A method for measuring serum levels of melanin-associated indole metabolites using LC-MS/MS and its application to malignant melanoma.

BACKGROUND AND AIMS: With the development of novel therapies for advanced malignant melanoma (MM), biomarkers that can accurately reflect the progression of MM are needed. Serum levels of melanin-related indole metabolites such as 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) are potential biomarkers for MM. Here, we describe the development of a mass spectrometry (MS)-based assay to determine serum levels of 5H6MI2C and 6H5MI2C. MATERIALS AND METHODS: We developed a stable isotope dilution-selective reaction monitoring-MS protocol using liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure human serum 5H6MI2C and 6H5MI2C levels. Analytical evaluations of the method were performed and the method was applied to serum samples from MM patients (n = 81). RESULTS: The method established in this study showed high reproducibility and linearity. This novel method also found that serum 6H5MI2C levels were significantly elevated in patients with metastatic MM compared to those with non-metastatic MM. Unfortunately, 5H6MI2C did not show a comparable significant difference. CONCLUSION: We successfully established measurement methods for serum 5H6MI2C and 6H5MI2C levels using LC-MS/MS. Serum 6H5MI2C levels offer a potential marker for MM.

A Case-Control Study of the APELA Gene and Hypertensive Disorders of Pregnancy.

Hypertensive disorders of pregnancy (HDPs) are believed to comprise a group of multifactorial genetic diseases. Recently, it was reported that APELA-knockout mice exhibited HDP-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. The aim of the present study is to determine the associations between HDPs and single-nucleotide variants or haplotypes in the human APELA gene through a case-control study. The subjects were 196 pregnant women with HDPs and a control group of 254 women without HDPs. Six single-nucleotide variants (rs2068792, rs13120303, rs4541465, rs13152225, rs78639146, and rs67448487) were selected from the APELA gene region. Although there were no significant differences for each single-nucleotide polymorphism in the case-control study, the frequency of the T-A haplotypes rs4541465-rs67448487 was significantly higher in the HDP group, especially in those with gestational hypertension, than in the control group. The results suggest that the APELA gene may be a disease-susceptibility gene for HDP.

Identification of a novel frameshift c.930delG MEN1 germline mutation (p.Glu273LysfsTer7) in a sporadic case of multiple endocrine neoplasia type 1: A case report.

Multiple endocrine neoplasia type 1 (MEN1) is a rare genetic disorder that is inherited in an autosomal dominant manner. The characteristics of the disease are the combined occurrence of tumors in glands of the endocrine system, such as the parathyroid glands, pituitary gland and endocrine pancreas. Germline mutations in the MEN1 gene are associated with the occurrence of MEN1 and genetic testing for this gene is generally used as a basis for diagnosis. In this paper, a case of MEN1 in a middle­aged Japanese woman is reported. Direct sequencing analysis of the patient's DNA was performed and it revealed a MEN1 gene heterozygous germline (NM_130799.2:c.930delG) mutation in exon 5. This deletion/frameshift mutation produced a stop codon in the downstream sequence (NP_570711.1:p.Glu273LysfsTer7). To the best of our knowledge, this is the first report describing the NM_130799.2:c.930delG mutation as the basis for MEN1.

Attitudes toward and current status of disclosure of secondary findings from next-generation sequencing: a nation-wide survey of clinical genetics professionals in Japan.

The management of secondary findings (SFs), which are beyond the intended purpose of the analysis, from clinical comprehensive genomic analysis using next generation sequencing (NGS) presents challenges. Policy statements regarding their clinical management have been announced in Japan and other countries. In Japan, however, the current status of and attitudes of clinical genetics professionals toward reporting them are unclear. We conducted a questionnaire survey of clinical genetics professionals at two time points (2013 and 2019) to determine the enforcement of the SF management policy in cases of comprehensive genetic analysis of intractable diseases and clinical cancer genome profiling testing. According to the survey findings, 40% and 70% of the respondents stated in the 2013 and 2019 surveys, respectively, that they had an SF policy in the field of intractable diseases, indicating that SF policy awareness in Japan has changed significantly in recent years. Furthermore, a total of 80% of respondents stated that their facility had established a policy for clinical cancer genome profiling testing in the 2019 survey. In both surveys, the policies included the selection criteria for genes to be disclosed and the procedure to return SFs, followed by recommendations and proposals regarding SFs in Japan and other countries. To create a better list of the genes to be disclosed, further examination is needed considering the characteristics of each analysis.

Serum 5-S-cysteinyl-dopa levels as a predictive marker for the efficacy of nivolumab in advanced malignant melanoma.

PURPOSE: With the recent developments in novel molecular targeted therapy such as immune-checkpoint blockades, serine/threonine-protein kinase B-Raf, and mitogen-activated protein kinase kinase inhibitors, the prognosis of advanced malignant melanoma has been improving. 5-S-cysteinyl-dopa (5-S-CD), a precursor of pheomelanin, has been previously revealed to be a useful biomarker for advanced-stage malignant melanoma, especially in patients with distant metastases. Here, we aimed to assess and compare the utility of serum 5-S-CD and lactate dehydrogenase levels as markers for predicting the effects of nivolumab in advanced malignant melanoma. METHODS: Baseline serum 5-S-CD and lactate dehydrogenase levels in patients with unresectable stage IIIC and IV malignant melanoma treated with nivolumab (n = 21) were analyzed to determine their utility as predictive markers for survival. We also analyzed the prognostic value of these markers among patients with only stage IV malignant melanoma (n = 17). RESULTS: Our analysis showed that patients with baseline serum 5-S-CD levels >25.0 nmol/L had significantly poor prognosis. In contrast, serum lactate dehydrogenase levels at the upper limit of the normal range did not exhibit such changes. CONCLUSIONS: Serum 5-S-CD levels have the potential to be an excellent predictive marker for the efficacy of nivolumab therapy in patients with advanced malignant melanoma.

Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma.

The expression of immune checkpoint proteins such as programmed cell death protein 1 (PD-1) and its ligand (PD-L1) has been shown to correlate with patient prognosis in many malignant cancers. The expression of PD-L1 is controlled by c-Myc; however, further upstream regulation of PD-L1 expression is largely unknown. We have previously shown that atypical protein kinase C lambda/iota (aPKCλ) phosphorylates the Forkhead box protein O1 (FoxO1) transcription factor at Ser218 to suppress its DNA-binding ability, thereby regulating c-Myc expression and controlling physiologic and pathologic endothelial proliferation. The presence of phosphorylation of FoxO1 at Ser218 (pSer218 FoxO1) in cutaneous angiosarcoma (CAS) strongly correlates with poor patient prognosis. Here, we reported that patients with PD-L1+ cells in CAS lesions showed significantly worse prognosis compared to those that were PD-L1- . Expression of PD-L1 correlated with that of aPKCλ or the presence of pSer218FoxO1. Moreover, suppression of aPKCλ expression or inhibition of its activity in HUVECs or AS-M, an established human angiosarcoma cell line, resulted in decreased PD-L1 expression. Our results suggest that combined treatment with immune checkpoint inhibitors and aPKCλ inhibitors could be a novel treatment strategy for CAS patients.

aPKC controls endothelial growth by modulating c-Myc via FoxO1 DNA-binding ability.

Strict regulation of proliferation is vital for development, whereas unregulated cell proliferation is a fundamental characteristic of cancer. The polarity protein atypical protein kinase C lambda/iota (aPKCλ) is associated with cell proliferation through unknown mechanisms. In endothelial cells, suppression of aPKCλ impairs proliferation despite hyperactivated mitogenic signaling. Here we show that aPKCλ phosphorylates the DNA binding domain of forkhead box O1 (FoxO1) transcription factor, a gatekeeper of endothelial growth. Although mitogenic signaling excludes FoxO1 from the nucleus, consequently increasing c-Myc abundance and proliferation, aPKCλ controls c-Myc expression via FoxO1/miR-34c signaling without affecting its localization. We find this pathway is strongly activated in the malignant vascular sarcoma, angiosarcoma, and aPKC inhibition reduces c-Myc expression and proliferation of angiosarcoma cells. Moreover, FoxO1 phosphorylation at Ser218 and aPKC expression correlates with poor patient prognosis. Our findings may provide a potential therapeutic strategy for treatment of malignant cancers, like angiosarcoma.

Leukocytoclastic vasculitis associated with immunoglobulin A lambda monoclonal gammopathy of undetermined significance: A case report and review of previously reported cases.

Leukocytoclastic vasculitis is often associated with immunoglobulin (Ig)A deposition on the vascular walls. IgA-associated leukocytoclastic vasculitis comprises various underlying diseases. Hematological disorders that can be minor triggers include multiple myeloma and monoclonal gammopathy of undetermined significance. Here, we present the case of a 78-year-old woman with leukocytoclastic vasculitis associated with monoclonal gammopathy of undetermined significance of the IgA lambda chain. Oral steroid administration initially showed remission of vasculitis; however, the condition recurred after four attempts of treatment withdrawal. We also reviewed previous reports of 14 cases of IgA-associated leukocytoclastic vasculitis, of which 11 were associated with multiple myeloma and three with monoclonal gammopathy of undetermined significance.

Assessment by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of the Effects of Preanalytical Variables on Serum Peptidome Profiles Following Long-Term Sample Storage.

PURPOSE: Human serum and plasma are often used as clinical specimens in proteomics analyses, and peptidome profiling of human serum is a promising tool for identifying novel disease-associated biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for peptidomic biomarker discovery. Careful sample collection and handling are required as either can have a profound impact on serum peptidome patterns, yet the effects of preanalytical variables on serum peptidome profiles have not been completely elucidated. The present study investigated the effects of preanalytical variables, including storage temperature, duration (up to 12 months), and thawing methods, on MALDI-TOF MS-based serum peptidome patterns. EXPERIMENTAL DESIGN: Aliquots of serum samples were pretreated with weak cation exchanger magnetic beads using an automated ClinProtRobot system and then analyzed by MALDI-TOF MS. RESULTS: A number of significant differences in peak intensities were observed depending on sample processing variables. CONCLUSIONS AND CLINICAL RELEVANCE: These peaks can be used as sample quality markers to assess the effects of long-term storage on serum peptidome profiles using MALDI-TOF MS.

Prognostic value of 18 F-fluorodeoxyglucose positron emission tomography/computed tomography in patients with cutaneous angiosarcoma: A retrospective study of 18 cases.

Cutaneous angiosarcoma (CAS) is a rare soft tissue sarcoma with rapid growth and poor prognosis. We retrospectively analyzed the data of 18 patients with CAS who underwent 18 F-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) at the initial visit to the Department of Dermatology, Okayama University Hospital from September 2006 to March 2016. In the univariate survival analysis, patients with high standardized uptake values (SUVmax ) of the primary tumor showed significantly poorer prognosis than those with low SUVmax . Early assessment of prognosis using PET/CT may predict patient survival and is useful in the selection of therapeutic strategies.

Two arginine residues in the COOH-terminal of human ß-defensin-3 constitute an essential motif for antimicrobial activity and IL-6 production.

Human ß-defensin-3 (HBD-3) possesses antimicrobial activities and the potential to induce proinflammatory cytokines. HBD-3 contains a unique motif of two arginine residues (Arg or R) in the COOH-terminal region. To understand the bioactive properties of the Arg residues of HBD-3, we examined antimicrobial activities against Staphylococcus aureus and Pseudomonas aeruginosa using synthetic HBD-2, HBD-3 and two variant peptides of HBD-3: the Arg-truncated variant designated desR HBD-3 and NRR HBD-3, in which both Arg residues were shifted to the N-terminal region. IL-6 production from keratinocytes was studied using the peptides. HBD-3 possessed approximately five-fold more potent antimicrobial activities, evaluated as the minimum inhibitory concentration (MIC), against S. aureus compared with desR and NRR HBD-3, while no significant activity was observed in HBD-2. The antimicrobial activity of HBD-3 against S. aureus was well preserved even at high sodium chloride concentrations, but was attenuated in desR and NRR HBD-3. All the peptides exhibited similar antimicrobial activities against P. aeruginosa, but HBD-2 and desR HBD-3 showed diminished antimicrobial activities against P. aeruginosa at high salt concentrations. IL-6 production was significantly induced in keratinocytes with HBD-3, but not remarkably with stimulation by other peptide. These Arg residues are essential for the antimicrobial and biological properties of HBD-3.

Comparative study on driver mutations in primary and metastatic melanomas at a single Japanese institute: A clue for intra- and inter-tumor heterogeneity.

BACKGROUND: Searching for driver mutations in melanoma is critical to understanding melanoma genesis, progression and response to therapy. OBJECTIVES: We aimed to investigate the frequency and pattern of driver mutations in Japanese primary and metastatic melanomas including cases of unknown primary origin, in relation to their clinicopathologic manifestations. METHODS: Seventy-seven samples from 60 patients with melanoma were screened for 70 driver mutations of 20 oncogenes by Sequenom MelaCarta MassARRAY, and the results for primary and metastatic melanomas were compared. RESULTS: Of 77 tissue samples, BRAF V600E was detected in 21 samples (27%), CDK4 R24C in 7, EPHB6 G404S in 6, BRAF V600K in 2, NEK10 E379K in 2, and CDK4 R24H, NRAS Q61K, NRAS Q61R, KRAS G12A, KIT L576P, KIT V559A, ERBB4 E452K, and PDGFRA E996K in one sample each. No driver mutations related to the MAPK cascade including RAS and BRAF were detected in the chronically sun-damaged (CSD) group of melanoma. Dual or triple driver mutations were found in four of 40 (10%) samples from the primary melanomas, and three of 37 (8%) of the metastatic melanomas. Fourteen of 26 (54%) samples of non-CSD melanoma, and 3 of 6 (50%) melanomas of unknown primary origin had the BRAF V600E mutation. Mutations in membrane-bound receptors including KIT, ERBB4 and EPHB6 were detected in 8 of 77 (10%) samples. Of 17 pairs of primary and metastatic melanomas from the same patient, the primary mutation pattern was changed to a novel one in three cases, and only one of the plural mutations in the primary melanoma was found in the metastatic lesions in two cases. CONCLUSIONS: BRAF V600E is a predominant mutation in non-CSD melanoma and melanomas of unknown primary origin. Mutational heterogeneity may exist in the primary melanoma (intra-tumor heterogeneity), and between the primary and metastatic lesions (inter-tumor heterogeneity).

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.

Increased concentrations of apo A-I and apo A-II fragments in the serum of patients with hepatocellular carcinoma by magnetic beads-assisted MALDI-TOF mass spectrometry.

OBJECTIVES: Recent advances in sophisticated technologies in proteomics should provide promising ways to discover novel markers for hepatocellular carcinoma (HCC) in the early diagnosis. METHODS: Serum peptide and protein profiling was conducted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Profiling was carried out in a training set of 16 patients with HCC and a testing set of 15 patients with cirrhosis without HCC. All the patients were hepatitis C virus positive. Candidate peaks were processed to partial purification, followed by protein identification by amino acid sequence analysis. Immunoprecipitation was conducted to confirm the protein identity. RESULTS: Partial purification and protein identification revealed that one peak that was up-regulated in HCC sera both in the training and the testing sets was a fragment of apolipoprotein A-I (apo A-I). Immunoprecipitation confirmed this result. CONCLUSIONS: MALDI-TOF MS analysis revealed that apo A-I is a potential novel serum marker of HCC. Combination of these pretreatments and the current magnet bead-assisted MALDI-TOF MS will further enhance the efficiency of biomarker discovery for HCC.

Increase of DC-LAMP+ mature dendritic cell subsets in dermatopathic lymphadenitis of mycosis fungoides.

BACKGROUND: Little is known about the immunological milieu of the skin-draining lymph nodes (LNs) in mycosis fungoides (MF). OBJECTIVES: We studied dendritic cell (DC) subsets in the dermatopathic lymphadenitis of MF patients. METHODS: We immunohistochemically examined DC subsets and their distribution in 16 LN samples from 14 patients with MF (N1 LN, eight patients; N2, four; and N3, four), and we compared them with non-metastatic sentinel LNs from eight patients with melanoma. RESULTS: The number of S-100 protein+ DCs was markedly increased in the LNs from the MF patients and the major component was DC-LAMP+ mature DCs in the outer and paracortex areas, where DC-SIGN+ immature DCs were relatively decreased in proportion. In contrast, DC-SIGN+ cells were relatively increased in proportion compared to DC-LAMP+ cells in the medulla. Although no significant difference was observed in the proportions of CD1a+ or Langerin+ DCs among the N1, N2, and N3 nodes, CD163+ M2-type macrophages were increased in number in the N2 and N3 nodes. CONCLUSIONS: Our observations indicate that mature DCs accumulate in the outer and paracortex areas in dermatopathic lymphadenitis and M2-type macrophages might increase in number during disease progression.