Potential utility of pretreatment serum miRNAs for optimal treatment selection in advanced high-grade serous ovarian cancer.

OBJECTIVE: The primary treatment of patients with advanced ovarian cancer is selected from whether primary debulking surgery or neoadjuvant chemotherapy. We investigated whether pretreatment serum microRNA profiles are useful for selecting patients with advanced high-grade serous ovarian cancer who obtain better outcomes from undergoing primary debulking surgery or neoadjuvant chemotherapy. METHODS: Consecutive patients with clinical stage IIIB-IVB and serum microRNA data were selected. Patients who underwent primary debulking surgery or neoadjuvant chemotherapy were subjected to 1:1 propensity score matching before comparing their progression-free survival using Cox modelling. Progression-free probabilities for the selected microRNA profiles were calculated, and the estimated progression-free survival with the recommended primary treatment was determined and compared with the actual progression-free survival of the patients. RESULTS: Of the 108 patients with stage IIIB-IVB disease, the data of 24 who underwent primary debulking surgery or neoadjuvant chemotherapy were compared. Eleven and three microRNAs were independent predictors of progression-free survival in patients who underwent primary debulking surgery and neoadjuvant chemotherapy, respectively. Two microRNAs correlated significantly with complete resection of the tumours in primary debulking surgery. No differences were found between the actual and estimated progression-free survival in the primary debulking surgery and neoadjuvant chemotherapy groups (P > 0.05). The recommended and actual primary treatments were identical in 27 (56.3%) of the 48 patients. The median improved survival times between recommended and actual treatment were 11.7 and 32.6 months for patients with actual primary debulking surgery and neoadjuvant chemotherapy, respectively. CONCLUSIONS: Pretreatment microRNA profiles could be used to select subgroups of patients who benefited more from primary debulking surgery or neoadjuvant chemotherapy and might contribute to selecting the optimal primary treatment modality in advanced high-grade serous ovarian cancer patients.

Assessment of the Diagnostic Utility of Serum MicroRNA Classification in Patients With Diffuse Glioma.

Importance: A blood-based screening tool for detecting diffuse glioma is necessary to improve clinical outcomes. Objectives: To establish models using serum microRNAs to distinguish patients with diffuse glioma from control individuals without cancer (the Glioma Index) and to differentiate glioblastoma (GBM), primary central nervous system lymphoma (PCNSL), and metastatic brain tumors (the 3-Tumor Index). Design, Setting, and Participants: This retrospective, case-control diagnostic study included 157 patients with diffuse glioma and 109 patients with central nervous system (CNS) diseases other than diffuse glioma diagnosed from August 1, 2008, through May 1, 2016, and 314 sex- and age-matched controls without cancer. Samples of patients with diffuse glioma and controls were randomly divided into training and validation set 1, and those of patients with CNS diseases other than diffuse glioma were allocated to an exploratory set. Samples of patients with GBM, PCNSL, and metastatic brain tumors were randomly divided into training and validation set 2. Data were analyzed from April 1, 2018, to March 31, 2019. Main Outcomes and Measures: The expression of 2565 microRNAs was assessed, and the diagnostic performance was evaluated by calculating the area under the receiver operating characteristics curve (AUC), sensitivity, specificity, and accuracy. Results: A total of 580 patients were included in the analysis (309 [53.3%] male; median age, 57 years [range, 10-87 years]). In training set 1, 100 patients with diffuse glioma (median age, 56 years [range, 14-87 years]; 55 male [55.0%]) were compared with 200 control patients (median age, 56 years [range, 14-87 years]; 105 male [52.5%]), and the Glioma Index was constructed using 3 microRNAs (miR-4763-3p, miR-1915-3p, and miR-3679-5p). In validation set 1, the AUC was 0.99 (95% CI, 0.99-1.00); sensitivity, 0.95 (95% CI, 0.89-1.00); and specificity, 0.97 (95% CI, 0.93-1.00). The Glioma Index classified 39 of 42 PCNSL samples (92.9%) and 25 of 28 metastatic brain tumor samples (89.3%) as positive and 2 of 2 spinal tumors (100%) as negative in the exploratory set. In training set 2, 68 patients with GBM, 34 with PCNSL, and 23 with metastatic brain tumor were compared, and the 3-Tumor Index was constructed using 48 microRNAs. The 3-Tumor Index had an accuracy of 0.80, positively detecting 16 of 17 GBM samples (94.1%), 4 of 5 metastatic brain tumor samples (80.0%), and 4 of 8 PCNSL samples (50.0%) in validation set 2. Conclusions and Relevance: This study appears to have identified promising serum microRNA combinations for detecting diffuse glioma and for assessing histologic features of brain tumors.

Brain metastasis-related microRNAs in patients with advanced breast cancer.

Brain metastasis is a major distant metastasis occurring in patients with advanced breast cancer, and is associated with poor prognosis. MicroRNAs (miRNAs) have a strong influence on various oncological functions and have been reported as potential biomarkers for detecting distant metastasis. Specific biomarkers and unique miRNAs for brain metastasis have yet to be reported. The aim of this study was to identify novel miRNAs in serum, to assist in the diagnosis of brain metastasis in patients with advanced breast cancer. We retrospectively analyzed the medical records of patients with breast cancer and collected clinical data. In addition, we evaluated serum miRNA profiles in patients with breast cancer, with and without brain metastasis, using high-sensitivity microarrays. All patients underwent computed tomography or magnetic resonance imaging brain imaging tests. A total of 51 serum samples from patients with breast cancer and brain metastasis, stored in the National Cancer Center Biobank, were used, and 28 serum samples were obtained from controls without brain metastasis. Two miRNAs, miR-4428 and miR-4480, could significantly distinguish patients with brain metastasis, with area under the receiver operating characteristic curve (AUC) values of 0.779 and 0.781, respectively, while a combination of miR-4428 and progesterone receptor had an AUC value of 0.884. No significant correlations were identified between the expression levels of these two miRNAs in serum and clinical data. We conclude that serum miR-4428 and miR-4480 may be useful as biomarkers for predicting brain metastasis in patients with breast cancer.

Serum microRNA-based prediction of responsiveness to eribulin in metastatic breast cancer.

The identification of biomarkers for predicting the responsiveness to eribulin in patients with metastatic breast cancer pretreated with an anthracycline and a taxane remains an unmet need. Here, we established a serum microRNA (miRNA)-based prediction model for the emergence of new distant metastases after eribulin treatment. Serum samples were collected from metastatic breast cancer patients prior to eribulin treatment and comprehensively evaluated by miRNA microarray. The prediction model for estimating eribulin efficacy was established using the logistic LASSO regression model. Serum samples were collected from 147 patients, of which 52 developed at least one new distant metastasis after eribulin monotherapy and 95 did not develop new distant metastases. A combination of eight serum miRNAs (miR-4483, miR-8089, miR-4755-3p, miR-296-3p, miR-575, miR-4710, miR-5698 and miR-3160-5p) predicted the appearance of new distant metastases with an area under the curve of 0.79, sensitivity of 0.69 and specificity of 0.82. The serum levels of miR-8089 and miR-5698 were significantly associated with overall survival after the initiation of eribulin treatment. The present study provides evidence that serum miRNA profiling may serve as a biomarker for the responsiveness to eribulin and for predicting the development of new distant metastases in metastatic breast cancer.

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.

Serum miRNA-based Prediction of Axillary Lymph Node Metastasis in Breast Cancer.

PURPOSE: Sentinel lymph node biopsy (SLNB) is the gold-standard procedure for evaluating axillary lymph node (ALN) status in patients with breast cancer. However, the morbidity of SLNB is not negligible, and the procedure is invasive for patients without ALN metastasis. Here, we developed a diagnostic model for evaluating ALN status using a combination of serum miRNAs and clinicopathologic factors as a novel less-invasive biomarker.Experimental Design: Preoperative serum samples were collected from patients who underwent surgery for primary breast cancer or breast benign diseases between 2008 and 2014. A total of 958 serum samples (921 cases of primary breast cancer, including 630 cases in the no ALN metastasis group and 291 cases in the ALN metastasis group, and 37 patients with benign breast diseases) were analyzed by miRNA microarray. Samples were randomly divided into training and test sets. Logistic LASSO regression analysis was used to construct diagnostic models in the training set, which were validated in the test set. RESULTS: An optimal diagnostic model was identified using a combination of two miRNAs (miR-629-3p and miR-4710) and three clinicopathologic factors (T stage, lymphovascular invasion, and ultrasound findings), which showed a sensitivity of 0.88 (0.84-0.92), a specificity of 0.69 (0.61-0.76), an accuracy of 0.818, and an area under the receiver operating characteristic curve of 0.86 in the test set. CONCLUSIONS: Serum miRNA profiles may be useful for the diagnosis of ALN metastasis before surgery in a less-invasive manner than SLNB.

MicroRNA-8073: Tumor suppressor and potential therapeutic treatment.

The comprehensive screening of intracellular and extracellular microRNAs was performed to identify novel tumor suppressors. We found that miR-8073 was present in exosome and predominantly exported from colorectal cancer cells. Treatment with a synthetic miR-8073 mimic resulted in a dramatic decrease in the proliferation of various types of cancer cells, which was not observed in similarly treated normal cells. As little is known about the biological functions of miR-8073, its target mRNAs were analyzed by both mRNA expression and in silico sequence analyses, leading to five probable target candidates (FOXM1, MBD3, CCND1, KLK10, and CASP2) that enhance survival during the regulation of the cell cycle, cell proliferation, and apoptosis. We experimentally confirmed that miR-8073 binds the 3'-UTR of each of these mRNA target candidates and that the introduction of a synthetic miR-8073 mimic into cancer cells reduced levels of protein expression. Finally, the antiproliferative effects of miR-8073 were validated in vivo: the subcutaneous injection of a synthetic miR-8073 mimic suppressed colorectal tumor volume to 43% in tumor-bearing xenografted mice. These results suggest that because miR-8073 binds, and thus reduces the levels of, these oncogenic targets, cancer cells must actively downregulate miR-8073 as a survival mechanism. The introduction of miR-8073 into tumors could thus inhibit tumor growth, indicating its great potential for cancer therapeutics.

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.

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).

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.

Gene expression analysis using a highly sensitive DNA microarray for colorectal cancer screening.

BACKGROUND/AIM: Half of all patients with small, right-sided, non-metastatic colorectal cancer (CRC) have negative results for the fecal occult blood test (FOBT). In the present study, the usefulness of CRC screening with a highly sensitive DNA microarray was evaluated in comparison with that by FOBT using fecal samples. MATERIALS AND METHODS: A total of 53 patients with CRC and 61 healthy controls were divided into "training" and "validation sets". For the gene profiling, total RNA extracted from 0.5 g of feces was hybridized to a highly sensitive DNA chip. RESULTS: The expressions of 43 genes were significantly higher in the patients with CRC than in healthy controls (p<0.05). In the training set, the sensitivity and specificity of the DNA chip assay using six genes were 85.4% and 85.2%, respectively. On the other hand, in the validation set, the sensitivity and specificity of the DNA chip assay were 85.2% and 85.7%, respectively. The sensitivities of the DNA chip assay were higher than those of FOBT in cases of the small, right-sided, early-CRC, tumor invading up to the muscularis propria (i.e. surface tumor) subgroups. In particular, the sensitivities of the DNA chip assay in the surface tumor and early-CRC subgroups were significantly higher than those of FOBT (p=0.023 and 0.019, respectively.). CONCLUSION: Gene profiling assay using a highly sensitive DNA chip was more effective than FOBT at detecting patients with small, right-sided, surface tumor, and early-stage CRC.

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.

The Dugesia ryukyuensis database as a molecular resource for studying switching of the reproductive system.

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value<1E -20) with sequences deposited in NCBI. Highly redundant sequences encoded granulin and actin, which are expressed in the whole body, and other redundant sequences encoded a Vasa-like protein, which is known to be a component of germ-line cells and is expressed in the ovary, and Y-protein, which is expressed in the testis. The sexualized planarian expressed sequence tag database (http://planaria.bio.keio.ac.jp/planaria/) is an open-access, online resource providing access to sequence, classification, clustering, and annotation data. This database should constitute a powerful tool for analyzing sexualization in planarians.