Circulating miRNA panels for specific and early detection in bladder cancer.

Bladder cancer is the 9th leading cause of cancer death worldwide. The major problem in bladder cancer is primarily the high recurrence rate after drug treatment and resection. Although conventional screening methods, such as cystoscopy, urinary cytology and ultrasound sonography, have become widely used in clinical settings, the diagnostic performance of these modalities is unsatisfactory due to low accuracy or high invasiveness. Because circulating micro RNA (miRNA) profiles have recently been reported as an attractive tool for liquid biopsy in cancer screening, here, we performed global miRNA profiling of 392 serum samples of bladder cancer patients with 100 non-cancer samples and 480 samples of other types of cancer as controls. We randomly classified the bladder cancer and control samples into 2 cohorts, a training set (N = 486) and a validation set (N = 486). By comparing both controls, we identified specific miRNA, such as miR-6087, for diagnosing bladder cancer in the training and validation sets. Furthermore, we found that a combination of 7 miRNA (7-miRNA panel: miR-6087, miR-6724-5p, miR-3960, miR-1343-5p, miR-1185-1-3p, miR-6831-5p and miR-4695-5p) could discriminate bladder cancer from non-cancer and other types of tumors with the highest accuracy (AUC: .97; sensitivity: 95%; specificity: 87%). The diagnostic accuracy was high, regardless of the stage and grade of bladder cancer. Our data demonstrated that the 7-miRNA panel could be a biomarker for the specific and early detection of bladder cancer.

Identification of circulating microRNAs as potential biomarkers for hepatic necroinflammation in patients with autoimmune hepatitis.

OBJECTIVE: MicroRNAs (miRNAs) are implicated in the pathogenesis of autoimmune diseases and could be biomarkers of disease activity. This study aimed to identify highly expressed circulating miRNAs in patients with autoimmune hepatitis (AIH) and to evaluate their association with clinical characteristics. METHODS: Microarray analyses were performed, and miRNA expression profiling for AIH, primary biliary cholangitis (PBC) and overlap syndrome (OS) using the serum of patients and healthy individuals was done. Samples were divided into discovery and test sets to identify candidate miRNAs that could discriminate AIH from PBC; the former included 21 AIH and 23 PBC samples, while the latter included five AIH and eight PBC samples. RESULTS: Among 11 candidate miRNAs extracted in the discovery set, 4 (miR-3196, miR-6125, miR-4725-3 p and miR-4634) were specifically and highly expressed in patients with AIH in the test set. These four miRNAs discriminated AIH from PBC with high sensitivity (0.80-1.00) and specificity (0.88-1.00). In situ hybridisation analysis revealed that these miRNAs were expressed in the cytoplasm of hepatocytes in patients with AIH. Their expression levels were highest in untreated patients with AIH, followed by those in untreated patients with OS. They drastically or moderately decreased after prednisolone treatment. Histological analysis demonstrated that the expression levels of miR-3196, miR-6125 and miR-4634 in patients with AIH and OS were correlated with severe hepatic necroinflammatory activity. CONCLUSION: These circulating miRNAs are suggested to reflect hepatic necroinflammatory activity and serve as AIH-related and treatment-responsive biomarkers. These miRNAs could be beneficial in developing new therapeutic strategies for AIH.

[Anesthetic management of a child with moyamoya disease combined with von Gierke's disease].

We report on a child with moyamoya disease combined with von Gierke's disease. A 7-year-old girl with von Gierke's disease had a stroke associated with moyamoya disease. She had had many episodes of hypoglycemia and severe metabolic acidosis before surgery. General anesthesia was induced with midazolam 3 mg and fentanyl 100 microg followed by rocuronium 12 mg. After tracheal intubation, anesthesia was maintained with sevoflurane 2.5% in 33% oxygen and 66% nitrous oxide. We used mainly mixture of saline and glucose as intraoperative fluid instead of acetated Ringer solution, and controlled administration of glucose according to blood glucose levels. The patient's plasma lactate levels and base excess during operation showed changes compared with those before operation, because sodium bicarbonate was used during the surgery. The duration of anesthesia was 374 minutes. The patient woke up and spontaneous respiration returned, and the trachea was extubated in the operating room. We were able to manage this case safely without any complications.

Risk prediction models for dementia constructed by supervised principal component analysis using miRNA expression data.

Alzheimer's disease (AD) is the most common subtype of dementia, followed by Vascular Dementia (VaD), and Dementia with Lewy Bodies (DLB). Recently, microRNAs (miRNAs) have received a lot of attention as the novel biomarkers for dementia. Here, using serum miRNA expression of 1,601 Japanese individuals, we investigated potential miRNA biomarkers and constructed risk prediction models, based on a supervised principal component analysis (PCA) logistic regression method, according to the subtype of dementia. The final risk prediction model achieved a high accuracy of 0.873 on a validation cohort in AD, when using 78 miRNAs: Accuracy = 0.836 with 86 miRNAs in VaD; Accuracy = 0.825 with 110 miRNAs in DLB. To our knowledge, this is the first report applying miRNA-based risk prediction models to a dementia prospective cohort. Our study demonstrates our models to be effective in prospective disease risk prediction, and with further improvement may contribute to practical clinical use in dementia.

Large-scale Circulating microRNA Profiling for the Liquid Biopsy of Prostate Cancer.

PURPOSE: The high false-positive rate of prostate-specific antigen (PSA) may lead to unnecessary prostate biopsies. Therefore, the United States Preventive Services Task Force recommends that decisions regarding PSA-based screening of prostate cancer should be made with caution in men ages 55-69 years, and that men ≥70 years should not undergo PSA screening. Here, we investigated the potential of serum miRNAs as an accurate diagnostic method in patients with suspected prostate cancer. EXPERIMENTAL DESIGN: Serum samples of 809 patients with prostate cancer, 241 negative prostate biopsies, and 500 patients with other cancer types were obtained from the National Cancer Center, Japan. Forty-one healthy control samples were obtained from two other hospitals in Japan. Comprehensive microarray analysis was performed for all samples. Samples were divided into three sets. Candidate miRNAs for prostate cancer detection were identified in the discovery set (n = 123). A diagnostic model was constructed using combinations of candidate miRNAs in the training set (n = 484). The performance of the diagnostic model was evaluated in the validation set (n = 484). RESULTS: In the discovery set, 18 candidate miRNAs were identified. A robust diagnostic model was constructed using the combination of two miRNAs (miR-17-3p and miR-1185-2-3p) in the training set. High diagnostic performance with a sensitivity of 90% and a specificity of 90% was achieved in the validation set regardless of the Gleason score and clinical tumor-node-metastasis stage. CONCLUSIONS: The model developed in this study may help improve the diagnosis of prostate cancer and reduce the number of unnecessary prostate biopsies.

A serum microRNA classifier for the diagnosis of sarcomas of various histological subtypes.

Due to their rarity and diversity, sarcomas are difficult to diagnose. Consequently, there is an urgent demand for a novel diagnostic test for these cancers. In this study, we investigated serum miRNA profiles from 1002 patients with bone and soft tissue tumors representing more than 43 histological subtypes, including sarcomas, intermediate tumors, and benign tumors, to determine whether serum miRNA profiles could be used to specifically detect sarcomas. Circulating serum miRNA profiles in sarcoma patients were clearly distinct from those in patients with other types of tumors. Using the serum levels of seven miRNAs, we developed a molecular detector, Index VI, that could distinguish sarcoma patients from benign and healthy controls with remarkably high sensitivity (90%) and specificity (95%), regardless of histological subtype. Index VI provides an approach to the early and precise detection of sarcomas, potentially leading to curative treatment and longer survival.

Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification.

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.

[A case of heparin-induced thrombocytopenia associated with unexpected excessive argatroban anticoagulation after abdominal aortic aneurysm resection].

We experienced excessive anticoagulation induced by argatroban for the treatment of heparin-induced thrombocytopenia (HIT). A 74-year-old man was scheduled for elective abdominal aortic aneurysm resection. During the surgery, both femoral arteries were found non-pulsatile requiring thrombectomy. The next day, second laparotomy was needed because of superior mesenteric artery occlusion. After the surgery, acute renal failure and hypoxemia continued with progressive thrombocytopenia necessitating frequent administration of platelet concentrates. Considering possibility of HIT, we stopped heparin and began argatroban. Due to his mild liver dysfunction, we initiated argatroban at 0.5 microg x kg(-1) x min(-1) one-fourth of standard initial dose, according to its drug information approved by FDA. Although expected APTT level was from 50 to 60 sec, it increased immediately up to 93 sec. Excessive anticoagulation continued more than 24 hours after cessation of argatroban and bleeding occurred from the tracheostomy site. When APTT decreased to the target range, we restarted argatroban and found the adequate dosage at 0.08 microg x kg(-1) x min(-1). After argatroban treatment, platelet count recovered immediately and no thromboembolism was observed. We recommend that argatroban should be initiated at a lower dosage than the dose shown in its drug information for HIT patients after cardiovascular surgery with frequent monitoring of APTT.

[Successful management of a patient with rhabdomyolysis and marked elevation of serum creatine kinase level].

We experienced successful management of a patient with severe rhabdomyolysis by conservative treatment. A 41-year-old man developed Stanford-A-type acute aortic dissection and underwent an emergent replacement of the aortic root and arch. After the weaning from cardiopulmonary bypass, his left femoral artery was found non-pulsatile, probably due to extension of the aortic dissection, and femoro-femoral artery bypass surgery was added. Estimated ischemia time of the lower extremities was 7 hours. On admission to the intensive care unit (ICU), his left lower extremity showed signs of reperfusion injury accompanied with marked elevation of serum creatine kinase (12,397 IU x l(-1)) and myoglobin (19,980 ng x ml(-1)), and impaired oxygenation (a ratio of PaO2 to FIO2, 130 mmHg). We performed (1) moderately aggressive infusion treatment, (2) maintenance of hyperdynamic states using catecholamine, (3) diuresis therapy using atrial natriuretic peptide and furosemide, and (4) lung protective strategy. Although serum creatinine increased to 2.0 mg x dl(-1) on postoperative day (POD) 1, diuresis was maintained and the level of creatinine returned to normal on POD 6. He was extubated on POD 6 and discharged on POD 7. The early start of these combined therapies seems to have prevented acute renal failure without blood purification.

A combination of extraction reagent and DNA microarray that allows for the detection of global miRNA profiles from serum/plasma.

In recent years, miRNAs have been shown to exist stably in serum (plasma) and have drawn attention particularly as markers for diagnosis of diseases, evaluation of therapeutic effects, selection of treatment in clinical studies, and others.However, RNAs in serum (plasma) exist only in low amounts (0.1-1 ng/mL), and analysis with reproducibility is difficult. Therefore, we have developed a combination of an extraction reagent and a unique miRNA screening platform which allows for the rapid analysis and high-throughput detection of alterations in miRNA levels in serum/plasma samples. This offers the potential for the identification of novel biomarkers to specific diseases or conditions which may inform upon future diagnostic approaches. The features of this prescription include (1) an extraction method that can obtain high-purity RNA (high reproducibility and stability), (2) a straightforward, easy-to-use extraction procedure a simple method without complicated extraction operations, and (3) increased number of detected genes and data reproducibility using high sensitivity DNA chips.

Identification of AhR-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-Gene Human Immunity and Metabolic Syndrome 9k.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants with various toxic effects including immune suppression. However, the molecular mechanism of their toxicity has not been fully clarified. The purpose of this study was to identify novel aryl hydrocarbon receptor (AhR)-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-Gene(TM) Human Immunity & Metabolic Syndrome 9k. Leucine-rich repeat-containing protein 25, glucosaminyl (N-acetyl) transferase 3 (GCNT3), thyroxine-binding globulin, aldehyde dehydrogenase 8A1, diacylglycerol O-acyltransferase homolog 2 (DGAT2), haptoglobin, neuron navigator 2 isoform 1, hemopexin and bile acid receptor were found to be up- or down-regulated by PAHs via AhR. Among these genes, GCTN3 and DGAT2 were responsible for immune responses. Therefore, disruption of the expression of these genes via AhR may be one of the causes of the immunotoxicity of PAHs.

Time-saving multiplex detection of single nucleotide polymorphisms by ultrasensitive DNA microarray.

Rapid and multiplex detection system using an ultrasensitive DNA microarray was developed and utilized for the analysis of six pharmacokinetically relevant single nucleotide polymorphisms (SNPs) (MDR1-C1236T, MDR1-G2677TA, MDR1-C3435T, CYP3A5-A6986G, CYP2C19-G681A, CYP2C19-G636A) from blood samples derived from liver transplant patients. The SNP detection system is comprised of three processes: multiplex PCR, single base extension with fluorescently labelled di-deoxy-nucleotides and detection by DNA microarray. The entire workflow of this system completes within 5 h. The final genotype call was obtained statistically by Mahalanobis distance which was calculated from the bi-coloured fluorescent signals detected by the microarray. In order to detect the six SNPs, this system required only 50 copies of genomic DNA, and the obtained detection calls completely matched with the results by the sequencing-based genotyping method. With the high sensitivity and rapid processing, our SNP detection system utilizing ultrasensitive microarray is a promising device applicable for diagnostic utility.