44-2017 A Biomarker Ocular Circulating MicroRNAs Toward Diagnostics for Acute Ischemic Stroke.

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Editorial

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espite the routine use of computed tomographic scans and magnetic resonance imaging, the diagnosis of stroke remains challenging because ≈50% of patients with acute ischemic stroke (IS) lack abnormalities in the computed to-mographic and magnetic resonance imaging scans. There is an unmet need for new markers that support clinicians in the diagnosis of IS. Moreover, markers of prognosis would be of considerable interest because of the potential to guide second-ary preventive therapies enabling personalized treatment.

Article, see p 970

Biomarkers play an important role in the definition, the acute triage, and the prognostic implications in clinical prac-tice. A biomarker is a characteristic that is objectively mea-sured and evaluated as an indicator of normal and pathogenic processes or pharmacological responses, and the expectation of a biomarker is to enhance the ability of the clinician to op-timally manage the patients.1 Tremendous efforts have been put into in the identification and implementation of novel bio-markers in the vascular field,2 not only for proteins but also for emerging molecules such as noncoding RNAs, in particu-lar microRNAs (miRNAs).3,4 miRNAs are small sequences of endogenous RNA molecules that function as important global regulators of gene expression and play key roles in many bio-logical processes and diseases.5 Because of their remarkable stability in biological fluids 6 and their tissue specificity, there is a great interest in the establishment of miRNAs as circulat-ing biomarkers.

In this issue of Circulation Research , Tiedt et al 7 evalu-ated circulating miRNAs as diagnostic biomarkers for acute IS and comprehensively investigated the potential clinical use of selected miRNAs. The authors used a step-wise approach of discovery, validation, and replication in patient samples and combined these human data with the knowledge generated from experimental settings and animal models.

First, a relatively small number of patients were inves-tigated in the discovery (20 ischemic stroke patients versus 20 healthy controls) and validation step (40 ischemic stroke

patients versus 40 healthy controls). Both groups were matched for vascular risk factors and antiplatelet medica-tion. A total of 32 miRNAs were significantly deregulated on IS. Of these, 3 miRNAs were validated in an indepen-dent sample: miR-143-3p, miR-125a-5p, and miR-125b-5p. Subsequently, the 3 miRNAs were replicated in an indepen-dent, larger sample consisting of 200 IS patients and 100 healthy controls and proved significantly upregulated in IS. Moreover, specificity for IS was tested in samples of patients suffering a transitory ischemic attack in whom the 3 miRNAs were not dysregulated.

To investigate longitudinal changes, the miRNAs were measured in serial samples—at baseline, the second, third, and seventh day, and also 90 days after the event: all 3 miR-NAs peaked at a very early time point after the stroke event.Interestingly, miRNAs levels were similar across sub-groups of stroke (eg, large-vessel stroke, cardioembolic stroke, stroke of undetermined cause), and there was no significant correlation between miRNA levels in the acute phase and in-farct volume.

Although the concept to explore miRNAs for diagnosis is very attractive, and the authors have conducted a wealth of analyses, relevant questions on the translation of miRNAs into clinical practice are yet to be answered. Because of the het-erogeneity of miRNA studies conducted to date, there is slow progress toward a clinical implementation.5 This heterogene-ity includes, among others, the pathophysiological concept, the study design, the choice of material, isolation, detection and processing techniques, as well as normalization strate-gies, and possible confounding factors. Accordingly, there are several issues that need to be considered before translation of such data into clinical practice.

First, in the current study, the number of end points is fairly low, and the studies are of cross-sectional design; thus, overestimation of the results might be an issue. Although Tiedt et al 7 used an ideal 3-stage approach of discovery, vali-dation, and replication, comparatively small groups of cases and controls in each step were investigated. Furthermore, sub-jects included consisted of a highly selected group, as a large set of exclusion criteria was used including active malignant disease, inflammatory or infectious diseases, previous surgery and medication with heparin, antiplatelet medication, history of myocardial infarction, stroke or transient ischemic attack, or signs for silent central nervous system infarction. This se-lection does not reflect the real-world scenario of nonselected patients, which most likely would have yielded additional in-formation. Consequently, independent and prospective repli-cation of the data, first and foremost, is needed, using an even larger sample comprising a truly representative population of consecutive patients admitted with suspected IS.A Biomarker Ocular

Circulating MicroRNAs Toward Diagnostics for Acute Ischemic Stroke

Mahir Karakas, Tanja Zeller

(Circ Res . 2017;121:905-907.

DOI: 10.1161/CIRCRESAHA.117.311758.)? 2017 American Heart Association, Inc.Circulation Research is available at https://www.wendangku.net/doc/db2861650.html, DOI: 10.1161/CIRCRESAHA.117.311758

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.

From the Clinic for General and Interventional Cardiology, University Heart Center Hamburg, Germany; and German Center for Cardiovascular Research (DZHK e.V .), Partner Site Hamburg/Lübeck/Kiel, Germany.Correspondence to Tanja Zeller, PhD, Clinic for General and Interventional Cardiology (Genomics and Systems Biology), University H eart Center Hamburg, Hamburg 20246, Germany. E-mail t.zeller@uke.de by guest on October 1, 2017

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906 Circulation Research September 29, 2017

Second, questions on the underlying pathophysiology are remaining. Several pathophysiological processes are involved in the evolution of IS, including oxidative stress, inflamma-tion, apoptosis, and atherosclerosis. Of interest, miR-143, one of the miRNAs identified in the current study, has recently been implicated to be dysregulated in atherosclerosis.8

One of the main questions in circulating biomarker re-search is the question where do the markers come from, that is, what is the source of the markers? Using experimental set-tings and models, the data of Tiedt et al 7 suggest that platelets rather than brain cells are the major source of the miRNAs, miR-143-3p, miR-125a-5p, and miR-125b-5p. This is in line with the finding that the miRNA levels were not correlated with the infarct volume, thereby indicating that these miRNAs do not mirror neuronal cell death, but rather acute ischemic or thrombotic processes. On the other hand, this leads to the question why the 3 miRNAs did not evolve in large-scale pro-spective studies of acute coronary syndrome.9 In this context, in the future, care needs to be taken to establish the specificity of identified miRNAs and to show their potential for discrimi-nation of ischemic stroke from other ischemic or platelet-as-sociated conditions.

Third, from an analytic point of view, the detection of miRNAs still depends on the limitations raised by the lack of standardized isolation, detection, and normalization strate-gies. Many studies have sought to identify differentially regu-lated circulating miRNAs in stroke patients and validate them as biomarkers in which the existence of multiple stroke causes and classifications has complicated the challenge.10 Different preanalytical approaches might explain why such miRNAs which have been implicated in the pathogenesis of stroke did not evolve in the current study. To avoid technical and analytic variability and thereby artificial data generation, consensus on standard methods for all steps is imperative.

Fourth, to clinically evaluate a biomarker, its ability to dis-criminate patients with a certain condition and a comparison to the current standard marker need to be tested. In the current study,7 the clinical utilization of the 3 miRNAs as diagnostic tools was determined by combining them into a signature. In re-ceiver operating characteristic analyses, this miRNA signature provided a better discriminatory ability with high sensitivity and specificity compared with the current standard—comput-ed tomographic scans—and previously reported protein-based biomarkers. H owever, in addition to performance measures such as receiver operating characteristic analyses, reclassifica-tion strategies have to be incorporated into the statistical analy-sis plan to path the way toward potential clinical application.In conclusion, we have to ask what the clinical implica-tions of such improved risk stratification are and if the chal-lenges of translating miRNAs into clinical practice can be solved (Figure). Although first steps in this direction have al-ready been made by the development of point-of-care devices for easy and fast miRNA detection,11 the important question remains whether patients would be triaged, processed, and treated differently? Will such data lead to more or less diag-nostic testings? More, because biomarkers have a fix rate of false-positive test results or less, because patients with miR-NAs below a given threshold would no longer be referred to

computed tomographic and magnetic resonance imaging? Ultimately, only well-powered, randomized clinical trials will provide the adequate answer. Until then, we should be aware that patients with symptoms suggestive of an IS have a high risk of morbidity and mortality and deserve full clinical atten-tion and optimal medical treatment.

Sources of Funding

This work was supported by the German Center for Cardiovascular Research (DZHK e.V .; No. 81Z1710101 and 81438/152).

Disclosures

None.

References

1. Vasan RS. Biomarkers of cardiovascular disease: molecular basis

and practical considerations. Circulation . 2006;113:2335–2362. doi: 10.1161/CIRCULATIONAHA.104.482570.

2. H oefer IE, Steffens S, Ala-Korpela M, et al; ESC Working Group

Atherosclerosis and Vascular Biology. Novel methodologies for bio-marker discovery in atherosclerosis. Eur Heart J . 2015;36:2635–2642. doi: 10.1093/eurheartj/ehv236.

3. Thum T, Condorelli G. Long noncoding RNAs and microRNAs in

cardiovascular pathophysiology. Circ Res . 2015;116:751–762. doi: 10.1161/CIRCRESAHA.116.303549.

4. Poller W, Dimmeler S, Heymans S, Zeller T, Haas J, Karakas M, Leistner

DM, Jakob P, Nakagawa S, Blankenberg S, Engelhardt S, Thum T, Weber C, Meder B, Hajjar R, Landmesser U. Non-coding RNAs in car-diovascular diseases: diagnostic and therapeutic perspectives [published online ahead of print April 18, 2017]. Eur Heart J . doi: 10.1093/eur-heartj/ehx165. https://https://www.wendangku.net/doc/db2861650.html,/10.1093/eurheartj/ehx165.

5. Keller A, Meese E. Can circulating miRNAs live up to the promise of

being minimal invasive biomarkers in clinical settings? Wiley Interdiscip Rev RNA . 2016;7:148–156. doi: 10.1002/wrna.1320.

6. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA

. 2008;105:10513–10518.

Figure . Steps and challenges toward the clinical application of microRNAs as diagnostic tools.

by guest on October 1, 2017

https://www.wendangku.net/doc/db2861650.html,/Downloaded from

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7. Tiedt S, Prestel M, Malik R, et al. RNA-Seq identifies circulating

miR-125a-5p, miR-125b-5p, and miR-143-3p as potential biomarkers for acute ischemic stroke. Circ Res . 2017;121:970–980. doi: 10.1161/CIRCRESAHA.117.311572.

8. Wei YS, Xiang Y , Liao PH, Wang JL, Peng YF. An rs4705342 T>C poly-morphism in the promoter of miR-143/145 is associated with a decreased risk of ischemic stroke. Sci Rep . 2016;6:34620. doi: 10.1038/srep34620. 9. Karakas M, Schulte C, Appelbaum S, Ojeda F, Lackner KJ, Münzel T,

Schnabel RB, Blankenberg S, Zeller T. Circulating microRNAs strongly predict cardiovascular death in patients with coronary artery disease-results from the large AtheroGene study. Eur Heart J . 2017;38:516–523. doi: 10.1093/eurheartj/ehw250.

10. Sepramaniam S, Tan JR, Tan KS, DeSilva DA, Tavintharan S, Woon FP,

Wang CW, Yong FL, Karolina DS, Kaur P, Liu FJ, Lim KY , Armugam A, Jeyaseelan K. Circulating microRNAs as biomarkers of acute stroke. Int J Mol Sci . 2014;15:1418–1432. doi: 10.3390/ijms15011418.

11. Williams MR, Stedtfeld RD, Stedtfeld TM, Tiedje JM, H ashsham

SA. Quantification of microRNAs directly from body fluids us-ing a base-stacking isothermal amplification method in a point-of-care device. Biomed Microdevices . 2017;19:45. doi: 10.1007/s10544-017-0191-2.K ey W ords : Editorials ■ biomarkers ■ diagnosis ■ gene expression ■ ischemic stroke ■ microRNAs ■ prognosis

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Mahir Karakas and Tanja Zeller

Stroke

A Biomarker Ocular: Circulating MicroRNAs Toward Diagnostics for Acute Ischemic

Print ISSN: 0009-7330. Online ISSN: 1524-4571

Copyright ? 2017 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation Research

doi: 10.1161/CIRCRESAHA.117.311758

2017;121:905-907

Circ Res.

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