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Circulating microRNAs as Promising Tumor Biomarkers Meng Chen*, George A. Calin†, Qing H. Meng*,1 *Department of Laboratory Medicine, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA † Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 1 Corresponding author: e-mail address:
[email protected] Contents 1. 2. 3. 4. 5.
Introduction miRNA Biogenesis miRNAs and Cancer Origin and Function of Circulating miRNAs Detection of Circulating miRNAs 5.1 qRT-PCR 5.2 miRNA microarray 5.3 Deep sequencing 6. Circulating miRNAs as Cancer Biomarkers 6.1 Prostate cancer 6.2 Breast cancer 6.3 Lung cancer 6.4 Colorectal cancer 6.5 Hematologic cancers 7. Conclusions References
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Abstract microRNAs (miRNAs) are small, nonprotein-coding RNAs that function as posttranscriptional regulators of target genes. miRNAs are involved in multiple cell differentiation, proliferation, and apoptosis processes that are closely related to tumorigenesis. Circulating miRNAs are promising cancer biomarkers under development with great translational potential in personalized medicine. Here, we describe the origin and function of circulating miRNAs and compare the current new high-throughput technology applied to miRNA quantitation. The latest publications on circulating miRNAs were summarized, indicating that miRNAs are potential biomarkers of diagnosis, prognosis, and treatment response of major cancer types including prostate, breast, lung, colorectal, and hematological cancers. We addressed the strengths and limitations of applying circulating miRNAs in clinical laboratory and several issues associated with the accurate measurement of circulating miRNAs. Advances in Clinical Chemistry ISSN 0065-2423 http://dx.doi.org/10.1016/bs.acc.2014.09.007
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2014 Elsevier Inc. All rights reserved.
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1. INTRODUCTION microRNAs (miRNAs) are a class of endogenous, single-stranded noncoding small (17–25 nucleotides, typically 22 nucleotides) RNAs that are involved in regulating gene expression at the posttranscriptional level [1]. miRNAs were first discovered in Caenorhabditis elegans in 1993 and have since been found to be highly conserved in nearly all organisms [2]. In humans, miRNAs account for only approximately 1% of the human genome, but they have been estimated to regulate the translation and stability of up to 50–60% of mRNAs [3,4], demonstrating the importance of their functions in gene expression.
2. miRNA BIOGENESIS miRNAs are transcribed in the same way protein-coding genes are transcribed, and their expression is strictly regulated by the processing machinery in their biogenesis pathway [4]. Long primary transcripts of miRNAs (pri-miRNAs) are mainly transcribed by RNA polymerase II and partially by RNA polymerase III. Pri-miRNAs are hundreds of nucleotides long and modified by adding a 50 cap and 30 poly-A tail. These primiRNAs are processed by a RNase III endonuclease-Drosha-into hairpin precursor miRNAs (pre-miRNAs) that are about 70 nucleotides long and are exported from the nucleus to the cytoplasm. The pre-miRNAs are subsequently processed by another RNase III endonuclease—Dicer—to form a mature miRNA duplex with 17–25 nucleotides. One strand (guide strand) of the mature miRNA duplex is incorporated into a miRNA-induced silencing complex that is composed of the argonaute (AGO) protein family and the transactivation-responsive RNA-binding proteins. miRNAs mediate the target mRNA repression or degradation via partial or complete base pairing to the 30 untranslated region of the target mRNA. Limited evidence suggests that miRNAs may also activate gene expression by binding the 50 promoter region of certain genes [5]. The other strand (passenger strand) of pre-miRNAs is either degraded or exported from cells by exosomes, microvesicles, high-density lipoprotein (HDL), and low-density lipoprotein (LDL), or RNA-binding proteins and then released into the circulation [6].
3. miRNAs AND CANCER The link between miRNAs and human diseases, particularly cancer, has been well established. Shortly after miRNA expression was detected in human
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cells in 2000, Calin et al. first reported that the deletion and downregulation of miR-15 and miR-16 were associated with high occurrence of chronic lymphocytic leukemia (CLL) [7]. miRNA genes may be located in either proteincoding genes (70%) or intergenic regions (30%) [8], but more than 50% of known miRNAs are located inside or near the cancer-related regions, such as fragile sites, regions of loss of heterozygosity, amplification, rearrangement, and common break points [9]. miRNA profiling of various tumor tissues and corresponding normal tissues has been performed in numerous studies [10]. miRNA expression signatures are tissue-, developmental stage-, and diseasespecific. Thus, miRNAs can not only be applied to discover unknown cancer origins, identify therapeutic targets, and classify cancer subtypes but can also be correlated with tumorigenesis, cancer progression, prognosis, and treatment response [11,12]. miRNAs in cancers can function as either tumor suppressors or oncogenes (oncomirs) depending on the tissue type, mRNA target, and microenvironment [12]. For example, miR-17-5p can act as a tumor suppressor in breast cancer by inhibiting AIB1 translation and inhibiting breast cancer cell proliferation, but in many other cancers, this miRNA is upregulated and acts as an oncogene [13]. miRNA dysregulation—either the decrease of tumor suppressor miRNAs or the overexpression of oncomirs—in cancer can be induced by a number of genetic and epigenetic mechanisms, including deletion, mutation, amplification, hypermethylation, and miRNA processing alterations [12]. miRNA dysregulation is involved in many critical pathways in cancer development such as those that regulate apoptosis, cell proliferation, epithelial-to-mesenchymal transition, and metastasis.
4. ORIGIN AND FUNCTION OF CIRCULATING miRNAs Although most miRNAs are intracellular, a large number of miRNAs have been observed outside cells in various body fluids [14–16]. The first evidence of circulating miRNAs was reported by Lawrie et al.[17] who showed that high levels of miR-155, miR-210, and miR-21 in serum were associated with diffuse large B-cell lymphoma (DLBCL), and high-serum levels of miR-21 were also associated with longer relapse-free survival. At the same time, Mitchell et al. found that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity, and provided direct evidence that tumor-derived miRNAs can enter the circulation [18]. Moreover, Chen et al. conducted a comprehensive analysis of serum miRNA expression pattern and demonstrated that miRNAs are present in the serum and plasma of humans and many animals. The miRNAs in serum are stable, reproducible, and consistent among
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individuals of the same species [19]. The global correlation between different tissue-specific miRNAs and circulating miRNAs in the normal population has been investigated, and liver miRNAs were more closely correlated with circulating miRNAs than were any other tissue-specific miRNAs (miRNAs from the placenta, testis, and brain tissue). On the other hand, Pritchard et al. reported a 50-fold increase of plasma/serum miRNA levels during perturbations and hemolysis of blood cells, indicating that blood cells are a major source of circulating miRNAs [20]. However, miRNAs may be released via a cellular selection mechanism, which would account for different extracellular and cellular miRNA profiles [21]. The origin of miRNAs remains elusive. Two major theories about the source of circulating miRNAs have been proposed: (1) passive leakage of cellular miRNAs into the circulation and (2) active and selective secretion of miRNAs in response to various stimuli as microvesicle-free miRNAs or via binding to cell-derived microvesicles. Passive leakage of miRNAs from broken cells may occur under pathological conditions such as tissue damage, apoptosis, inflammation, or tumor metastasis [16]. The majority of circulating miRNAs are chaperoned by various carriers including exosomes and other larger membrane-bound particles (e.g., apoptotic bodies, shedding vesicles, etc.) [22], HDL and LDL [23], and proteins such as Ago2 [24,25], which explains the stability of circulating miRNAs. These vesicle carriers are believed to protect circulating miRNAs from RNase degradation, mediate cell–cell communication via ligand–receptor interaction, and transport intracellular components, including miRNAs, to recipient cells via fusion or endocytosis [22]. In addition to regulating mRNAs posttranscriptionally, circulating miRNAs interact with the toll-like receptors of immune cells to stimulate the production of prometastatic inflammatory cytokines and to induce the protumor inflammatory [26]. Tumor-derived exosomal miRNAs were first isolated by Taylor et al. and were closely correlated with tissue miRNAs in paired samples, indicating that circulating exosomal miRNAs may be used as surrogate diagnostic markers for biopsy profiling [27,28]. The vast majority of circulating miRNAs are presented in the form of binding with Ago2 [25]. Moreover, posttranscriptional modification of miRNAs, such as methylation, adenylation, and uridylation has been reported [29–31], but whether these modifications exist in circulating miRNAs remains unclear.
5. DETECTION OF CIRCULATING miRNAs miRNAs are stable in cell culture supernatants and in many body fluids such as blood, urine, saliva, milk, and pleural effusions [18,19,32].
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Previous studies showed that higher concentrations of miRNAs can be extracted from whole blood samples than from serum and plasma, but whole blood sample may increase the background signal or interfere with immunoassays or downstream molecular assays [24]. Therefore, serum and plasma miRNAs are more commonly measured in current biomarker studies [33]. A pilot study reported that circulating miRNA expression levels in plasma were highly correlated with those in serum [34]. However, another paper demonstrated a considerable discrepancy between the circulating miRNA expression pattern in plasma and serum [35]. A recent study reported higher miRNA concentrations in sera than in corresponding plasma samples, suggesting that the coagulation process may affect the spectrum of circulating miRNAs in blood [36]. The correlation between plasma and serum miRNAs remains controversial. Therefore, specimen types should not be mixed, and the type of specimen should be consistent in the same study. Many techniques have been applied in measuring miRNA levels, including Northern blotting, in situ hybridization, quantitative reverse transcription polymerase chain reaction (qRT-PCR), microarray, and deep sequencing; the latter three techniques have been widely adopted in recent studies. These three techniques are summarized in Table 1 [37,38] and described in further detail in subsequent sections. The detection of circulating miRNAs in serum or plasma deals with extremely low initial amount of samples, which demands high performance in sample preparation and RNA extractions. Many preanalytical factors may affect circulating miRNA levels. For example, the centrifugal protocols, abnormal increases of blood cell and white blood cell counts in disease status, and hemolysis have remarkable influence on circulating miRNA measurements [20,25]. Current commercially available products for RNA extraction include TRIzol (Life Technologies), miRNeasy (QIAGEN), and mirVana (Life Technologies). Enrichment of small RNAs is required for sequencing, during which the samples are subjected to fractionation and recovered by electrophoresis and purification.
5.1. qRT-PCR qRT-PCR is the gold standard for quantifying circulating miRNAs with high sensitivity and specificity and with a wide analytical measurement range [34,39]. Two methods, stem loop primer RT-PCR and poly-A tailed RT-PCR, were developed to amplify circulating miRNAs. Normalization is critical for miRNA quantification, and there is no standard internal control for circulating miRNAs. Some controls, such as U6 snRNA or housekeeping miRNAs, do not exist in cell-free conditions. Invariant miRNAs such as
Table 1 Comparison of main technologies in circulating miRNA detection Methods
Advantages
Disadvantages
Throughput Assay or platform
Vendor
Quantitative reverse transcriptionPCR
High sensitivity and specificity. Can be used for absolute quantification
Only medium throughput with respect to the number of samples processed per day
Semi-high
TaqMan individual assays
ABI
miRCURY LNA qPCR
Exiqon
TaqMan OpenArray
ABI
RNA required Cost
μg
Low/miR High/sample
2–5 days
Discovery and confirmation
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HiSeq 2000 (or Genome Analyzer IIX)
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miR-16 are commonly selected as endogenous controls because they are consistently and highly expressed in plasma and serum [17,40,41]. However, miR-16 levels may increase during metastasis [42]. Recently, spike-in nonhuman mature miRNA controls have been applied as normalization controls [18,34].
5.2. miRNA microarray High-throughput microarray-based analysis is less expensive than qRTPCR and miRNA sequencing but has lower specificity [43]. This method can be used to predict the function of each miRNA in combination with the gene expression data. The miRNAs are enzymatically or chemically labeled with fluorescent dyes and hybridized to capture probes on the microarray plate. The signals from hybridized probes are detected by a scanner. The hybridization is sequence specific, and the efficiency depends on the guanine–cytosine content and DNA-melting temperature. Cross-hybridization may occur between different miRNAs. This method can measure the relative abundance of miRNAs but not the absolute value.
5.3. Deep sequencing Massive parallel sequencing technology enables the profiling of all expressed miRNAs and the discovery of novel miRNAs and isomiRNAs that are generated from alternative processing [44–46]. Many sequencing platforms from various manufacturers are currently commercially available (Table 1) [47]. This complicated method requires multistep sample purification, a series of reactions including miRNA reverse transcription, enzymatic ligation of adaptors, and amplification before sequencing, as well as sophisticated computational analysis after sequencing [48,49]. qRT-PCR, microarray, and deep sequencing are compared in Table 1. The correlations between miRNA measurements from different platforms or even from the same platform using different products are not optimal [36,50,51]. More accurate and reproducible methods need to be developed, reagents need to be improved, and sample preparation and normalization should be standardized in the future.
6. CIRCULATING miRNAs AS CANCER BIOMARKERS The associations between circulating miRNAs and cancer development, progression, and treatment have been heavily investigated; more than 250 related articles have been published since 2008. In this section, we
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summarize the recent findings on miRNAs in serum or plasma as potential biomarkers for the diagnosis, prognosis, and treatment response of major cancer types. The diagnostic biomarkers include biomarkers for monitoring high-risk populations, detecting early stage cancer, and discriminating between benign and malignant diseases. The prognostic biomarkers are used for predicting disease outcome and estimating progression-/recurrence-free survival and overall survival. The treatment response predictive biomarkers can monitor sensitivity to therapy and treatment response and aid in making treatment decisions.
6.1. Prostate cancer The first evidence of circulating miRNAs as cancer biomarkers was observed in prostate cancer in 2008. Mitchell et al. found that miR-141 levels were higher in men with advanced prostate cancer than in healthy men [18]. A subsequent study showed that plasma miR-141 levels were correlated with the levels of prostate specific antigen (PSA), circulating tumor cells, and lactate dehydrogenase [52]. The findings on miRNAs in prostate cancer are summarized in Table 2[18,53–64]. There is a significant differential expression pattern of miRNAs in prostate cancer. For example, miR-141 [18,55,57,62,64] and miR-375 [55,57,62,64] are upregulated in metastatic prostate cancer whereas elevated miR-21 and miR-221 levels are significantly associated with localized prostate cancer [54,58,61,63]. We have recently demonstrated that miR-221 and miR-222 are highly expressed in PC-3 cells. Inhibition of miR-221 or miR-222 leads to reduced cell proliferation and migration and increased apoptosis in prostate cancer cells [65]. Currently, prostate cancer prognosis is still based on PSA levels, Gleason score, and tumor stage [66]. In 2011, Brase et al. first reported that miR-141, miR-200b, and miR-375 levels were elevated in men with increasing stage and Gleason score [62]. miRNAs have shown promise in refining clinical variables for predicting prognosis in prostate cancer patients. Yet evidence of the utility of miRNAs in the early diagnosis of prostate cancer and monitoring therapeutic response is limited. Further studies are needed to identify those miRNAs and determine their quantitative cutoff values for predicting the prognosis of prostate cancer.
6.2. Breast cancer Two serum-based tumor biomarkers (CA15-3 and carcinoembryonic antigen, CEA) [67] and circulating tumor cells [68] are used for the prognostic assessment of advanced breast cancer, but no circulating biomarker has yet
Table 2 Potential circulating miRNAs as biomarkers of prostate cancer Type of biomarker
Upregulated miRNAs
Downregulated miRNAs
Sample Method
Study design
References
Serum qRT-PCR
25 metastatic PCa vs. 25 controls
[18]
miR-16, -92a, -103, -107, -197, -34b, -328, -485-3p, -486-5p, -92b, -574-3p, -636, -640, -766, -885-5p
Serum MiR microarray/ RT-PCR
5 PCa vs. 8 controls (other cancers tested)
[53]
miR-221
Plasma qRT-PCR
28 PCa vs. 20 controls
[54]
miR-141, -298, -346, -375
Serum qRT-PCR
25 metastatic PCa vs. 25 controls
[55]
Diagnostic miR-100, -125b, -141, -143, -296
let-7e, let-7c
Plasma Microarray/ qRT-PCR
25 PCa vs. 17 BPH, then validation in [56] 80 PCa, 44 BPH, and 54 HI
miR-375, -141
miR-181a-2
Plasma qRT-PCR
78 PCa vs. 28 controls, then 16 metastatic vs. 55 nonmetastatic
[57]
Plasma qRT-PCR
51 PCa (18 localized, 8 locally advanced, 25 metastatic) vs. 20 HI
[58]
miR-21, -221 miR-93, -106a, -874, -1207
miR-24, -26b, Serum Multiplexed -30c, -223 qRT-PCR
36 PCa (12 low risk, 12 medium risk, [59] 12 high risk) vs. 12 controls
miR-26a, -195, let-7i, -16
Serum qRT-PCR
45 (37 localized, 8 metastatic) PCa vs. [60] 38 controls (18 BPH, 20 HI)
miR-20a, -21
Plasma qRT-PCR
82 PCa assessing association with CAPRA score
Serum qRT-PCR
7 metastatic PCa vs. 14 localized PCa [62]
Serum qRT-PCR
50 PCa (20 localized, 20 ADPC, 10 HRPC) vs. 6 BPH
[63] [57]
Prognostic miR-9*, -141, -200b, -375, -516a miR-21
[61]
miR-375, -141
miR-181a-2
Plasma qRT-PCR
78 PCa vs. 28 controls, then 16 metastatic vs. 55 nonmetastatic
miR-375, -141, -378*
miR-409-3p
Serum microarray, qRT-PCR
26 metastatic PCa vs. 28 localized Pca [64]
*Where one hairpin miR precursor gives rise to two mature miRs, one from each arm, an asterisk is used to denote the least predominant form. ADPC: androgen-dependent prostate cancer; HRPC: hormone refractory prostate cancer; BPH: benign prostatic hypertrophy; HI: healthy individuals; PCa: prostate cancer; CAPRA: Cancer of the Prostate Risk Assessment.
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been recommended for determining diagnosis, assessing prognosis, or monitoring therapeutic response. Mammography [69] remains the primary choice for breast cancer screening, but the costs are high and the sensitivity is low. Mammography is limited to high-risk populations only and is not recommend for young women without a family history of cancer. There is an urgent need for rapid and reliable blood-based assays for assessing diagnosis, prognosis, and treatment response in breast cancer. Circulating miRNAs have been extensively studied in breast cancer (Table 3) [70–87]. In particular, miR-155 [76,77,84] and miR-21 [71,73] were found to be elevated in the serum of primary breast cancer patients. Moreover, miR-155 is related to progesterone receptor status [76] and can predict chemotherapy response [77]. Recent in vitro evidence indicates that suppression of miR-221 and miR-222 increases the sensitivity of estrogen receptorpositive MCF-7 breast cancer cells to tamoxifen [88]. In addition, circulating miR-125b and miR-210 were reported associated with the chemoresistance in breast cancer patients. Another emerging area in breast cancer is the identification of miRNAs in circulating tumor cells; a dozen miRNAs were found to be more abundant in circulating tumor cells, which may be associated with metastasis [83,89].
6.3. Lung cancer Lung cancer is the leading cause of cancer mortality, and most studies of lung cancer focus on nonsmall cell lung cancer (NSCLC), which accounts for more than 80% of cases. Two studies have been conducted to comprehensively screen and identify circulating miRNAs as early detection markers in asymptomatic patients [90,91]. Bianchi et al. developed a test based on 34 miRNAs from serum to detect NSCLC [90]. Boeri et al. identified a group of plasma miRNAs that could both predict the development of lung cancer in asymptomatic patients (1–2 years prior to diagnosis) and detect new cases [91]. The study by Boeri et al. also showed that miR-197, miR-221, miR-486-5p, miR-140-5p, miR-106a, and miR-16 were associated with the aggressiveness of the disease. There was no significant correlation between plasma miRNAs and serum miRNAs, and this discrepancy was also reported in a study by Heegaard et al.[35]. Numerous case–control studies have been conducted in lung cancer, and the levels of several miRNAs were shown to be either higher or lower in cases than in controls, although these results were not entirely consistent and need to be validated in independent populations (Table 4). miR-21 appears to be the most
Table 3 Potential circulating miRNAs as biomarkers of breast cancer Type of biomarker
Upregulated miRNAs
Diagnostic
Downregulated miRNAs
Method
Study design
References
miR-376c, -409-3p, -801, -148b
Plasma
Microarray/ qRT-PCR
127 BC cases and 80 controls
[70]
Diagnostic
miR-10b, -21, -125b, -145, -155, -191, -382
Serum
qRT-PCR
61 breast cancer and 10 controls
[71]
Diagnostic
miR-451
miR-145
Plasma
qRT-PCR
240 breast cancer and 150 controls; correlated [72] to PBC
Diagnostic
miR-21
miR-92a
Diagnostic
miR-215, -299-5p, -411
Diagnostic
miR-30a
Serum
qRT-PCR
100 PBC vs. 20 controls
[73]
Serum
Microarray/ qRT-PCR
75 BC (including 16-untreated MBC vs. 20 controls); increased in MBC
[74]
Plasma
qRT-PCR
100 PBC vs. 64 controls; decreased in PBC; correlated to receptor status
[75]
Diagnostic
miR-155
Serum
qRT-PCR
8 BC; correlated to progesterone receptor status
[76]
Diagnostic
miR-155
Serum
qRT-PCR
103 PBC vs. 55 controls; increased in PBC
[77]
Diagnostic
miR-195, let-7a
Whole blood
qRT-PCR
83 patients vs. 44 controls; increased in patients
[78]
let-7a
Correlated with lymph node positivity
miR-10b, -21 Diagnostic
miR-425*, -302b
Diagnostic
miR-202
Higher in ER negative compared to ER positive let-7c
Plasma
Microarray/ qRT-PCR
45 BC vs. 45 controls
[79]
Whole blood
Microarray/ qRT-PCR
92 early stage BC vs. 81 controls
[80]
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Sample
miR-122
Serum
Sequencing/ qRT-PCR
68 stages II–III and inflammatory BC patients [81] who received neoadjuvant chemotherapy; predicts BC metastasis in early stage patients
Diagnostic and prognostic
miR-21
Serum
qRT-PCR
102 BC patients with different disease stages vs. 20 controls; increased in BC patients; increased in metastasis
[82]
Diagnostic and prognostic
miR-141, -200a, -200b, miR-768-3p -200c, -203, -210, -375, -801
Plasma
Microarray/ qRT-PCR
61 CTC-positive, 72 CTC-negative; 60 CTC-low MBC cases, and 76 controls; increased in MBC; correlated to CTC status
[83]
Diagnostic and prognostic
miR-10b, -34a
Serum
Microarray/ qRT-PCR
59 PBC vs. 30 MBC vs. 29 controls; increased [84] in MBC compared to PBC and controls
Diagnostic and prognostic
miR-155
Prognostic
miR-10b, 373
Plasma
qRT-PCR
35 MBC, 25 PBC, 10 control; increased in MBC
[85]
Predictive
miR-155
Serum
qRT-PCR
29 BC patients with surgery and chemotherapy vs. 103 PBC decrease after surgery and chemotherapy
[77]
Predictive
miR-125b
Serum
qRT-PCR
56 invasive ductal carcinoma BC; correlated with chemoresistance
[86]
Predictive
miR-210
Plasma
qRT-PCR
39 preoperative; 30 postoperative; 43 PBC without treatment; correlated with trastuzumab resistance, tumor presence, and lymph node metastasis
[87]
Increased in PBC and MBC compared to controls; increased in PBC compared to MBC
*Where one hairpin miR precursor give rise to two mature miRs, one from each arm, an asterisk is used to denote the least predominant form. CTC: circulating tumor cells; BC: breast cancer; PBC: primary breast cancer; MBC: metastatic breast cancer; ER: estrogen receptor.
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Prognostic
Table 4 Potential circulating miRNAs as biomarkers of lung cancer Type of biomarker
Upregulated miRNAs
Downregulated miRNAs
Method
Sample
Microarray/ Plasma qRT-PCR
Study design
References
Diagnostic
miR-21, -19b, -30c, -92a, -17, -28-3p, -106a, -140-5p, -451, -660,
91 LC and 81 controls; increased in LC [91]
Prognostic
miR-197, -221, -486-5p, -140-5p, miR-16 -106a
Diagnostic
34 different miRNAs
Microarray/ Serum qRT-PCR
59 patients vs. 69 controls, identify [90] NSCLCs from asymptomatic population
Diagnostic
miR-25, -223
Sequencing/ Serum qRT-PCR
152 LC vs. 75 controls; increased in LC [19]
Diagnostic
miR-574-5p, -1254
qRT-PCR
Serum
33 NSCLC vs. 42 controls; increased in [92] NSCLC
Diagnostic
miR-29c
let-7a, miR-17-5p, -27a, -106a, -146b, -155, -221
qRT-PCR
Plasma and serum
220 early stage NSCLC vs. 220 controls; [35] correlated with NSCLC; differences between distinct ethnic groups
Prognostic
miR-30d, -486
miR-1, -499
Sequencing/ Serum qRT-PCR
303 patients and 113 death; correlated to [93] poorer overall survival
Diagnostic
miR-16, -452*, -518a-5p, -574-5p, -593*, -663, -718, -1228*, -1972, -2114*
qRT-PCR
Serum
8 cancer patients vs. 6 controls; decreased [94] in pre- and postdiagnostic samples
Diagnostic
miR-21, -24, -30d, -205
qRT-PCR
Serum
82 pre- vs. postoperative LC, 50 healthy [95] controls; increased in LC and early stage LC
Increased in aggressive disease
[91]
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Prognostic
miR-21, -30d
Prognostic
Correlated to poorer overall survival miR-21, -24 miR-141, -200c
Decrease in postoperative samples
Diagnostic
miR-21
qRT-PCR
Serum
70 NSCLC vs. 44 controls; correlated with NSCLC
Prognostic
miR-21
Diagnostic
miR-21, -182, -210
miR-126, -486-5p
Microarray/ Plasma qRT-PCR
28 stage I NSCLC, 58 NSCLC, vs. 29 controls
[97]
Diagnostic
miR-21, -155
miR-145
qRT-PCR
Plasma
62 patients vs. 60 controls; and 34 malignant patients vs. 30 benign vs. 32 controls
[98]
Diagnostic and predictive
miR-21
qRT-PCR
Plasma
63 NSCLC vs. 30 controls; increased in [99] NSCLC; predictive for platinum-based chemotherapy
Diagnostic, prognostic, and predictive
miR-155, -182, -197
qRT-PCR
Plasma
74 LC patients and 68 controls; increased [100] in cases; increased in patients with metastasis; decreased in patients responding to therapy
[96]
Lymph node metastasis and advanced clinical stage of NSCLC
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*Where one hairpin miR precursor give rise to two mature miRs, one from each arm, an asterisk is used to denote the least predominant form. NSCLC: nonsmall cell lung cancer; LC: lung cancer.
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prominent biomarker in lung cancer and has been shown in several papers including one of our unpublished papers to be correlated with the risk of NSCLC, advanced disease stage and metastasis, poor overall survival, and chemotherapy response [95–99].
6.4. Colorectal cancer Colorectal cancer (CRC) is the third most common and deadly cancer in the United States [101,102]. The current tumor marker for CRC is serum CEA levels, but this blood test is neither sensitive nor specific. Recent studies of circulating miRNAs have focused on early diagnosis and prognosis (Table 5). In these studies, miR-29a was differentially expressed in CRC patients compared to controls and was also associated with liver metastasis [41,103,108]. miR-141 was also significantly associated with metastasis and poor prognosis [105]. Altered levels of miR-92 can differentiate CRC from gastric cancer [104]. These miRNAs are promising diagnostic and prognostic biomarkers in CRC.
6.5. Hematologic cancers The first report of circulating miRNAs was from Lawrie et al.[17] who showed that serum levels of miR-21, miR-155, and miR-210 were significantly higher in DLBCL patients than in healthy controls (Table 6). Furthermore, higher serum levels of miR-21 were also correlated with longer relapse-free survival in these patients [17]. Of note, the finding of elevated miR-155 in DLBCL was recently validated by another group [110]. The prognostic value of miR-155 was reported by Ferrajoli et al., who found that higher serum levels of miR-155 were associated with increased disease progression in CLL [111]. miR-92a was significantly decreased in the plasma of patients with non-Hodgkin lymphoma [112] and acute myelogenous leukemia [113]. Three publications have indicated that circulating miRNAs may become predictive markers for response to chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone. Plasma levels of miR-155 were shown to be higher in CLL patients who did not achieve a complete response than in patients who did achieve a complete response [111]. Elevated plasma miR-221 levels were associated with shorter after chemotherapy in patients with natural killer/T-cell lymphoma, suggesting poor treatment response [114]. Low plasma levels of miR-92a were correlated with shorter relapse-free survival in non-Hodgkin lymphoma patients [113] (Table 6).
Table 5 Potential circulating miRNAs as biomarkers of colorectal cancer Type of biomarker
Upregulated miRNAs
Diagnostic
miR-15b, -18a, -19a, -29a, -335
Downregulated miRNAs
Method
Microarray/ Plasma 123 patients vs. 73 controls; increased in CRC qRT-PCR
miR-18a Diagnostic
miR-17-3p
Sample Study design
References
[103]
Increased advanced adenomas compared to controls qRT-PCR
[104]
Increase in CRC; decrease in postoperative samples; can differentiate CRC from gastric cancer IBD and controls
Prognostic
miR-141
qRT-PCR
Plasma 258 stage IV colon cancer; correlated to poor survival
Diagnostic
miR-92a, -29a
qRT-PCR
Plasma 130 CRC vs. 37 adenoma vs. 59 controls; increased in CRC [41] and advanced adenomas compared to controls
Diagnostic
miR-21
Microarray/ Plasma 50 CRC patients vs. 50 controls; increased in CRC qRT-PCR
Diagnostic and prognostic
miR-221
qRT-PCR
Plasma 103 CRC patients and 37 healthy normal controls; increased in [107] CRC patients; correlated to OS and p53 score
Diagnostic and prognostic
miR-29a
qRT-PCR
Serum 58 CRC with liver metastasis vs. 56 CRC without metastasis; [108] increased in CRC with liver metastasis vs. nonmetastatic CRC
Diagnostic
miR-601, -760 qRT-PCR
CRC: colorectal cancer; OS: overall survival; IBD: inflammatory bowel disease.
Plasma 100 CRC, 43 advanced adenoma, 68 controls; decreased in CRC and advanced adenomas compared to controls
[105]
[106]
[109]
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miR-92
Plasma 95 CRC, 20 gastric cancer, 20 IBD, 55 controls; increase in CRC; decrease in postoperative samples
Table 6 Potential circulating miRNAs as biomarkers of hematologic cancers Type of biomarker
Upregulated miRNAs
Downregulated miRNAs
Cancer
Sample Method
Study design
References
75 DLBCL vs. 75 controls; increased in DLBCL
[110]
Diagnostic
miR-15a, -16-1, -29c, -155
miR-34a
DLBCL
Serum qRT-PCR
Diagnostic
let-7b, miR- 523
let-7d, miR-150, -339, -342
AML
Plasma Microarray/ 20 AML without CR, 20 controls, 20 AML with qRT-PCR CR; correlated to AML
Prognostic
miR-150, -342
Prognostic and predictive
miR-155
CLL
Diagnostic, prognostic, and predictive
miR-221
NK/TPlasma qRT-PCR cell lymphoma
79 patients and 37 normal subjects; increased in lymphoma; correlated to shorter OS
[114]
Diagnostic
miR-21, -155, -210
Serum qRT-PCR
60 patients vs. 43 controls; increased in DLBCL
[17]
Prognostic
miR-21
De novo DLBCL AML
Plasma Microarray/ 77 AML patients vs. 16 controls; decreased in AML [112] qRT-PCR
AML
Serum Sequencing qRT-PCR
miR-92a
Diagnostic
miR-10a-5p, -93-5p, -129-5p, -155-5p, -181b-5p, -320d
Prognostic
miR-181b-5p
Diagnostic and predictive
Increased in cases with CR compared to those without CR Plasma qRT-PCR
228 CLL patients; increased in CLL progression and [111] poor treatment response
High levels correlated with improved RFS
140 AML patients and 135 controls; increased in AML patients
[116]
Correlates to shorter OS miR-92a
NHL
[113] Plasma Microarray/ 126 patients vs. 37 controls; decreased in NHL; qRT-PCR further decreased in patients with shorter RFS after chemotherapy
DLBCL: diffuse large B-cell lymphoma; RFS: relapse-free survival; OS: overall survival; CLL: chronic lymphocytic leukemia; NHL: non-Hodgkin lymphomas; AML: acute myelogenous leukemia; CR: complete remission.
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7. CONCLUSIONS Current cancer biomarkers available in clinics are mostly proteins, and the U.S. Food and Drug Administration has not approved any new biomarkers in more than a decade. These biomarkers have complex compositions and are rendered vulnerable by many posttranslational modifications. With the low abundance and numerous sequence variations of protein biomarkers, the development of assays for protein biomarkers is challenging, often resulting in low sensitivity and specificity for the assays. Circulating miRNAs are being developed as promising cancer biomarkers with great translational potential, although technical issues still exist. Circulating miRNAs are extremely stable in RNase-rich body fluids. With the rapid advances in measurement methods, circulating miRNAs are readily detected even in extremely small amounts. Numerous studies in a wide spectrum of cancers have provided solid evidence that circulating miRNAs are reliable and sensitive in predicting disease occurrence and detecting changes in pathology, disease recurrence and progression, and treatment response. Disease-associated miRNA changes may be detected years prior to disease onset in asymptomatic patients, suggesting the value of these miRNAs in screening and early detection. However, several issues still hamper the clinical application of circulating miRNAs in cancers. First, most differentially detected miRNAs are not limited to a single cancer type, but are detected consistently across different cancer types and various pathologic statuses. For example, serum miR-21 is elevated in breast, lung, colorectal, and hematologic cancer and is also correlated with prognosis and treatment response. These nontissue-, organ-, or cancer type-specific miRNAs raise concerns about the specificity of the test and limits to its application. Cancer typespecific circulating miRNAs are desired. In contrast, several aberrant miRNAs can be seen in one cancer type and even at different stages of same cancer. Most likely, the combination of a panel of miRNAs, rather than a single miRNA, will be needed to reach high specificity. Second, considering the heterogeneous origin of circulating miRNAs, distinguishing tumorderived miRNAs, and avoiding the interference of miRNAs from other resources such as blood cells and the liver is critical in establishing the concordance between miRNA expression in tumor tissue and in the circulation. Third, the reported association of circulating miRNAs with cancer diagnosis and prognosis is confounded by many factors, such as age, sex, ethnicity, and types of sample (i.e., plasma, serum, or whole blood). The vast majority of
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recent studies were conducted retrospectively with limited sample sizes, and the results are not consistent and need to be validated prospectively in large independent populations. Although the levels of circulating miRNAs have been shown to be either elevated or reduced in cancer patients, only those with elevated expression are likely to be used as early detection and diagnosis markers, considering the practical issues in clinical laboratories. Finally, various approaches have been applied to identify and quantify circulating miRNAs. There are still technical challenges to preparing samples, choosing normalization controls, and normalizing data. There is a lack of quality control program in monitoring the performance and proficiency of these assays. The assays will need to be standardized for different platforms to allow comparisons of the identified circulating miRNAs across different laboratories. The cutoff values of aberrant miRNAs in each type of cancer need to be established for the ease of clinical interpretation.
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