Satoko Takizawa

MicroRNA profile predicts recurrence after resection in patients with hepatocellular carcinoma within the Milan Criteria.

Objective Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection. Methods We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out cross-validation method, and the time-averaged area under the ROC curve (ta-AUROC). Results The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P<0.0001). The best ta-AUROC using the whole dataset, T-miRs, N-miRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P<0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the ‘field effect’. Conclusion microRNA profiling can predict HCC recurrence in Milan criteria cases.

MicroRNA Markers for the Diagnosis of Pancreatic and Biliary-Tract Cancers

It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With “3D-Gene”, a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.

Novel combination of serum microRNA for detecting breast cancer in the early stage.

MicroRNA (miRNA), which are stably present in serum, have been reported to be potentially useful for detecting cancer. In the present study, we examined the expression profiles of serum miRNA in several large cohorts to identify novel miRNA that can be used to detect early stage breast cancer. We comprehensively evaluated the serum miRNA expression profiles using highly sensitive microarray analysis. A total of 1280 serum samples of breast cancer patients stored in the National Cancer Center Biobank were used. In addition, 2836 serum samples were obtained from non‐cancer controls, 451 from patients with other types of cancers, and 63 from patients with non‐breast benign diseases. The samples were divided into a training cohort including non‐cancer controls, other cancers and breast cancer, and a test cohort including non‐cancer controls and breast cancer. The training cohort was used to identify a combination of miRNA that could detect breast cancer, and the test cohort was used to validate that combination. miRNA expressions were compared between patients with breast cancer and non‐breast cancer, and a combination of five miRNA (miR‐1246, miR‐1307‐3p, miR‐4634, miR‐6861‐5p and miR‐6875‐5p) was found to be able to detect breast cancer. This combination had a sensitivity of 97.3%, specificity of 82.9% and accuracy of 89.7% for breast cancer in the test cohort. In addition, this combination could detect early stage breast cancer (sensitivity of 98.0% for Tis).

Identification of AhR-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-GeneTM 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.

Use of Non-Amplified RNA Samples for Microarray Analysis of Gene Expression

Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also technical disadvantages such as amplification bias and the long, laborious protocol. Using RNA derived from human brain, breast and colon, we demonstrate that a non-amplification method, which was previously shown to be inferior, could be transformed to a highly quantitative method with a dynamic range of five orders of magnitude. Furthermore, the correlation coefficient calculated by comparing microarray assays using non-amplified samples with qRT-PCR assays was approximately 0.9, a value much higher than when samples were prepared using amplification methods. Our results were also compared with data from various microarray platforms studied in the MicroArray Quality Control (MAQC) project. In combination with micro-columnar 3D-Gene™ microarray, this non-amplification method is applicable to a variety of genetic analyses, including biomarker screening and diagnostic tests for cancer.

A possible mechanism for hepatotoxicity induced by BIRB‐796, an orally active p38 mitogen‐activated protein kinase inhibitor

BIRB‐796, a selective inhibitor of p38 mitogen‐activated protein kinase, has entered clinical trials for the treatment of autoimmune diseases. Levels of alanine transaminase, a biomarker of hepatic toxicity in clinical pathology, were found to be increased in Crohns disease patients treated with BIRB‐796. The purpose of the present study was to clarify the molecular mechanism(s) of this hepatotoxicity. A toxicogenomic analysis using a highly sensitive DNA chip, 3D‐Gene™ Mouse Oligo chip 24k, indicated that BIRB‐796 treatment activated the nuclear factor (erythroid‐derived 2)‐like 2 signaling pathway, which plays a key role in the response to oxidative stress. A reactive intermediate of BIRB‐796 was detected by the glutathione‐trapping method using mouse and human liver microsomes. The production of this reactive metabolite in the liver may be one of the causes of BIRB‐796s hepatotoxicity. Copyright

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.

Integrated extracellular microRNA profiling for ovarian cancer screening

A major obstacle to improving prognoses in ovarian cancer is the lack of effective screening methods for early detection. Circulating microRNAs (miRNAs) have been recognized as promising biomarkers that could lead to clinical applications. Here, to develop an optimal detection method, we use microarrays to obtain comprehensive miRNA profiles from 4046 serum samples, including 428 patients with ovarian tumors. A diagnostic model based on expression levels of ten miRNAs is constructed in the discovery set. Validation in an independent cohort reveals that the model is very accurate (sensitivity, 0.99; specificity, 1.00), and the diagnostic accuracy is maintained even in early-stage ovarian cancers. Furthermore, we construct two additional models, each using 9–10 serum miRNAs, aimed at discriminating ovarian cancers from the other types of solid tumors or benign ovarian tumors. Our findings provide robust evidence that the serum miRNA profile represents a promising diagnostic biomarker for ovarian cancer.Screening methods for early detection of ovarian cancer is technically difficult. Here, the authors investigated circulating microRNA in human blood serum and developed a model using 10 microRNAs to discern between ovarian cancer and being ovarian tumors, solid tumors, and non-cancer patients.

Abstract P4-07-04: Detecting early breast cancer by the combination of five serum microRNAs and its possibility of prediction of pathological complete response in neoadjuvant chemotherapy

[Background] It is recently reported that microRNAs (miRNAs) are stably present in serum and potentially useful in the diagnosis and evaluation of treatment of cancer. [Materials and Methods] Serum samples of breast cancer before treatment (n=1280) between 2008 and 2014 were obtained from National Cancer Center Hospital and controls (n=3348) were obtained from collaborative institutes. Additionally, the serum sample of patients who received neoadjuvant chemotherapy (NAC) and surgery between last chemotherapy administration and surgery were collected. A comprehensive quantitative expression analysis of miRNA was performed using the by DNA chip “3D-Gene® (Toray Industries Inc.)” Clinicopathological data was retrieved from medical records. Pathological complete response (pCR) was defined as the absence of residual invasive and in situ cancer of the resected breast specimen and all sampled regional lymph nodes. [Results] Serum samples before treatment of breast cancer patients (n=74), non-cancer controls and patients with other cancers were used (n=2007) in a training set. The rest except for samples after NAC were used in a test set. The formula with the combination of five miRNAs (miR-1246, miR-1307-3p, miR-4634, miR-6861-5p, and miR-6875-5p) was found to be able to detect breast cancer (BCmiR set). BCmiR set had a sensitivity of 97.3%, specificity of 82.9% and accuracy of 89.7% in the test cohort. BCmiR set could detect breast cancer in the non-invasive stage (sensitivity of 98.0% for Tis). In the breast cancer patients, 91 patients received NAC and surgery. Median age of NAC patients was 49 years (range 28-77). Forty-two patients were hormone receptor-positive (HR+) and HER2-negative (HER2-), 24 were HR+ and HER2-positive (HER2+), 11 were hormone receptor-negative (HR-) and HER2+ and 14 were HR- and HER2-. pCR was observed in 19 (20.9%) of NAC patients. pCR in each subtypes were 3 (7.7%) in HR+ and HER2-, 6 (33.3%) in HR+ and HER2+, 4 (57.1%) in HR- and HER2+ and 3 (27.3%) in HR- and HER2-. Serum after NAC were obtained from 19 pCR patients and 71 non-pCR patients. When we applied BCmiR set to the serum samples after NAC, the average diagnostic index (cut-off value=0) in pCR patients was significantly lower than that in non-pCR patients (pCR, -0.30±0.84; non-pCR, 0.31±1.15; p=0.03). In fact, 57.9% of pCR patients were classified into non-breast cancer. However, 40.8% of non-pCR patients were misclassified into non-breast cancer. [Conclusion] The combination of five miRNAs (BCmiR set) measured from serum can be used to detect breast cancer. BCmiR set has potential to predict pCR in patients who received NAC. The further analysis to predict pCR is underway and further results will be presented in the symposium. Citation Format: Shimomura A, Shiino S, Kawauchi J, Takizawa S, Sakamoto H, Shimizu C, Takeshita F, Niida S, Kinoshita T, Tamura K, Ochiya T. Detecting early breast cancer by the combination of five serum microRNAs and its possibility of prediction of pathological complete response in neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-04.

Abstract 5678: Novel serum microRNAs that enable liquid biopsy for colorectal cancer: validation study of large cohort

Introduction: Recent studies have reported that serum microRNAs (miRNAs) are potentially useful biomarkers for cancer. However, the detection system using serum miRNAs is not established in colorectal cancer (CRC). The aims of this study are 1) to identify specific serum miRNAs and establish the discriminant model for CRC detection and 2) to validate the miRNAs and the discriminant model using a large cohort. Methods: First, we identified serum miRNAs related with the presence of CRC and constructed the discriminant model for CRC detection using the publicly-released serum miRNA database including 50 patients with CRC and 90 healthy individuals (GSE59856 and GSE73002). Comparing serum miRNA levels between patients with CRC and healthy individuals, we picked up miRNAs (p 2). Using these miRNAs, Fisher’s linear discriminant analysis was performed and the diagnostic models were constructed using less than 7 miRNAs. Subsequently, 1117 patients of National Cancer Center Hospital with CRC were enrolled as a validation cohort. Patients with the following criteria were excluded; (i) patients simultaneously or previously diagnosed as the other cancers, (ii) patients who were treated for CRC previously and (iii) patients with familial adenomatous polyposis or Lynch syndrome. Control blood samples were obtained from patients without any history of cancer who were admitted or referred to National Center for Geriatrics and Gerontology or Yokohama Minoru Clinic between 2010 and 2015. A total of 1013 CRC patients and 4384 non-cancerous patients were analyzed. Total RNA was extracted from 300 micro L of serum and comprehensive miRNA expression analysis was performed using a 3D-Gene microarray. ROC analysis was performed to evaluate the previous discriminant models. The sensitivity in each pathological stage and location of CRC was also investigated.This research is partially supported by the “Development of Diagnostic Technology for Detection of miRNA in Body Fluids” grant from the Japan Agency for Medical Research and Development (AMED). Results:First we picked up 30 miRNAs for CRC diagnosis. Fisher’s analysis revealed 107611 candidates of numerical formulas, that showed > 80% of sensitivity and specificity. We could narrow these candidates to 43 formulas with validation analysis. By ROC analysis, the AUC, sensitivity and specificity of the discriminant formula with best performance was 0.904, 79.6% and 86.5%, respectively. The sensitivities of each pathological stage were as follows; pStage 0: 79.0%, pStage I: 90.7%, pStage II: 85.1%, pStage III: 73.7%, pStage IV: 62.2%. The sensitivity of CRC in the right side of the colon was 79.4%, whereas that in the left side was 79.7%. Conclusions:We identified novel serum miRNAs for CRC detection. Our discriminant using these miRNAs can diagnose CRC including early stage. Furthermore, the sensitivity was high irrespective of the tumor location. Citation Format: Hiroyuki Takamaru, Yutaka Saito, Taku Sakamoto, Seiichiro Abe, Masayoshi Yamada, Takeshi Nakajima, Kazuki Sudo, Ken Kato, Junpei Kawauchi, Satoko Takizawa, Hiromi Sakamoto, Motohiro Kojima, Atsushi Ochiai, Shumpei Niida, Hideshi Ishii, Tomoko Takamaru, Juntaro Matsuzaki, Takahisa Matsuda, Takahiro Ochiya. Novel serum microRNAs that enable liquid biopsy for colorectal cancer: validation study of large cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5678. doi:10.1158/1538-7445.AM2017-5678