Circulating Exosomal microRNAs as Biomarkers of Colon Cancer

Purpose

Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined.

Experimental Design

Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients.

Results

The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis.

Conclusion

Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.     

Circulating microRNAs as Biomarkers for Detection of Autologous Blood Transfusion

MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs measured in blood plasma have emerged as specific and sensitive markers of physiological processes and disease. In this study, we investigated whether circulating miRNAs can serve as biomarkers for the detection of autologous blood transfusion, a major doping technique that is still undetectable. Plasma miRNA levels were analyzed using high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after autologous blood transfusion (blood bag storage time 42 days) in 10 healthy subjects and 10 controls without transfusion. Other serum markers of erythropoiesis were determined in the same samples. Our results revealed a distinct change in the pattern of circulating miRNAs. Ten miRNAs were upregulated in transfusion samples compared with control samples. Among these, miR-30b, miR-30c, and miR-26b increased significantly and showed a 3.9-, 4.0-, and 3.0-fold change, respectively. The origin of these miRNAs was related to pulmonary and liver tissues. Erythropoietin (EPO) concentration decreased after blood reinfusion. A combination of miRNAs and EPO measurement in a mathematical model enhanced the efficiency of autologous transfusion detection through miRNA analysis. Therefore, our results lay the foundation for the development of miRNAs as novel blood-based biomarkers to detect autologous transfusion.     

Circulating Tumor DNA as Biomarkers for Cancer Detection

Detection of circulating tumor DNAs (ctDNAs) in cancer patients is an important component of cancer precision medicine ctDNAs. Compared to the traditional physical and biochemical methods, blood-based ctDNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ctDNA detection in various types of cancers has been attracting much attention, due to the great potential of ctDNA as blood-based biomarkers for early diagnosis and treatment of cancers. ctDNAs are detected and tracked primarily based on tumor-related genetic and epigenetic alterations. In this article, we reviewed the available studies on ctDNA detection and described the representative methods. We also discussed the current understanding of ctDNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ctDNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.     

Circulating MicroRNAs as Non-Invasive Biomarkers for Early Detection of Non-Small-Cell Lung Cancer

BackgroundDetection of lung cancer at an early stage by sensitive screening tests could be an important strategy to improving prognosis. Our objective was to identify a panel of circulating microRNAs in plasma that will contribute to early detection of lung cancer.ResultsWe identified a panel of 24 microRNAs with optimum classification performance. The combination of these 24 microRNAs alone could discriminate lung cancer cases from non-cancer controls with an AUC of 0.92 (95% CI: 0.87-0.95). This classification improved to an AUC of 0.94 (95% CI: 0.90-0.97) following addition of sex, age and smoking status to the model. Internal validation of the model suggests that the discriminatory power of the panel will be high when applied to independent samples with a corrected AUC of 0.78 for the 24-miRNA panel alone.ConclusionOur 24-microRNA predictor improves lung cancer prediction beyond that of known risk factors.     

Relationship between Circulating and Tissue microRNAs in a Murine Model of Breast Cancer

MiRNAs are key regulators of tumorigenesis that are aberrantly expressed in the circulation and tissue of patients with cancer. The aim of this study was to determine whether miRNA dysregulation in the circulation reflected similar changes in tumour tissue. Athymic nude mice (n = 20) received either a mammary fat pad (n = 8, MFP), or subcutaneous (n = 7, SC) injection of MDA-MB-231 cells. Controls received no tumour cells (n = 5). Tumour volume was monitored weekly and blood sampling performed at weeks 1, 3 and 6 following tumour induction (total n = 60). Animals were sacrificed at week 6 and tumour tissue (n = 15), lungs (n = 20) and enlarged lymph nodes (n = 3) harvested. MicroRNAs were extracted from all samples (n = 98) and relative expression quantified using RQ-PCR. MiR-221 expression was significantly increased in tumour compared to healthy tissue (p<0.001). MiR-10b expression was significantly higher in MFP compared to SC tumours (p<0.05), with the highest levels detected in diseased lymph nodes (p<0.05). MiR-10b was undetectable in the circulation, with no significant change in circulating miR-221 expression detected during disease progression. MiR-195 and miR-497 were significantly decreased in tumour tissue (p<0.05), and also in the circulation of animals 3 weeks following tumour induction (p<0.05). At both tissue and circulating level, a positive correlation was observed between miR-497 and miR-195 (r = 0.61, p<0.001; r = 0.41, p<0.01 respectively). This study highlights the distinct roles of miRNAs in circulation and tissue. It also implicates miRNAs in disease dissemination and progression, which may be important in systemic therapy and biomarker development.     

Salivary MicroRNAs as Promising Biomarkers for Detection of Esophageal Cancer

Background and Purpose

Tissue microRNAs (miRNAs) can detect cancers and predict prognosis. Several recent studies reported that tissue, plasma, and saliva miRNAs share similar expression profiles. In this study, we investigated the discriminatory power of salivary miRNAs (including whole saliva and saliva supernatant) for detection of esophageal cancer.

Materials and Methods

By Agilent microarray, six deregulated miRNAs from whole saliva samples from seven patients with esophageal cancer and three healthy controls were selected. The six selected miRNAs were subjected to validation of their expression levels by RT-qPCR using both whole saliva and saliva supernatant samples from an independent set of 39 patients with esophageal cancer and 19 healthy controls.

Results

Six miRNAs (miR-10b*, miR-144, miR-21, miR-451, miR-486-5p, and miR-634) were identified as targets by Agilent microarray. After validation by RT-qPCR, miR-10b*, miR-144, and miR-451 in whole saliva and miR-10b*, miR-144, miR-21, and miR-451 in saliva supernatant were significantly upregulated in patients, with sensitivities of 89.7, 92.3, 84.6, 79.5, 43.6, 89.7, and 51.3% and specificities of 57.9, 47.4, 57.9%, 57.9, 89.5, 47.4, and 84.2%, respectively.

Conclusions

We found distinctive miRNAs for esophageal cancer in both whole saliva and saliva supernatant. These miRNAs possess discriminatory power for detection of esophageal cancer. Because saliva collection is noninvasive and convenient, salivary miRNAs show great promise as biomarkers for detection of esophageal cancer in areas at high risk.     

Circulating microRNAs as a Fingerprint for Liver Cirrhosis

Background

Sensitive and specific detection of liver cirrhosis is an urgent need for optimal individualized management of disease activity. Substantial studies have identified circulation miRNAs as biomarkers for diverse diseases including chronic liver diseases. In this study, we investigated the plasma miRNA signature to serve as a potential diagnostic biomarker for silent liver cirrhosis.

Methods

A genome-wide miRNA microarray was first performed in 80 plasma specimens. Six candidate miRNAs were selected and then trained in CHB-related cirrhosis and controls by qPCR. A classifier, miR-106b and miR-181b, was validated finally in two independent cohorts including CHB-related silent cirrhosis and controls, as well as non−CHB-related cirrhosis and controls as validation sets, respectively.

Results

A profile of 2 miRNAs (miR-106b and miR-181b) was identified as liver cirrhosis biomarkers irrespective of etiology. The classifier constructed by the two miRNAs provided a high diagnostic accuracy for cirrhosis (AUC = 0.882 for CHB-related cirrhosis in the training set, 0.774 for CHB-related silent cirrhosis in one validation set, and 0.915 for non−CHB-related cirrhosis in another validation set).

Conclusion

Our study demonstrated that the combined detection of miR-106b and miR-181b has a considerable clinical value to diagnose patients with liver cirrhosis, especially those at early stage.     

Dampening Enthusiasm for Circulating MicroRNA in Breast Cancer

Genome-wide platforms for high-throughput profiling of circulating miRNA (oligoarray or miR-Seq) offer enormous promise for agnostic discovery of circulating miRNA biomarkers as a pathway for development in breast cancer detection. By harmonizing data from 15 previous reports, we found widespread inconsistencies across prior studies. Whether this arises from differences in study design, such as sample source or profiling platform, is unclear. As a reproducibility experiment, we generated a genome-wide plasma miRNA dataset using the Illumina oligoarray and compared this to a publically available dataset generated using an identical sample size, substrate and profiling platform. Samples from 20 breast cancer patients, 20 mammography-screened controls, as well as 20 breast cancer patients after surgical resection and 10 female lung or colorectal cancer patients were included. After filtering for miRNAs derived from blood cells, and for low abundance miRNAs (non-detectable in over 10% of samples), a set of 522 plasma miRNAs remained, of which 46 were found to be differentially expressed between breast cancer patients and healthy controls (p<0.05), of which only 3 normalized to baseline levels in post-resection cases and were unique to breast cancer vs. lung or colorectal cancer (miR-708*, miR-92b* and miR-568, none previously reported). We were unable to demonstrate reproducibility by various measures between the two datasets. This finding, along with widespread inconsistencies across prior studies, highlight the need for better understanding of factors influencing circulating miRNA levels as prerequisites to progress in this area of translational research.     

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF_19-27) and NY-ESO-1_157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.     

Circulating microRNAs as potential biomarkers for smoking-related interstitial fibrosis

Numerous efforts have been made to indentify reliable and predictive biomarkers to detect the early signs of smoking-induced lung disease. Using 6-month cigarette smoking in mice, we have established smoking-related interstitial fibrosis (SRIF). Microarray analyses and cytokine/chemokine biomarker measurements were made to select circulating microRNAs (miRNAs) biomarkers. We have demonstrated that specific miRNAs species (miR-125b-5p, miR-128, miR-30e, and miR-20b) were significantly changed, both in the lung tissue and in plasma, and exhibited mainstream (MS) exposure duration-dependent pathological changes in the lung. These findings suggested a potential use of specific circulating miRNAs as sensitive and informative biomarkers for smoking-induced lung disease.     

Circulating microRNAs as potential biomarkers for smoking-related interstitial fibrosis

Numerous efforts have been made to indentify reliable and predictive biomarkers to detect the early signs of smoking-induced lung disease. Using 6-month cigarette smoking in mice, we have established smoking-related interstitial fibrosis (SRIF). Microarray analyses and cytokine/chemokine biomarker measurements were made to select circulating microRNAs (miRNAs) biomarkers. We have demonstrated that specific miRNAs species (miR-125b-5p, miR-128, miR-30e, and miR-20b) were significantly changed, both in the lung tissue and in plasma, and exhibited mainstream (MS) exposure duration-dependent pathological changes in the lung. These findings suggested a potential use of specific circulating miRNAs as sensitive and informative biomarkers for smoking-induced lung disease.     

乳腺癌患者循环肿瘤细胞检测的研究进展

血行播散是乳腺癌转移的重要途径,检测腋窝淋巴结已经不能完全准确判断乳腺癌患者是否存在转移。肿瘤细胞进入外周血是肿瘤远处转移的前提,对乳腺癌患者循环肿瘤细胞的检测将有助于判断预后,指导治疗,监测治疗效果。简要综述了乳腺癌循环肿瘤细胞及其检测方法的研究进展。     

A Novel Color Change Mechanism for Breast Cancer Biomarker Detection: Naphthoquinones as Specific Ligands of Human Arylamine N-Acetyltransferase 1

Human arylamine N-acetyltransferase 1 (hNAT1) has become an attractive potential biomarker for estrogen-receptor-positive breast cancers. We describe here the mechanism of action of a selective non-covalent colorimetric biosensor for the recognition of hNAT1 and its murine homologue, mNat2, over their respective isoenzymes, leading to new opportunities in diagnosis. On interaction with the enzyme, the naphthoquinone probe undergoes an instantaneous and striking visible color change from red to blue. Spectroscopic, chemical, molecular modelling and biochemical studies reported here show that the color change is mediated by selective recognition between the conjugate base of the sulfonamide group within the probe and the conjugate acid of the arginine residue within the active site of both hNAT1 and mNat2. This represents a new mechanism for selective biomarker sensing and may be exploited as a general approach to the specific detection of biomarkers in disease.     

Circulating MicroRNAs as Easy-to-Measure Aging Biomarkers in Older Breast Cancer Patients: Correlation with Chronological Age but Not with Fitness/Frailty Status

Circulating microRNAs (miRNAs) hold great promise as easily accessible biomarkers for diverse (patho)physiological processes, including aging. We have compared miRNA expression profiles in cell-free blood from older versus young breast cancer patients, in order to identify “aging miRNAs” that can be used in the future to monitor the impact of chemotherapy on the patient’s biological age. First, we assessed 175 miRNAs that may possibly be present in serum/plasma in an exploratory screening in 10 young and 10 older patients. The top-15 ranking miRNAs showing differential expression between young and older subjects were further investigated in an independent cohort consisting of another 10 young and 20 older subjects. Plasma levels of miR-20a-3p, miR-30b-5p, miR106b, miR191 and miR-301a were confirmed to show significant age-related decreases (all p≤0.004). The remaining miRNAs included in the validation study (miR-21, miR-210, miR-320b, miR-378, miR-423-5p, let-7d, miR-140-5p, miR-200c, miR-374a, miR376a) all showed similar trends as observed in the exploratory screening but these differences did not reach statistical significance. Interestingly, the age-associated miRNAs did not show differential expression between fit/healthy and non-fit/frail subjects within the older breast cancer cohort of the validation study and thus merit further investigation as true aging markers that not merely reflect frailty.     

Analysis of Detection Processes for Breast Cancer

Most existing records of detection processes for breast cancer are in the form of cancer registries or are results of large clinical trials. Statistical modelling can be applied to these data sets to study various properties of breast cancer. In particular we estimate the probability of cure given the size of the tumour at detection, the distribution of tumour growth rates and the distribution of the size of the tumour at detection. There has been a strong recent interest in early detection methods. These consist of giving regular examinations, called screenings. The effect of screening design on the probability of cure is considered. The results of an existing screening trial are used to derive another estimate of the tumour growth rate distribution which agrees well both with our earlier estimate and the most widely used empirical estimate in the literature. The calculation of lead time, which is the time gained in detection when screenings are given, is also discussed.     

Rare Circulating MicroRNAs as Biomarkers of Colorectal Neoplasia

Background

MicroRNAs (miRNAs) are regulatory RNAs, stable in circulation, and implicated in colorectal cancer (CRC) etiology and progression. Therefore they are promising as early detection biomarkers of colorectal neoplasia. However, many circulating miRNAs are highly expressed in blood cells, and therefore may not be specific to colorectal neoplasia.

Methods

We selected 7 miRNA candidates with previously reported elevated expression in adenoma tissue but low expression in blood cells (“rare” miRNAs), 2 previously proposed as adenoma biomarkers, and 3 implicated in CRC. We conducted a colonoscopy-based case-control study including 48 polyp-free controls, 43 advanced adenomas, 73 non-advanced adenomas, and 8 CRC cases. miRNAs from plasma were quantified by qRT-PCR. Correlations between miRNA expression levels, adjusted for age and sex, were assessed. We used polytomous logistic regression to estimate odds ratios (ORs) and 95% confidence intervals quantifying the association between expression levels of miRNAs and case groups. We also conducted nonparametric receiver operating characteristic (ROC) analyses and estimated area under the curve (AUC).

Results

miRNAs with high expression levels were statistically significantly correlated with one another. No miRNAs were significantly associated with non-advanced or advanced adenomas. Strong (ORs >5) and significant associations with CRC were observed for 6 miRNA candidates, with corresponding AUCs significantly >0.5.

Conclusions

These candidate miRNAs, assayed by qRT-PCR, are probably unsuitable as blood-based adenoma biomarkers. Strong associations between miRNAs and CRC were observed, but primarily with miRNAs highly expressed in blood cells. These results suggest that rare miRNAs will require new detection methods to serve as circulating biomarkers of adenomas.     

加权ceRNA网络筛选乳腺癌生物标志物

竞争性内源RNA (competing endogenous RNA,ceRNA)作为生物标志物和潜在的治疗靶点,在探究肿瘤的发病机制中,表现出了巨大的研究价值和临床应用前景.本文对乳腺癌ceRNA网络进行了系统的分析,首先通过差异分析获得差异miRNA、mRNA和lncRNA,其次利用网络的边聚集系数(edge cl...