Cell-free miRNAs Research Articles

Circulating microRNAs in cancer: Hope or hype?

Circulating miRNAs are a novel class of stable, minimally invasive disease biomarkers that are considered to be valuable in diagnosis, prognosis and treatment response monitoring. Unlike intracellular miRNAs, circulating miRNAs are released from their producer cells and, based on their targeted functions, they may shuttle in and out of circulation. Their discovery has opened up new avenues for clinical realms and led to a quest for targeted biomarkers. Subsequently, as more cell-free miRNAs are being discovered, their expression is expected to provide precise information regarding disease progression and treatment outcomes, thereby fostering personalized therapeutic strategies. The significance of circulating miRNAs capitalizes on the fact that they are highly stable in body fluids and their expression levels can be detected by common techniques such as qPCR and microarray. However, discrepancies have started to emerge in terms of their reliability and their response under physiological and pathological conditions. Functional studies are still pending, which may determine whether circulating miRNAs play a role as a central component or just as an auxiliary tuner. Also, the distinct clinical signatures that they display have never been subjected to an extensive critical review and experimental validation. As a consequence, the applicability of circulating miRNAs remains a matter of deliberation, despite many intriguing perspectives about their competency. In this review, we highlight some ambiguous issues with the application of circulating miRNAs, which may warrant an immediate consideration. We propose that the circulating miRNA domain needs to be reevaluated to authenticate their specific role and to probe whether they actually carry any clinical weightage.

Abstract 1666: Biomarkers for early detection of radiation nephropathy

Abstract Purpose/Background: Chronic kidney disease is among the common late effects detected in patients receiving chemotherapy and/or radiotherapy. Currently, there are no biomarkers that allow detection of normal tissue toxicity in late responding organs such as kidney. Developing predictive biomarkers could help identify individuals that are at high risk for close follow-up and prophylactic treatments. Our studies show that miRNAs detectable in cell-free body fluids can be developed as biomarkers of acute radiation syndromes. In the present study, we sought to investigate the potential of serum and urine miRNAs as indicators of radiation induced kidney damage in rodent models. Methods and Results: An amplification-free hybridization based nCounter assay was optimized for evaluating the changes of miRNAs in body fluids. Comparison of cell-free miRNAs in urine as well as serum, collected from control versus irradiated mice, enabled us to identify candidate biomarkers that are potential indicators of radiation response. Over 40 miRNAs were detectable in urine samples collected from mice and over 80 miRNAs were detectable in serum. Molecules such as miR-1224, miR-804, miR-714 and miR-709 showed significant increase in their urine level after radiation, which peaked 6-8 hours after exposure to acute doses. On the other hand, markers such as miR-378 that are also abundant in urine did not show significant change after radiation, hence serving as an internal control. Comparison of urinary response in mice exposed to 2, 4, 6 and 8 Gy TBI revealed good dose response. Furthermore, the same markers exhibited a dose and time dependent changes following exposure to fractionated myeloablative regimen (6 × 2 Gy, bid) that is commonly used in leukaemia patients, prior to stem cell transplantation. Specifically, two of these biomarkers, miR-714 and miR-1224 exhibited a dose dependent increase in their serum level after fractionated or acute single dose exposure. In-situ hybridization displayed an overall increase in signals of miR-1224 and miR-714 at outer medulla after radiation. Increased signals detected in tubular epithelial cells indicate the possible tubular origin of the urinary miRNAs that are responsive to radiation. Conclusion: We have developed a panel of miRNAs for non-/minimally invasive detection of radiation response in kidney. Several of the urinary miRNA biomarkers exhibited a dose and time dependent changes, suggesting their potential as biomarkers for non-invasive evaluation of radiation response in organs such as kidney for which early detection of damage is a major challenge. Citation Format: Feifei Song, Jidhin Jacob, Arnab Chakravarti, Naduparambil K. Jacob. Biomarkers for early detection of radiation nephropathy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1666.

Clinical relevance of circulating cell-free microRNAs in ovarian cancer.

Ovarian cancer is the leading cause of death among gynecologic malignancies. Since ovarian cancer develops asymptomatically, it is often diagnosed at an advanced and incurable stage. Despite many years of research, there is still a lack of reliable diagnostic markers and methods for early detection and screening. Recently, it was discovered that cell-free microRNAs (miRNAs) circulate in the body fluids of healthy and diseased patients, suggesting that they may serve as a novel diagnostic marker. This review summarizes the current knowledge regarding the potential clinical relevance of circulating cell-free miRNA for ovarian cancer diagnosis, prognosis, and therapeutics. Despite the high levels of ribonucleases in many types of body fluids, most of the circulating miRNAs are packaged in microvesicles, exosomes, or apoptotic bodies, are binding to RNA-binding protein such as argonaute 2 or lipoprotein complexes, and are thus highly stable. Cell-free miRNA signatures are known to be parallel to those from the originating tumor cells, indicating that circulating miRNA profiles accurately reflect the tumor profiles. Since it is well established that the dysregulation of miRNAs is involved in the tumorigenesis of ovarian cancer, cell-free miRNAs circulating in body fluids such as serum, plasma, whole blood, and urine may reflect not only the existence of ovarian cancer but also tumor histology, stage, and prognoses of the patients. Several groups have successfully demonstrated that serum or plasma miRNAs are able to discriminate patients with ovarian cancer patients from healthy controls, suggesting that the addition of these miRNAs to current testing regimens may improve diagnosis accuracies for ovarian cancer. Furthermore, recent studies have revealed that changes in levels of cell-free circulating miRNAs are associated with the condition of cancer patients. Discrepancies between the results across studies due to the lack of an established endogenous miRNA control to normalize for circulating miRNA levels, as well as differing extraction and quantification methods, are the pitfalls to be resolved before clinical application. There is still a long way, however, before this can be achieved, and further evidence would make it possible to apply circulating cell-free miRNAs not only as biomarkers but also as potential therapeutic targets for ovarian cancer in the future.

Cell-free microRNAs in blood and other body fluids, as cancer biomarkers.

The discovery of cell-free microRNAs (miRNAs) in serum, plasma and other body fluids has yielded an invaluable potential source of non-invasive biomarkers for cancer and other non-malignant diseases. miRNAs in the blood and other body fluids are highly stable in biological samples and are resistant to environmental conditions, such as freezing, thawing or enzymatic degradation, which makes them convenient as potential biomarkers. In addition, they are more easily sampled than tissue miRNAs. Altered levels of cell-free miRNAs have been found in every type of cancer analysed, and increasing evidence indicates that they may participate in carcinogenesis by acting as cell-to-cell signalling molecules. This review summarizes the biological characteristics and mechanisms of release of cell-free miRNAs that make them promising candidates as non-invasive biomarkers of cancer.

Diagnostic, prognostic and predictive value of cell-free miRNAs in prostate cancer: a systematic review.

Prostate cancer, the second most frequently diagnosed cancer in males worldwide, is estimated to be diagnosed in 1.1 million men per year. Introduction of PSA testing substantially improved early detection of prostate cancer, however it also led to overdiagnosis and subsequent overtreatment of patients with an indolent disease. Treatment outcome and management of prostate cancer could be improved by the development of non-invasive biomarker assays that aid in increasing the sensitivity and specificity of prostate cancer screening, help to distinguish aggressive from indolent disease and guide therapeutic decisions. Prostate cancer cells release miRNAs into the bloodstream, where they exist incorporated into ribonucleoprotein complexes or extracellular vesicles. Later, cell-free miRNAs have been found in various other biofluids. The initial RNA sequencing studies suggested that most of the circulating cell-free miRNAs in healthy individuals are derived from blood cells, while specific disease-associated miRNA signatures may appear in the circulation of patients affected with various diseases, including cancer. This raised a hope that cell-free miRNAs may serve as non-invasive biomarkers for prostate cancer. Indeed, a number of cell-free miRNAs that potentially may serve as diagnostic, prognostic or predictive biomarkers have been discovered in blood or other biofluids of prostate cancer patients and need to be validated in appropriately designed longitudinal studies and clinical trials. In this review, we systematically summarise studies investigating cell-free miRNAs in biofluids of prostate cancer patients and discuss the utility of the identified biomarkers in various clinical scenarios. Furthermore, we discuss the possible mechanisms of miRNA release into biofluids and outline the biological questions and technical challenges that have arisen from these studies.

AB003. Urinary microRNAs of prostate cancer

BackgroundMicroRNAs (miRNAs) may be potential cancer biomarkers as stable miRNAs have been detected in biological fluids such as serum, plasma, and urine. The aims of the study were to investigate whether expression of urinary cell-free miRNAs is different in prostate cancer (CaP) patients and patients with benign prostatic hyperplasia (BPH).MethodsTotal 750 urines, 150 serum and 269 prostate tissues from patients with CaP, BPH, and prostate biopsy were used in the study. After miRNA array analysis from urine to select candidate urinary miRNAs, these were validated in independent cohort. Finally matched samples from cases and control were used to evaluate correlation between candidate miRNA in prostate tissue, urine and serum.ResultsThere were significant differences in the expression of five urinary miRNAs (hsa-miR-615-3p, ebv-miR-BART4, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316) between BPH controls and CaP patients. In particular, hsv1-miR-H18 and hsv2-miR-H9-5p levels were higher in urine from prostate cancer patients. Moreover, hsv2-miR-H9-5p showed a better diagnostic performance than serum PSA for patients in the PSA gray zone. There was a significant correlation between hsv1-miR-H18 and hsv2-miR-H9-5p in tissue and urine samples, and two miRNAs were highly expressed in cancer tissues and surrounding non-cancer tissues than BPH tissues. Finally, urinary hsv2-miR-H9-5prevealed comparable diagnostic power to serum PSA even in transrectal biopsy patients, and combined with serum PSA could reduce unnecessary biopsy.ConclusionsVirus-encoded miRNAs were strongly associated with CaP, and hsv1-miR-H18 and hsv2-miR-H9-5p may be important urinary diagnostic markers for CaP even in patients with PSA gray zone.

Circulating microRNAs as a Novel Class of Potential Diagnostic Biomarkers in Neuropsychiatric Disorders

Neuropsychiatric diseases, such as schizophrenia, bipolar disorder (BD), major depressive disorder (MDD) and autism spectrum disorder (ASD), are a huge burden on society, impairing the health of those affected, as well as their ability to learn and work. Biomarkers that reflect the dysregulations linked to neuropsychiatric diseases may potentially assist the diagnosis of these disorders. Most of these biomarkers are found in the brain tissue, which is not easily accessible. This is the challenge for the search of novel biomarkers that are present in various body fluids, including serum or plasma. As a group of important endogenous small noncoding RNAs that regulate gene expression at post-transcriptional level, microRNAs (miRNAs) play a crucial role in many physiological and pathological processes. Previously, researchers discovered that miRNAs contribute to the neurodevelopment and maturation, including neurite outgrowth, dendritogenesis and dendritic spine formation. These developments underline the significance of miRNAs as potential biomarkers for diagnosing and prognosing central nervous system diseases. Accumulated evidence indicates that there are considerable differences between the cell-free miRNA expression profiles of healthy subjects and those of patients. Therefore, circulating miRNAs are likely to become a new class of noninvasive, sensitive biomarkers. Despite the fact that little is known about the origin and functions of circulating miRNAs, their essential roles in the clinical diagnosis and prognosis of neuropsychiatric diseases make them attractive biomarkers. In this review we cover the increasing amounts of dataset that have accumulated in the last years on the use of circulating miRNAs and their values as potential biomarkers in most areas of neuropsychiatric diseases.

Circulating Cancer Biomarkers: The Macro-revolution of the Micro-RNA

MicroRNAs (miRNAs) are small non-coding RNAs that act as master regulators of many cellular processes. The expression of miRNAs is often deregulated in human tumors, causing the alteration of molecular mechanisms relevant for cancer progression. Importantly, miRNAs are detectable in the blood and their quantity fluctuations are the hallmark of pathogenic conditions, including cancer. Several groups reported the identification of circulating cell-free miRNAs (cf-miRNAs) in the human serum and plasma and demonstrated their diagnostic and prognostic utility. Other studies also shown that it may be feasible to apply such cf-miRNA signatures within screening programs in order to improve cancer early detection. Circulating cf-miRNAs therefore appear to be excellent candidates for blood-borne cancer biomarkers.

Supernatant versus exosomal urinary microRNAs. Two fractions with different outcomes in gynaecological cancers.

MicroRNAs (miRNAs) are key regulatory molecules implicated in fundamental cell processes. Recent investigations have been focused to investigate their diagnostic potential also in various body fluids. Plasma and serum are widely used for these purposes. Urinary miRNAs, as the easily available type of sample, have been explored particularly in urological diseases recently. However, we have shown previously that differential expression of urinary cell-free miRNAs may be observed also in gynaecological cancers, such as ovarian and endometrial cancers. In the present article, we focus on the differences in particular urine cell-free miRNA abundance among different samples including particularly ovarian and endometrial cancers and rare gynaecological diagnoses involved in the study. Using raw abundance miRNA expression data, we confirmed significant up-regulation of miR-92a in ovarian cancer, and significant down-regulation of miR-106b in endometrial cancers. As miR-21 appeared up-regulated in the endometrial cancer similarly as in the verification process, where also miR-106b resulted in significant down-regulation in ovarian cancer, these miRNAs may be good candidates for further evaluation as novel diagnostics. To find out why supernatant but not exosomal urine miRNAs fraction resulted in significant results in regards to de-regulation of expression, we performed acomparison of the same urine samples isolated by these two manners. We show that diagnostic potential of cell-free urinary miRNAs may depend on the urine fraction used for the isolation. While particular urinary miRNAs may be enriched, other may reveal unchanged or diminished expression in the exosomal fraction in comparison with supernatant fraction, giving differences also between cancer and control samples. More research will be needed to further explore which kind of cell-free samples would give better results for diagnostic purposes in various diagnoses using urinary samples and investigating cell-free miRNAs expression. Meanwhile, different urine fractions should be explored for their miRNA expression to establish novel diagnostic urinary miRNA markers.

Circulating cell-free DNAs and miRNAs as promising non-invasive biomarkers for early detection of gastric cancer

Early diagnosis of gastric cancer is critical to decrease the mortality of this globally fatal disease. Currently, endoscopic biopsy is the gold standard for diagnosis of gastric cancer. However, invasiveness and high expense limit its application. Hence, non-invasive and cost-effective biomarkers for early detection and subsequent management are crucial steps to monitor gastric cancer. Recent studies suggest that circulating cell-free nucleic acids, including circulating tumor DNAs and microRNAs (miRNAs), are promising for various applications. Development of such blood-based biomarkers is expected to facilitate detection, predict prognosis, monitor chemotherapeutic response and manage recurrence of human cancers. In this review, the characteristics of circulating cell-free DNAs and miRNAs will be elucidated, including their origin and dysregulation. Mutations and hypermethylation of circulating DNAs, abnormal alternations of expression of circulating miRNAs will be revealed as aberrant changes indicating gastric cancer. The roles of circulating DNAs and miRNAs for early detection of gastric cancer will be focused on, as well as the challenges of developing circulating nucleic acids as biomarkers.

Isolation and enrichment of circulating biomarkers for cancer screening, detection, and diagnostics.

Much research has been performed over the past several decades in an attempt to conquer cancer. Tissue biopsy is the conventional method for gathering biological materials to analyze cancer and has contributed greatly to the understanding of cancer. However, this method is limited because it is time-consuming (requires tissue sectioning, staining, and pathological analysis), costly, provides scarce starting materials for multiple tests, and is painful. A liquid biopsy, which analyzes cancer-derived materials from various body fluids using a minimally invasive procedure, is more practical for real-time monitoring of disease progression than tissue biopsy. Biomarkers analyzable through liquid biopsy include circulating tumor cells (CTCs), exosomes, circulating cell-free DNA (cfDNA), miRNA, and proteins. Research on CTCs has been actively conducted because CTCs provide information on the whole cell, unlike the other biomarkers mentioned above. However, owing to the rarity and heterogeneity of CTCs, CTC research faces many critical concerns. Although exosomes and cfDNA have some technical challenges, they are being highlighted as new target materials. That is because they also have genetic information on cancers. Even though the number of exosomes and cfDNA from early stage cancer patients are similar to healthy individuals, they are present in high concentrations after metastasis. In this article, we review several technologies for material analyses of cancer, discuss the critical concerns based on hands-on experience, and describe future directions for cancer screening, detection, and diagnostics.

Increased Levels of Cell-Free miR-517a and Decreased Levels of Cell-Free miR-518b in Maternal Plasma Samples From Placenta Previa Pregnancies at 32 Weeks of Gestation.

The aim of this study was to clarify the association between placenta previa and circulating levels of cell-free pregnancy-associated placenta-specific microRNAs (miRNAs) in maternal plasma. Twenty singleton pregnancies with placenta previa (placenta previa group) and 26 uncomplicated pregnancies (control group) were recruited. Blood sampling was performed at 32 weeks of gestation, and cesarean delivery in all cases of placenta previa was performed at a mean gestational age of 37 weeks. The maternal plasma concentrations of cell-free pregnancy-associated placenta-specific miRNAs (miR-517a and miR-518b) were measured by absolute quantitative real-time reverse transcription-polymerase chain reaction. Plasma concentrations of cell-free miR-517a were significantly higher in the placenta previa group than that in the control group (P = .011), while the plasma concentration of cell-free miR-518b was significantly lower in the placenta previa group than that in the control group (P = .004). Plasma concentrations of cell-free miR-517a in placenta previa were significantly higher in placenta previa with alert bleeding later group than those in placenta previa without alert bleeding group or control group (P = .030 or .047, respectively) and correlated with the volume of hemorrhage at delivery (R and P value: .512 and .025). Plasma concentrations of cell-free miR-517a and miR-518b at 32 weeks of gestation were altered in pregnant women with placenta previa, and the circulating level of cell-free miR-517a in placenta previa may be a predictive marker for the risks of alert bleeding later and massive hemorrhage at delivery.

P67: Use of extracellular RNA from urine for the diagnosis of prostate cancer

Differently sized microparticles, including exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100–1000 nm) were found in blood and urine. Exosomes from prostate cancer (PCa) patients can potentially contain cancer-specific nucleic acids, and thus can represent a valuable source of diagnostic material. In this study, we have investigated microvesicles and miRNA from urine of healthy donors and PCa patients. To isolate miRNAs from urine and microparticles, novel methods for miRNA isolation were elaborated (Rus. patent application No 2014137763, priority date 17.09.2014). The study population included 14 patients with PCa (63–82 years, T2-3NxMx1) and control group of 20 healthy volunteers with no previous history of prostate disease (48–73 years). Urine was clarified by two serial centrifugations at 400 g , 20 °C, 20 min and at 17000 g , 20 °C, 20 min. Microparticles were precipitated from the resulting supernatant by high-speed centrifugation at 100000 g , 18 °C, 90 min, the pellet was resuspended and pelleted by centrifugation under the same conditions. To isolate exosomes, total microparticles were filtered through 0.1 μm pore filters and reprecipitated. The resulting pellets were resuspended and exosome samples were investigated by transmission electron microscopy (TEM). MiRNAs were isolated by one-step single-phase protocol and purified using “BioSilica” spin- columns (Zaporozhchenko et al., Anal. Biochem, upcoming, doi: 10.1016/j.ab.2015.03.028) and by recently developed method based on precipitation of excess biopolymers, allowing to isolate miRNAs with better efficiency than commercially available kits. The size and quantity assessment of extracellular RNA were performed using capillary electrophoresis system on Agilent 2100 Bioanalyzer. Concentrations of miRNAs (miR19b, miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method. TEM demonstrated the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. Approximately 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by particles larger than 100 nm. (The pool of urine microvesicles consists of 50–70% exosomes and 30–50% particles larger than 100 nm.). The major part of extracellular RNA found both in exosomes and total microparticles fraction of healthy donors and patients with PCa, is 25–200 nt long and can include tRNA (73–93 n.), 5.8 rRNA (∼150 n.), snoRNK (10–20 n.), snRNA (60–300 n.) piRNAs (29–30 n.), miRNAs (20–25 n.), siRNA (21–25 n.). Concentration of extracellular urine RNA in exosomes and total microparticles of healthy donors and patients with PCa amounts to100 pg/ml of urine on average.Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not demonstrate any diagnostic significance. In contrast, in training cohort concentration of miR-19b in exosomes and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively. Thus, microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant miRNAs. To investigate miR-19b, a large testing cohort is required. Search for other potential miRNA-markers by microarray profiling of cell-free miRNA isolated from urine of PCa patients is also required to reveal a sold set of markers for confident PCa diagnostics.

P34: Plasma micro-RNAs as noninvasive biomarkers for diagnostics of lung cancer

Lung cancer (LC) is currently the world’s leading cause of cancer-related mortality with overall mortality to incidence ratio of 0.87. Only 15% of LC patients are diagnosed at early stage of disease and have 5-year survival rate of 54%, whereas 56% of patients have distant metastases and 5-year survival rate of about 4%. Current methods of lung cancer diagnostics are not efficient as screening tools due to high costs (CT), low sensitivity and health risks (radiology), invasiveness (biopsy) or low prevalence in population (genetic alterations). New strategies for preclinical lung cancer screening as well as monitoring of post therapy relapses are required. MiRNAs circulating in blood were shown to reflect the progression of disease and are thus considered as potential biomarkers for cancer diagnostics and theranostics. In this study we investigated seven-miRNA signature circulating in plasma of lung cancer patients and healthy individuals. All seven miRNAs were previously shown to be involved in either regulation of cell cycle and apoptosis and/or tumor development, invasion and vascularization. Blood samples of 50 healthy individuals were obtained from Center of New Medical Technologies (Novosibirsk, Russia) and Novosibirsk Research Institute of Circulation Pathology of E.N. Meshalkin (Novosibirsk, Russia). Samples of 75 lung cancer patients, including 52 patients with squamous cell carcinoma (SCC) and 18 patients with adenocarcinoma (AC) were obtained from Novosibirsk Research Institute of Circulation Pathology of E.N. Meshalkin (Novosibirsk, Russia) and Tomsk Cancer Research Institute RAMS (Tomsk, Russia). None of the patients have undergone surgical treatment or received chemotherapy prior to blood collection. Lung biopsy specimens and imaging techniques were applied to confirm the histopathological features and tumor stages of LC patients. Study was approved by ethical committees of all participating organizations and written informed consent was provided by all participants. Circulating miRNAs were isolated from blood plasma using a single-phase phenol-free protocol (Zaporozhchenko et al., Anal Biochem, 2015; Rus. patent application No. 2014137763, priority date 17.09.2014). Concentrations of miRNAs (miR-21, miR-19b, miR-126, miR-25, miR-205, miR-183, miR-125b) were measured by qRT-PCR and normalized to miR-16. Concentrations of four miRNAs (miR-19b, miR-21, miR-25, miR-183) were significantly different in lung cancer versus healthy individuals (p Receiver Operating Characteristic (ROC) curve analysis of differentially expressed miRNAs in total study population and subtype-based groups showed that miR-19b has highest predictive value for total population and SCC patients, while miR-183 was more effective in discriminating patients with AC. All miRNAs except miR-21 showed strong bias towards one of the subtypes. Presumably a combination of miRNAs with opposite bias should provide a more potent diagnostic tool for cancer detection in total population than individual miRNAs or panels of miRNAs that are specific to one cancer subtype. Indeed, stepwise binary logistic regression has identified the combination of miR-19b and miR-183 to be a strong prediction of disease in total population and yielded a solid increase in AUC: 0,990 (miR-19b+miR-183) versus 0,806 (miR-19b) or 0,924 (miR-183). This combination can be used to identify lung cancer with 94.7% sensitivity and 95.2% specificity. Thus, biological aspects such as tumor genetics and phenotype, stage of disease, response to therapy, and other meaningful tumor properties of such heterogeneous malignancies as LC can interfere with contents and composition of cell-free miRNA pool. Wide profiling of circulating miRNAs will specify biomarkers of LC phenotypes and improve non/less-invasive LC diagnostics.

Abstract 3964: How to fish a good micro-marker out from a worthless lake: The case of cell-free miR-181a-5p and breast cancer

Abstract The hypothesis to use microRNAs (miRNAs) circulating in blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exiting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs (i.e. that retrieved in serum or plasma) was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables (i.e. tissue preparation, storage condition, extraction method, quantification technique, normalization approach) have on the final result. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung and melanoma tumors, and healthy controls. Then, we used droplet digital PCR (ddPCR) technology to get an accurate absolute quantification of specific cell-free miRNAs. We assessed the level of 8 different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 180 samples from healthy controls and cancer patients. We identified miRNAs specifically modulated in one or more cancer types. Plasma and serum from the same patient provided different results in terms of absolute miRNA amount and modulation. The significant reduction of miR-181a-5p levels in serum of breast cancer patients was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90). This study finally powers the use miR-181a-5p as a breast cancer biomarker. Citation Format: Manuela Ferracin, Laura Lupini, Irene Salamon, Elena Saccenti, Barabara Zagatti, Alessandra Mangolini, Maria Vittoria Zanzi, Paolo Carcoforo, Andrea Rocchi, Giorgio Cavallesco, Antonio Frassoldati, Alan B. Hollingsworth, Massimo Negrini. How to fish a good micro-marker out from a worthless lake: The case of cell-free miR-181a-5p and breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2015-3964

Circulating microRNAs as Candidate Biomarkers for the Surveillance of Melanoma Patients

Circulating microRNAs as Candidate Biomarkers for the Surveillance of Melanoma Patients

Urinary MicroRNAs of Prostate Cancer: Virus-Encoded hsv1-miRH18 and hsv2-miR-H9-5p Could Be Valuable Diagnostic Markers.

Purpose:MicroRNAs (miRNAs) in biological fluids are potential biomarkers for the diagnosis and assessment of urological diseases such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The aim of the study was to identify and validate urinary cell-free miRNAs that can segregate patients with PCa from those with BPH. Methods:In total, 1,052 urine, 150 serum, and 150 prostate tissue samples from patients with PCa or BPH were used in the study. A urine-based miRNA microarray analysis suggested the presence of differentially expressed urinary miRNAs in patients with PCa, and these were further validated in three independent PCa cohorts, using a quantitative reverse transcriptionpolymerase chain reaction analysis. Results:The expression levels of hsa-miR-615-3p, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316 were significantly higher in urine samples of patients with PCa than in those of BPH controls. In particular, herpes simplex virus (hsv)-derived hsv1-miR-H18 and hsv2-miR-H9-5p showed better diagnostic performance than did the serum prostate-specific antigen (PSA) test for patients in the PSA gray zone. Furthermore, a combination of urinary hsv2-miR-H9-5p with serum PSA showed high sensitivity and specificity, providing a potential clinical benefit by reducing unnecessary biopsies. Conclusions:Our findings showed that hsv-encoded hsv1-miR-H18 and hsv2-miR-H9-5p are significantly associated with PCa and can facilitate early diagnosis of PCa for patients within the serum PSA gray zone.

An Endocrine Genetic Signal Between Blood Cells and Vascular Smooth Muscle Cells: Role of MicroRNA-223 in Smooth Muscle Function and Atherogenesis

BackgroundMicroRNA-223 (miR-223) is a hematopoietic lineage cell-specific microRNA. However, a significant amount of miR-223 has been identified in vascular smooth muscle cells (VSMCs) and vascular walls that should not have endogenous miR-223. ObjectivesThis study sought to determine the sources of miR-223 in normal and atherosclerotic arteries and the role of miR-223 in atherogenesis. MethodsThe levels and sources of miR-223 in blood cells (leukocytes and platelets), serum, blood microparticles, VSMCs, and vascular walls were determined. Both in vivo and in vitro studies were conducted to evaluate miR-223 secretion by blood cells and the ability of miR-223 to enter VSMCs and vascular walls. Subsequent changes in and the effects of miR-223 levels on serum and arteries in atherosclerotic animals and patients were investigated. ResultsBlood cells were able to secrete miR-223 into serum. MicroRNA-223 from blood cells was the most abundant cell-free miRNA in blood. Blood cell–secreted miR-223 could enter VSMCs and vascular walls, which produced strong biological effects via its target genes. In both atherosclerotic apolipoprotein-E knockout mice and patients with atherosclerosis, miR-223 levels were significantly increased in serum and atherosclerotic vascular walls. The atherosclerotic lesions in apolipoprotein-E knockout mice were exacerbated by miR-223 knockdown. The effect of miR-223 on atherogenesis was verified using miR-223 knockout mice. ConclusionsBlood cell–secreted miR-223 enters vascular cells and walls, and appears to play important roles in VSMC function and atherogenesis. As a novel endocrine genetic signal between blood cells and vascular cells, miR-223 may provide a novel mechanism and new therapeutic target for atherosclerosis.

Absolute quantification of cell-free microRNAs in cancer patients

The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker.

648. Non-Coding RNAs as Clinically Relevant Host Factors and Target Molecules in HIV-1 Infection

Over the past decade, non-coding RNAs comprising miRNAs and lncRNAs (long non-coding RNAs) have gained tremendous attention due to their fundamental biological role as gatekeepers of cellular protein expression. Concurrently, they have become attractive from a clinical standpoint based on their frequent dysregulation in human diseases including cancers and viral infections. Particularly interesting in this respect is accumulating evidence that miRNAs are secreted from somatic cells into various easily accessible body fluids where they likewise form signatures that may describe physiological or pathological states of the host. Ideally, a thorough characterization of non-coding RNAs that are dysregulated in cells and extra-cellular fluids will not only improve our knowledge of human pathologies, but also yield additional biomarkers for disease monitoring, novel targets for clinical intervention and new avenues for creation of therapeutic vectors that are sensitive to these perturbations. Towards these aims, we focused on a major pathogen, the human immunodeficiency virus HIV-1, and asked how HIV-1 infection would dysregulate intra- and extra-cellular non-coding RNA profiles in the blood of adult humans. We thus profiled a subset of human miRNAs in HIV-1-infected patients as well as healthy age- and gender-matched controls, using a combination of microarrays and qRT-PCR for miRNA detection and quantification. In more detail, we assembled a cohort of 20 HIV-1-infected subjects with a viral load of at least 103 HIV genomes per ml, and then extracted total RNA from whole blood and cellfree plasma for miRNA profiling. The resulting comprehensive signatures of HIV-associated intra- or extra-cellular miRNAs were next compared amongst each other, as well as to those from the control cohort of HIV-1-negative adults. Notably, we identified 11 miRNAs that were specifically and significantly upregulated in the blood cells of HIV-1-infected subjects versus controls, accompanied by wide-spread increases of further miRNAs in the plasma of this cohort. Interestingly, we moreover measured intra-cellular HIV-1-dependent elevations of ISR2, a lncRNA that we recently also found to be induced by interferons as well as other pathogens including hepatitis C and influenza virus (Carnero et al., Front Immunol 2014;5:548). As a whole, our data validate that HIV-1 infection triggers a massive dysregulation of miRNA and lncRNA expression in blood cells of affected patients and a concurrent perturbation of cellfree miRNA signatures. While some of these candidates may indicate a generalized response to infection, our hope is that these non-coding RNA fingerprints could ultimately provide new clinically relevant and specific biomarkers and targets for HIV-1 diagnosis, prognosis or therapy. In addition, the observed discrepancies between intra- and extra-cellular miRNA profiles yield numerous starting points for further investigation into fundamental miRNA biology in humans.