The Emergent Role of MicroRNAs in Molecular Diagnostics of Cancer
2008; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2008.080067
ISSN1943-7811
Autores Tópico(s)RNA Research and Splicing
ResumoA technical breakthrough that expands the scope of clinical samples amenable for global microRNA analysis is highlighted in this Commentary. A technical breakthrough that expands the scope of clinical samples amenable for global microRNA analysis is highlighted in this Commentary. When lin-4 was discovered in 1993 as a small noncoding RNA that regulates the development of the earthworm Caenorhabditis elegans,1Lee RC Feinbaum RL Ambros V The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (10120) Google Scholar,2Wightman B Ha I Ruvkun G Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.Cell. 1993; 75: 855-862Abstract Full Text PDF PubMed Scopus (3251) Google Scholar it was thought that this RNA was a species-specific peculiarity. Seven years later, another similar small developmentally regulated RNA, let-7, was identified to be evolutionarily conserved, suggesting that this type of RNA molecule has conserved biological functions.3Pasquinelli AE Reinhart BJ Slack F Martindale MQ Kuroda MI Maller B Hayward DC Ball EE Degnan B Muller P Spring J Srinivasan A Fishman M Finnerty J Corbo J Levine M Leahy P Davidson E Ruvkun G Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.Nature. 2000; 408: 86-89Crossref PubMed Scopus (1919) Google Scholar In 2001, multiple novel transcripts of about 22 nucleotides akin to lin-4 and let-7, termed microRNAs (miRNAs), were cloned from worms, flies, and human cells.4Lagos-Quintana M Rauhut R Lendeckel W Tuschl T Identification of novel genes coding for small expressed RNAs.Science. 2001; 294: 853-858Crossref PubMed Scopus (4046) Google Scholar5Lau NC Lim LP Weinstein EG Bartel DP An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.Science. 2001; 294: 858-862Crossref PubMed Scopus (2738) Google Scholar6Lee RC Ambros V An extensive class of small RNAs in Caenorhabditis elegans.Science. 2001; 294: 862-864Crossref PubMed Scopus (2363) Google Scholar These studies opened the door for numerous exciting scientific investigations that have important biological and clinical implications. miRNAs are processed from precursors and in their mature forms, serve as important gene regulators that have the capacity to down-regulate gene expression through translation inhibition and promotion of mRNA degradation mediated by specific target site binding to the 3′-untranslated region of target genes.7Zhang W Dahlberg JE Tam W MicroRNAs in tumorigenesis: a primer.Am J Pathol. 2007; 171: 728-738Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar According to the most recent version of miRBase (v.11.0),8Griffiths-Jones S Saini HK van Dongen S Enright AJ miRBase: tools for microRNA genomics.Nucleic Acids Res. 2008; 36: D154-D158Crossref PubMed Scopus (3705) Google Scholar 678 different mature miRNA sequences have been identified in humans to date. Primarily through clinico-pathological studies involving expression analysis of miRNAs in normal and diseased, especially cancerous, human tissues and cells, many of these molecules are emerging as potential markers in molecular diagnostics, particularly in the field of cancer diagnostics. As discussed below, identification of miRNA biomarkers has been facilitated by novel development and refinement of detection methodologies. The work by Szafranska et al,9Szafranska AE Davidson T Shingara J Doleshal M Riggenbach JA Morrison CD Jewell S Labourier E Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.J Mol Diagn. 2008; 10: 415-423Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar highlighted in this issue of The Journal of Molecular Diagnostics, details a technical advance that expands the scope of clinical samples amenable for global miRNA analysis. A unique attribute of miRNAs that renders them potentially useful for the molecular diagnosis of tumors is their tissue and cell lineage specificity.10Landgraf P Rusu M Sheridan R Sewer A Iovino N Aravin A Pfeffer S Rice A Kamphorst AO Landthaler M Lin C Socci ND Hermida L Fulci V Chiaretti S Foa R Schliwka J Fuchs U Novosel A Muller RU Schermer B Bissels U Inman J Phan Q Chien M Weir DB Choksi R De Vita G Frezzetti D Trompeter HI Hornung V Teng G Hartmann G Palkovits M Di Lauro R Wernet P Macino G Rogler CE Nagle JW Ju J Papavasiliou FN Benzing T Lichter P Tam W Brownstein MJ Bosio A Borkhardt A Russo JJ Sander C Zavolan M Tuschl T A mammalian microRNA expression atlas based on small RNA library sequencing.Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3099) Google Scholar Many of the miRNAs are highly specific in their expression in specific tissues and cell types, and this specificity is often retained in the corresponding tumor tissues. Identification of cell origin by profiling of miRNAs is more efficient compared with global analysis of mRNAs, because the former is not confounded by such a large pool of irrelevant genes because of the relatively small number of miRNA species. Therefore, miRNAs could facilitate the accurate diagnosis of tumors that are difficult to classify with respect to the tissue origin by conventional means, for example, metastatic cancer of unknown primary origin,11Jeffrey SS Cancer biomarker profiling with microRNAs.Nat Biotechnol. 2008; 26: 400-401Crossref PubMed Scopus (93) Google Scholar a highly aggressive malignancy that poses diagnostic and management difficulties.12Varadhachary GR Abbruzzese JL Lenzi R Diagnostic strategies for unknown primary cancer.Cancer. 2004; 100: 1776-1785Crossref PubMed Scopus (215) Google Scholar In an initial study by Lu et al,13Lu J Getz G Miska EA Alvarez-Saavedra E Lamb J Peck D Sweet-Cordero A Ebert BL Mak RH Ferrando AA Downing JR Jacks T Horvitz HR Golub TR MicroRNA expression profiles classify human cancers.Nature. 2005; 435: 834-838Crossref PubMed Scopus (8416) Google Scholar global microRNA profiling could accurately classify 12 of 17 poorly differentiated carcinomas. The diagnostic accuracy has been further increased with the use of a selected set of miRNAs. Recently, an miRNA classifier consisting of 48 miRNAs generated from 253 samples representing 22 different types of human cancers was found to predict tissue origin with an overall accuracy of 89% in an independent blinded test set of 83 samples using an algorithm in which one to five specific miRNAs determine the decision at each node of a binary decision tree.14Rosenfeld N Aharonov R Meiri E Rosenwald S Spector Y Zepeniuk M Benjamin H Shabes N Tabak S Levy A Lebanony D Goren Y Silberschein E Targan N Ben-Ari A Gilad S Sion-Vardy N Tobar A Feinmesser M Kharenko O Nativ O Nass D Perelman M Yosepovich A Shalmon B Polak-Charcon S Fridman E Avniel A Bentwich I Bentwich Z Cohen D Chajut A Barshack I MicroRNAs accurately identify cancer tissue origin.Nat Biotechnol. 2008; 26: 462-469Crossref PubMed Scopus (870) Google Scholar Moreover, the classification accuracy reached 100% in 6 of the 10 tissue types within the metastatic test set. However, the effectiveness of this set of miRNAs in identifying tissue origin in cases of cancers of unknown primary was not tested in this study. Nevertheless, this methodology compares very favorably with the mRNA-based methods in identifying tissue origins of cancer. Even when only a selected subset of genes is analyzed, the latter method requires a large number of genes (usually hundreds) in the classifier to achieve a similar accuracy as miRNA profiling.15Bloom G Yang IV Boulware D Kwong KY Coppola D Eschrich S Quackenbush J Yeatman TJ Multi-platform, multi-site, microarray-based human tumor classification.Am J Pathol. 2004; 164: 9-16Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar16Ma XJ Patel R Wang X Salunga R Murage J Desai R Tuggle JT Wang W Chu S Stecker K Raja R Robin H Moore M Baunoch D Sgroi D Erlander M Molecular classification of human cancers using a 92-gene real-time quantitative polymerase chain reaction assay.Arch Pathol Lab Med. 2006; 130: 465-473PubMed Google Scholar17Tothill RW Kowalczyk A Rischin D Bousioutas A Haviv I van Laar RK Waring PM Zalcberg J Ward R Biankin AV Sutherland RL Henshall SM Fong K Pollack JR Bowtell DD Holloway AJ An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin.Cancer Res. 2005; 65: 4031-4040Crossref PubMed Scopus (194) Google Scholar18Shedden KA Taylor JM Giordano TJ Kuick R Misek DE Rennert G Schwartz DR Gruber SB Logsdon C Simeone D Kardia SL Greenson JK Cho KR Beer DG Fearon ER Hanash S Accurate molecular classification of human cancers based on gene expression using a simple classifier with a pathological tree-based framework.Am J Pathol. 2003; 163: 1985-1995Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Moreover, it uses algorithms that average gene expression levels, which result in loss of diagnostic and potential pathogenetic information. Furthermore, it is more easily confounded by background noise related to natural variations in gene expression. As a result, mRNA-based methods may be less robust. The power of miRNAs in determining the tissue origin of tumors may be further enhanced by incorporating a subset of carefully selected mRNAs to generate a combined miRNA-mRNA classifier. The rationale behind this lies in the observation that cell-type-specific miRNA signatures correlate with mRNA expression patterns.19Sood P Krek A Zavolan M Macino G Rajewsky N Cell-type-specific signatures of microRNAs on target mRNA expression.Proc Natl Acad Sci USA. 2006; 103: 2746-2751Crossref PubMed Scopus (533) Google Scholar Highly expressed mRNAs tend to lack binding sites of highly expressed miRNAs in the same tissues, whereas levels of mRNAs targeted by certain miRNAs tend to be lower in tissues where those miRNAs are present at high levels. Thus, it may be possible to increase the predictive value of the miRNA classifier by simultaneously determining expression of a few selected mRNAs. Deregulation of miRNAs occurs frequently during tumorigenesis,7Zhang W Dahlberg JE Tam W MicroRNAs in tumorigenesis: a primer.Am J Pathol. 2007; 171: 728-738Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar making them attractive candidates for molecular detection of malignancy. A subset of miRNAs can often be found up- or down-regulated in tumors compared with the normal tissues in a specific tumor type or more globally in a number of different tumor types. Not only is the identification of these miRNAs critical in the understanding of the pathogenetic role of miRNAs in cancer development, it can also provide a diagnostic methodology for distinguishing tumors from normal tissues of different cell origins. In the current setting of clinical diagnostic practice, where morphological and antigenic evaluation appears to be adequate for accurate diagnostic separation between tumor and normal tissues in the vast majority of biopsies, the utility of miRNA analysis in this arena may not be too apparent. However, the differential expression of miRNAs between tumors and normal tissues may be exploited in the diagnosis of samples where cells are scant or poorly preserved, which may render diagnosis of a malignancy by traditional methods difficult, and in the noninvasive screening for cancer, provided that alterations in specific miRNAs in tumors can be similarly detected in uninvolved body fluids such as peripheral blood. The latter application is illustrated by the recent discovery that higher levels (about 2.5- to 5-fold) of miR-155, miR-210, and miR-21 can be found in the serum of patients with diffuse large B-cell lymphomas and that higher levels of miR-21 correlates with a longer relapse-free survival.20Lawrie CH Gal S Dunlop HM Pushkaran B Liggins AP Pulford K Banham AH Pezzella F Boultwood J Wainscoat JS Hatton CS Harris AL Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma.Br J Haematol. 2008; 141: 672-675Crossref PubMed Scopus (1528) Google Scholar Circulating nucleic acids have been implicated as biomarkers for various diseases.21Tsang JC Lo YM Circulating nucleic acids in plasma/serum.Pathology. 2007; 39: 197-207Crossref PubMed Scopus (147) Google Scholar This work documents as a proof-of-concept the presence of circulating miRNAs at detectable levels and their utility in cancer diagnosis and prognosis. Because miRNAs are frequently overexpressed in tumors, it is possible that alteration in the profile of circulating miRNAs, which are likely to be derived from tumor cells, is a common occurrence in cancer patients. Thus, detection of miRNAs in serum and other body fluids may serve as a noninvasive means of screening, diagnosing, and prognosing tumors. It may also be used in the detection of occult tumor or minimal residual neoplastic disease, assessment of tumor load, and monitoring of treatment response. miRNAs may also be useful in subclassifying tumors of a particular tissue origin. Expression of specific miRNAs has been shown to correlate with histological subtypes of certain types of cancer, for example, ductal and lobular breast carcinoma22Iorio MV Ferracin M Liu CG Veronese A Spizzo R Sabbioni S Magri E Pedriali M Fabbri M Campiglio M Menard S Palazzo JP Rosenberg A Musiani P Volinia S Nenci I Calin GA Querzoli P Negrini M Croce CM MicroRNA gene expression deregulation in human breast cancer.Cancer Res. 2005; 65: 7065-7070Crossref PubMed Scopus (3538) Google Scholar; mucinous and nonmucinous carcinoma of the lung23Garfield D let-7 microRNA expression and the distinction between nonmucinous and mucinous bronchioloalveolar carcinomas.Lung Cancer. 2008; 60: 307Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar,24Yanaihara N Caplen N Bowman E Seike M Kumamoto K Yi M Stephens RM Okamoto A Yokota J Tanaka T Calin GA Liu CG Croce CM Harris CC Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell. 2006; 9: 189-198Abstract Full Text Full Text PDF PubMed Scopus (2731) Google Scholar; and papillary, follicular, and anaplastic thyroid carcinomas.25He H Jazdzewski K Li W Liyanarachchi S Nagy R Volinia S Calin GA Liu CG Franssila K Suster S Kloos RT Croce CM de la Chapelle A The role of microRNA genes in papillary thyroid carcinoma.Proc Natl Acad Sci USA. 2005; 102: 19075-19080Crossref PubMed Scopus (1087) Google Scholar26Weber F Teresi RE Broelsch CE Frilling A Eng C A limited set of human MicroRNA is deregulated in follicular thyroid carcinoma.J Clin Endocrinol Metab. 2006; 91: 3584-3591Crossref PubMed Scopus (265) Google Scholar27Visone R Pallante P Vecchione A Cirombella R Ferracin M Ferraro A Volinia S Coluzzi S Leone V Borbone E Liu CG Petrocca F Troncone G Calin GA Scarpa A Colato C Tallini G Santoro M Croce CM Fusco A Specific microRNAs are down-regulated in human thyroid anaplastic carcinomas.Oncogene. 2007; 26: 7590-7595Crossref PubMed Scopus (347) Google Scholar These miRNAs are likely to play a role in the distinct pathogenetic pathways leading to the different histological subtypes, and may be useful as a diagnostic aid in unusually difficult cases. There is increasing evidence that miRNAs can be invaluable as a biomarker for patient prognosis. For example, a set of differentially expressed miRNAs were identified in chronic lymphocytic leukemia that can separate it into prognostic categories.28Calin GA Ferracin M Cimmino A Di Leva G Shimizu M Wojcik SE Iorio MV Visone R Sever NI Fabbri M Iuliano R Palumbo T Pichiorri F Roldo C Garzon R Sevignani C Rassenti L Alder H Volinia S Liu CG Kipps TJ Negrini M Croce CM A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia.N Engl J Med. 2005; 353: 1793-1801Crossref PubMed Scopus (2116) Google Scholar Reduced expression of let-7 and high expression of miR-155 were associated with poor survival in human lung cancers.29Takamizawa J Konishi H Yanagisawa K Tomida S Osada H Endoh H Harano T Yatabe Y Nagino M Nimura Y Mitsudomi T Takahashi T Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.Cancer Res. 2004; 64: 3753-3756Crossref PubMed Scopus (2185) Google Scholar High levels of miR-21 are associated with poor survival and poor therapeutic outcome in colon cancer.30Schetter AJ Leung SY Sohn JJ Zanetti KA Bowman ED Yanaihara N Yuen ST Chan TL Kwong DL Au GK Liu CG Calin GA Croce CM Harris CC MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.JAMA. 2008; 299: 425-436Crossref PubMed Scopus (1448) Google Scholar In addition, several miRNAs, including miR-10b, miR-126, miR-335, miR-373, and miR-520c were recently found to promote or suppress invasion and metastasis in breast cancer cells.31Huang Q Gumireddy K Schrier M le Sage C Nagel R Nair S Egan DA Li A Huang G Klein-Szanto AJ Gimotty PA Katsaros D Coukos G Zhang L Pure E Agami R The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis.Nat Cell Biol. 2008; 10: 202-210Crossref PubMed Scopus (888) Google Scholar32Ma L Teruya-Feldstein J Weinberg RA Tumour invasion and metastasis initiated by microRNA-10b in breast cancer.Nature. 2007; 449: 682-688Crossref PubMed Scopus (2271) Google Scholar33Tavazoie SF Alarcon C Oskarsson T Padua D Wang Q Bos PD Gerald WL Massague J Endogenous human microRNAs that suppress breast cancer metastasis.Nature. 2008; 451: 147-152Crossref PubMed Scopus (1654) Google Scholar Some of these miRNAs were also shown to correlate with clinical outcome and disease progression in breast cancer. Thus, levels of these miRNAs in primary breast tumors could be useful as predictive biomarkers of their metastatic potential. The application of miRNAs for molecular diagnostic purposes is critically dependent on the development of methods for their accurate and high-throughput quantification. At present, the most commonly used method for quantitative measurement of miRNAs is the real-time RT-PCR. This method is similar to the standard real-time RT-PCR for the detection of mRNA, except that the former makes use of a stem-loop reverse transcription primer for the initiation of cDNA templates.34Chen C Ridzon DA Broomer AJ Zhou Z Lee DH Nguyen JT Barbisin M Xu NL Mahuvakar VR Andersen MR Lao KQ Livak KJ Guegler KJ Real-time quantification of microRNAs by stem-loop RT-PCR.Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (4177) Google Scholar,35Raymond CK Roberts BS Garrett-Engele P Lim LP Johnson JM Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs.Rna. 2005; 11: 1737-1744Crossref PubMed Scopus (396) Google Scholar This method is relatively easy to use, highly sensitive and has a broad dynamic range. Another quantitative method is Invader assay,36Allawi HT Dahlberg JE Olson S Lund E Olson M Ma WP Takova T Neri BP Lyamichev VI Quantitation of microRNAs using a modified Invader assay.RNA. 2004; 10: 1153-1161Crossref PubMed Scopus (160) Google Scholar,37Eis PS Tam W Sun L Chadburn A Li Z Gomez MF Lund E Dahlberg JE Accumulation of miR-155 and BIC RNA in human B cell lymphomas.Proc Natl Acad Sci USA. 2005; 102: 3627-3632Crossref PubMed Scopus (1216) Google Scholar which directly detects specific RNA molecules using an isothermal amplification process with a fluorescent read-out. Since its initial development, the Invader assay for miRNA quantification has undergone modifications (Third Wave Technology, Madison, WI, personal communication). In its current format, it offers comparable sensitivity, specificity, dynamic range, and ease of use as real-time RT-PCR. However, neither of these techniques is optimal for the simultaneous analysis of hundreds of different miRNAs. The method of choice for this type of analysis is global expression profiling, which is mostly performed on glass slide microarrays38Nelson PT Baldwin DA Scearce LM Oberholtzer JC Tobias JW Mourelatos Z Microarray-based, high-throughput gene expression profiling of microRNAs.Nat Methods. 2004; 1: 155-161Crossref PubMed Scopus (548) Google Scholar,39Thomson JM Parker J Perou CM Hammond SM A custom microarray platform for analysis of microRNA gene expression.Nat Methods. 2004; 1: 47-53Crossref PubMed Scopus (679) Google Scholar but also can be performed using bead-based flow cytometry.13Lu J Getz G Miska EA Alvarez-Saavedra E Lamb J Peck D Sweet-Cordero A Ebert BL Mak RH Ferrando AA Downing JR Jacks T Horvitz HR Golub TR MicroRNA expression profiles classify human cancers.Nature. 2005; 435: 834-838Crossref PubMed Scopus (8416) Google Scholar The use of locked nucleic acid-modified probes has enhanced the sensitivity and specificity of miRNA microarrays.40Castoldi M Schmidt S Benes V Hentze MW Muckenthaler MU miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes.Nat Protoc. 2008; 3: 321-329Crossref PubMed Scopus (119) Google Scholar Detection of miRNAs can be performed not only with fresh or frozen tissues but also with formalin-fixed, paraffin-embedded (FFPE) samples. miRNA has been shown by RT-PCR to be a superior analyte compared with mRNA when FFPE materials are used because miRNAs are small and therefore less subject to RNA degradation.41Doleshal M Magotra AA Choudhury B Cannon BD Labourier E Szafranska AE Evaluation and validation of total RNA extraction methods for MicroRNA expression analyses in formalin-fixed, paraffin-embedded tissues.J Mol Diagn. 2008; 10: 203-211Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar FFPE materials have also been used successfully in expression profiling studies of miRNA on different microarray platforms.14Rosenfeld N Aharonov R Meiri E Rosenwald S Spector Y Zepeniuk M Benjamin H Shabes N Tabak S Levy A Lebanony D Goren Y Silberschein E Targan N Ben-Ari A Gilad S Sion-Vardy N Tobar A Feinmesser M Kharenko O Nativ O Nass D Perelman M Yosepovich A Shalmon B Polak-Charcon S Fridman E Avniel A Bentwich I Bentwich Z Cohen D Chajut A Barshack I MicroRNAs accurately identify cancer tissue origin.Nat Biotechnol. 2008; 26: 462-469Crossref PubMed Scopus (870) Google Scholar,42Nelson PT Baldwin DA Kloosterman WP Kauppinen S Plasterk RH Mourelatos Z RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain.RNA. 2006; 12: 187-191Crossref PubMed Scopus (265) Google Scholar,43Xi Y Nakajima G Gavin E Morris CG Kudo K Hayashi K Ju J Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples.RNA. 2007; 13: 1668-1674Crossref PubMed Scopus (508) Google Scholar The current study by Szafranska et al in this issue of The JMD systematically compared the use of FFPE and frozen materials and demonstrated that expression profiling of miRNAs can be performed in an accurate and reproducible fashion on FFPE materials using a commercially available, one-color, non-locked nucleic acid-based array platform and optimized RNA extraction methods. Their results provide solid evidence for the feasibility of FFPE as a suitable source in miRNA profiling, which would enable a wider collection of clinically annotated patient materials to be studied retrospectively. However, several observations in the study suggest potential pitfalls. Although miRNA expression profiles generated from FFPE materials are highly similar to those derived from frozen materials, there is some loss of signals in the former, particularly when the materials are fixed for a longer duration and when the miRNAs are present in low abundance. Profiling using FFPE materials is also more susceptible to nonspecific signals generated by nonspecific hybridization to degraded products of mRNA. As a result, false negatives and positives can arise more frequently when comparing miRNA expression patterns between two FFPE samples. Fortunately, the detection of the most differentially expressed genes does not appear to be compromised based on a limited comparison. Thus, global comparative expression profiling of FFPE materials may be most suitable for the initial identification of the more differentially expressed and relatively more abundant miRNAs. Reliable identification of less differentially expressed miRNAs of low to medium abundance may require the use of frozen materials, or other quantitative methods if specific miRNAs are known to be involved. Because the concordance of miRNA profiles between FFPE and frozen materials can vary depending on tissue type and block age, a comparison between these two sources for miRNA expression profiling, if feasible, is recommended before embarking a large-scale study on FFPE materials. Armed with the ability to analyze global miRNA gene expression in FFPE samples, we are in a better position to perform large-scale studies to identify additional miRNAs with potential in cancer diagnostics and to confirm the existing sets of miRNAs as candidates for clinical biomarkers. Besides cancer diagnostics, it is likely that miRNAs will also be useful in molecular diagnostics of other diseases as we begin to elucidate the role of miRNAs in the normal physiology of different organs and their alterations in various disease processes. For example, it is conceivable that circulating miRNAs may be used for risk stratification of chronic diseases like cardiovascular disease or diabetes. Despite the many exciting developments that demonstrate the potential capacity of miRNAs for tumor classification and prognosis, their practical use as biomarkers in a routine clinical setting is still in its infancy. To ascertain and accelerate its advancement beyond the developmental phase, efforts should be made to perform retrospective and prospective studies in global and gene-specific analysis of miRNAs on multiple independent cohorts of patient samples using standardized methodologies. Robust methods for accurate detection of specific or multiple miRNAs using very limited quantities of input RNA need to be developed to enable reproducible and reliable miRNA quantification on small samples, for instance, cytology specimen or laser-capture microdissected tumor cells. To improve accuracy in diagnostic and prognostic classifications and in predicting clinical outcome, miRNA may be evaluated together with a selected set of mRNA and protein markers to develop a "collaborative" classifier. Finally, because miRNAs function as micromanagers in gene regulatory networks, many miRNAs that have been identified as potential markers in neoplasms may have a pathogenetic role instead of being a by-product of the disease states. This immediate pathogenetic relevance makes miRNAs distinct from many other biomarkers. Continued investigations into the biochemical functions of miRNAs through target identification will uncover important mechanistic insights into their potential as biomarkers and therapeutic targets and offer opportunities for discovery of additional biomarkers. Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression ProfilingThe Journal of Molecular DiagnosticsVol. 10Issue 5PreviewFormalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalin-fixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. Full-Text PDF
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