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.

Use of finer needles for venipuncture increases in vitro haemolysis despite reducing persistent pain and nerve injury: A retrospective study.

BACKGROUND: Although venipuncture is minimally invasive, and is the most frequently performed medical procedure, it carries the small risk of causing persistent pain, including nerve damage. Recently, our hospital stopped using 22-gauge needles for venipuncture in outpatients and switched to using only 23- and 25-gauge needles. We investigated the impact of using only the finer needles on the incidence of persistent or neuropathic pain and the prevalence of haemolysis, as well as the impact of haemolysis associated with the needle change on other laboratory data. METHODS: We retrospectively collected and analysed data on venipuncture-associated pain complaints made during the 1-year period prior and 1-year period after the change in needles, as well as the frequency of haemolysis before and after the change. We also focused on 90 cases that showed significant haemolysis after the needle change and compared the serum aspartate aminotransferase, lactate dehydrogenase, and potassium levels before and after the needle change. RESULTS: The incidence of persistent pain was significantly reduced from 1 in 10,825 venipunctures before the change to 1 in 29,747 venipunctures after the change. Notably, no patients experienced neuropathic pain after the change. However, the prevalence of haemolysis was significantly increased. Additionally, the serum aspartate aminotransferase, lactate dehydrogenase, and potassium levels were significantly elevated in the cases that showed moderate to gross haemolysis after the needle change. CONCLUSION: Using finer needles involves both advantages and disadvantages, and careful consideration is needed to determine which type of needle is in the best interests of the patient.

Sex differences in the evaluation of proteinuria using the urine dipstick test.

Background: The urine protein dipstick test is widely used, but false-positive and false-negative results may occur. This study aimed to compare the urine protein dipstick test with a urine protein quantification method. Methods: The data were extracted using the Abbott Diagnostic Support System, which analyzes the inspection results using multiple parameters. This study included 41,058 specimens tested using the urine dipstick test and protein creatinine ratio from patients aged ≥18 years. The proteinuria creatinine ratio was classified according to the Kidney Disease Outcomes Quality Initiative guidelines. Results: Urine protein on the dipstick test was negative in 15,548 samples (37.9%), trace in 6,422 samples (15.6%), and ≥1+ in 19,088 samples (46.5%). Among the trace proteinuria samples, A1 (<0.15 g/gCr), A2 (0.15-0.49 g/gCr), and A3 (≥0.5 g/gCr) category proteinuria accounted for 31.2, 44.8, and 24.0% of samples, respectively. All trace proteinuria specimens with a specific gravity of <1.010 were classified as A2 and A3 category proteinuria. In the trace proteinuria cases, women had a lower specific gravity and a higher percentage of A2 or A3 category proteinuria than men. The sensitivity in the "dipstick proteinuria" ≥ trace" group was higher than that in the "dipstick proteinuria ≥ 1+" group within the lower specific gravity group. The sensitivity in the "dipstick proteinuria ≥ 1+" group was higher for men than for women, and the sensitivity in the "dipstick proteinuria ≥ trace" group was higher than that in the "dipstick proteinuria ≥ 1+" group for women. Conclusion: Pathological proteinuria assessment requires caution; this study suggests that evaluating the specific gravity of urine specimens with trace proteinuria is essential. Particularly for women, the sensitivity of the urine dipstick test is low, and caution is needed even with trace specimens.

Evaluation of a novel sample preparation method for identifying Aspergillus fumigatus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: Combining Yatalase and silica beads treatment.

Aspergillus spp. belong to filamentous fungi and sometimes cause invasive aspergillosis which has high mortality. Filamentous fungi are generally identified morphologically. However, morphologic identification is time consuming and requires advanced skills. It is difficult to train technicians and ensure a high level of quality. Therefore, an identification technique that is both accurate and relatively easy to learn is needed. In the present study, we focused on the effects of Yatalase and silica beads, which enable the efficient extraction of proteins via cell wall disruption of Aspergillus spp., and aimed to establish a novel sample preparation method using Yatalase and silica beads to enhance the efficiency of Aspergillus spp. identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sample preparation method using the combination of Yatalase and silica beads showed higher accuracy for the identification of Aspergillus spp. compared with Yatalase or silica beads alone. The Yatalase/silica beads method also resulted in significantly higher identification scores compared with the conventional method for the identification of Aspergillus fumigatus (n = 33). These findings indicate that our novel Yatalase/silica beads method provides more reliable identification of A. fumigatus than does the conventional method.

Pharmacokinetic/pharmacodynamic analysis of vancomycin in patients with Enterococcus faecium bacteraemia: a retrospective cohort study.

OBJECTIVES: The trough concentration of vancomycin and the area under the concentration-time curve (AUC)/minimum inhibitory concentration (MIC) ratio are crucial in determining vancomycin efficacy against methicillin-resistant Staphylococcus aureus. However, the use of similar pharmacokinetic principles in determining antibiotic efficacy against other gram-positive cocci is lacking. We performed a pharmacokinetic/pharmacodynamic analysis (association of target trough concentration values and AUC/MIC with therapeutic outcome) of vancomycin in patients with Enterococcus faecium bacteraemia. METHODS: Between January 2014 and December 2021 we performed a retrospective cohort study of patients with E. faecium bacteraemia treated with vancomycin. Patients who received renal replacement therapy or had chronic kidney disease were excluded. Clinical failure, the primary outcome, was defined as a composite of 30-day all-cause mortality, vancomycin-susceptible infection requiring change of treatment, and/or recurrence. AUC24 was estimated using a Bayesian estimation approach based on an individual vancomycin trough concentration. The MIC for vancomycin was determined using a standardised agar dilution method. Additionally, classification was used to identify the vancomycin AUC24/MIC ratio associated with clinical failure. RESULTS: Of the 151 patients identified, 69 were enrolled. All MICs of vancomycin for E. faecium were ≤1.0 µg/mL. The AUC24 and AUC24/MIC ratio were not significantly different between the clinical failure group and the clinical success group (432±123 µg/mL/hour vs 488±92 µg/mL/hour; p=0.075). However, 7 of 12 patients (58.3%) in the clinical failure group and 49 of 57 patients (86.0%) in the clinical success group had a vancomycin AUC24/MIC ratio ≥389 (p=0.041). No significant association between trough concentration or AUC24 ≥600 µg/mL×hour and acute kidney injury was observed (p=0.365 and p=0.487, respectively). CONCLUSION: The AUC24/MIC ratio is associated with the clinical outcome of vancomycin administration in E. faecium bacteraemia. In Japan, where vancomycin-resistant enterococcal infection is rare, empirical therapy with a target AUC24 ≥389 should be recommended.

Elucidation of the Mechanism and Significance of the Erythrocyte Sedimentation Rate from Clinical Laboratory Data.

The erythrocyte sedimentation rate (ESR) is a widely used marker of inflammation, but the detailed mechanisms underlying the ESR remain unclear. We retrospectively collected laboratory data from our hospital's laboratory information system, and performed multiple linear regression analysis and correlation analysis to determine relationships between the ESR and other laboratory test parameters. The alpha-2, beta-2, and gamma fractions from serum protein electrophoresis, serum immunoglobulin (Ig) G, IgA, IgM, and complement C3 levels, plasma fibrinogen levels, and platelet count showed positive effects on the ESR; however, the serum albumin level showed negative effects. Since erythrocytes are negatively charged, an increase in positively charged proteins and a decrease in negatively charged albumin were suggested to increase the ESR. Notably, C-reactive protein (CRP) showed the third-strongest correlation with the ESR despite having no significant effect on the ESR. We also reviewed cases with discordant ESR and CRP levels to compare the disease profiles of high ESR/low CRP patients and low ESR/high CRP patients. The patients with high ESR/low CRP had a completely different disease profile from those with low ESR/high CRP. Since the ESR and CRP have different roles, they should be used as markers in a context-dependent manner.

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.

Effect of humidity during sample preparation on bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10-20%, 30-40%, and 50-60%). As a result, higher identification scores were obtained at 30-40% humidity than at 10-20% or 50-60% humidity. At 30-40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30-40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.

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.

Current Status of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in Clinical Diagnostic Microbiology.

Mass spectrometry (MS), a core technology for proteomics and metabolomics, is currently being developed for clinical applications. The identification of microorganisms in clinical samples using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a representative MS-based proteomics application that is relevant to daily clinical practice. This technology has the advantages of convenience, speed, and accuracy when compared with conventional biochemical methods. MALDI-TOF MS can shorten the time used for microbial identification by about 1 day in routine workflows. Sample preparation from microbial colonies has been improved, increasing the accuracy and speed of identification. MALDI-TOF MS is also used for testing blood, cerebrospinal fluid, and urine, because it can directly identify the microorganisms in these liquid samples without prior culture or subculture. Thus, MALDI-TOF MS has the potential to improve patient prognosis and decrease the length of hospitalization and is therefore currently considered an essential tool in clinical microbiology. Furthermore, MALDI-TOF MS is currently being combined with other technologies, such as flow cytometry, to expand the scope of clinical applications.

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.

Identification of a Novel ACVRL1 Gene Mutation (c.100T>A, p.Cys34Ser) in a Japanese Patient with Possible Hereditary Hemorrhagic Telangiectasia (Osler-Weber-Rendu Disease).

Hereditary hemorrhagic telangiectasia (HHT; also known as Osler-Weber-Rendu disease) is an autosomal dominant genetic disorder that causes frequent epistaxis, mucocutaneous telangiectasia, and visceral arteriovenous malformations. Four genes (ENG, ACVRL1, SMAD4, and GDF2) have been identified as pathogenic in HHT. We describe the case of a 50-year-old Japanese man highly suspected of having HHT due to recurrent epistaxis, mucocutaneous telangiectasia, and a family history. Genomic analysis revealed a novel missense mutation of c.100T>A, p.Cys34Ser in the patient's ACVRL1 gene. We used 6 freeware programs to perform an in silico analysis of this mutation. The results demonstrated the mutation's high pathogenicity.

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.