been determined [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the present study, we examined the sensitivity of 6H5MI2C to each stage of MM at three different cut-off values (0.80, 0,90, and 1.00 ng/mL). With reference to the sensitivity of 5-S-CD and LD to MM in the non-metastatic group, a cut-off value of 1.00 ng/mL was considered appropriate for serum 6H5MI2C levels.\u003c/p\u003e \u003cp\u003eAmong the three markers examined in the present study, the serum 6H5MI2C level appeared to best reflect MM progression. Similarly, Hara \u003cem\u003eet al.\u003c/em\u003e previously reported that the plasma 6H5MI2C levels determined by HPLC reflected MM progression better and more reliably than the plasma 5-S-CD levels [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, there have been papers showing that the serum and urinary levels of 5-S-CD reflect MM progression better than those of 6H5MI2C [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although it remains to be determined whether 6H5MI2C or 5-S-CD is a better marker for MM, in the future, comparisons should be made with a larger number of cases.\u003c/p\u003e \u003cp\u003eAlthough various tumor markers for MM have been developed, only LD has been widely used worldwide as a serum biomarker. Serum LD is also included in the staging criteria for MM, and increased baseline LD levels reflect the prognosis of patients receiving nivolumab or pembrolizumab therapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. LD can be measured with commonly available apparatuses for laboratory testing, and is thus relatively inexpensive and does not require specialized knowledge and skills to measure. In contrast, the HPLC for measuring 5-S-CD and the LC-MS/MS for measuring 6H5MI2C have the disadvantages of being expensive and requiring specialized knowledge and skills to perform, limiting their application. We have previously reported that the serum 5-S-CD levels may be a better predictive marker of nivolumab treatment efficacy than LD in patients with advanced MM [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Taken together with the results of the present study, serum melanin metabolites, especially 6H5MI2C measured by LC-MS/MS, appear to have the potential to be good markers for MM. Therefore, further experiments to examine their usefulness, optimize the measurement technology, and decrease costs are essential for the practical clinical application of melanin metabolites, including 6H5MI2C, as markers of MM.\u003c/p\u003e\n\u003ch3\u003eLimitations\u003c/h3\u003e\n\u003cp\u003eSamples from healthy volunteers are necessary to obtain precise cut-off values, sensitivity, and specificity of tumor markers. In the present study, however, the cut-off values and sensitivity of 6H5MI2C could only be determined using samples from MM patients only, because serum samples from healthy volunteers were not available.\u003c/p\u003e \u003cp\u003eAdditionally, the present study compared the serum 6H5MI2C levels measured by LC-MS/MS to the serum 5-S-CD levels measured by HPLC, and it is possible that the difference in measurement methods affected the results; thus, the development of a technique for measuring the serum 5-S-CD levels by LC-MS/MS is also desirable.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e6H5MI2C \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 6-hydroxy-5-methoxyindole-2-carboxylic acid\u003c/p\u003e\n\u003cp\u003e5-S-CD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 5-S-cysteinyl-dopa\u003c/p\u003e\n\u003cp\u003eHPLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; High-performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eLC-MS/MS \u0026nbsp; \u0026nbsp; \u0026nbsp;Liquid chromatography tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003eLD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eMM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Malignant melanoma\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr. Mamoru Satoh and Prof. Fumio Nomura (Division of Clinical Mass Spectrometry, Chiba University, Chiba, Japan) for providing insightful comments and suggestions for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHiroshi Umemura, Kentaro Abe, Kiyotaka Fujino and Osamu Yamasaki designed the study concept. Masaki Takiwaki, Yoshikuni Kikutani and Seketsu Fukuzawa performed the experiments. Hiroshi Umemura and Masaki Takiwaki drafted data analysis and manuscript. Satoru Sugihara, Kota Tachibana, Shin Morizane and Osamu Yamasaki collected serum samples and data from patients. All authors researched and collated this paper as a group effort. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by JSPS KAKENHI Grant Number 23K07796, and the 28\u003csup\u003eth\u003c/sup\u003e Research Grant of the Kurozumi Medical Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethics committees of the Nihon University Itabashi Hospital (no. RK-210910-1), Nihon University School of Medicine (no. 30-9-0), and Okayama University Hospital (no. 1902-011). Written informed consent was obtained from each participant. Participation was entirely voluntary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGershenwald JE, Scolyer RA, Hess KR, Sondak VK, Long GV, Ross MI, et al. Melanoma staging: Evidence-based changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA Cancer J Clin. 2017;67:472\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3322/caac.21409\u003c/span\u003e\u003cspan address=\"10.3322/caac.21409\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQueirolo P, Spagnolo F. 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Serum 5-S-cysteinyl-dopa levels as a predictive marker for the efficacy of nivolumab in advanced malignant melanoma. Int J Biol Markers. 2019;34:414\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/1724600819883658\u003c/span\u003e\u003cspan address=\"10.1177/1724600819883658\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"5-S-cysteinyl-dopa, 6-hydroxy-5-methoxyindole-2-carboxylic acid, biomarker, lactate dehydrogenase, melanoma","lastPublishedDoi":"10.21203/rs.3.rs-4470628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4470628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWith the development of novel therapies for advanced malignant melanoma (MM), biomarkers that can accurately reflect the progression of MM are needed. Recently, we have developed a method to measure the serum levels of 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), a melanin-related metabolite, using mass spectrometry and reported that it may be a potential marker for MM.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eIn the present study, we compared the usefulness of serum levels of 6H5MI2C, 5-S-cysteinyl-dopa (5-S-CD), and lactate dehydrogenase (LD) as tumor markers for MM (n\u0026thinsp;=\u0026thinsp;63).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the three markers, only 6H5MI2C was significantly elevated in the metastatic group (stages III and IV) compared to the non-metastatic group (stages 0 to II). Additionally, 6H5MI2C with a cut-off level of 1.00 ng/mL showed higher sensitivity for metastatic MM than 5-S-CD and LD.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThus, serum 6H5MI2C level has the potential to be a good marker for MM in comparison to 5-S-CD and LD.\u003c/p\u003e","manuscriptTitle":"Comparative study of serum 6-hydroxy-5-methoxyindole-2-carboxylic acid, 5-S-cysteinyl-dopa, and lactate dehydrogenase levels as markers for malignant melanoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 11:55:51","doi":"10.21203/rs.3.rs-4470628/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d2bf1e6-3593-427f-8ca7-cc1f69194878","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T12:39:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 11:55:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4470628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4470628","identity":"rs-4470628","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}