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Circulating microRNAs: potential biomarkers for common malignancies

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs (ncRNAs), which regulate gene expression at the post-transcriptional level. They are approximately 22 nucleotide sequences in length and have been predicted to control expression of up to 30–60% of all protein-coding genes in mammals. Considering this wide involvement in gene control, aberrant miRNA expression has a strong association with the presence and progression of a disease, hence generating much anticipation in using miRNAs as biomarkers for the diagnosis and prognosis of human cancers. The majority of these miRNAs are intracellular, but recently they have been discovered in bodily fluids. This review will provide an insight into these circulatory miRNA molecules and discuss their potential as cancer biomarkers. Keywords: cancer • circulating microRNA • diagnostic biomarkers • prognostic biomarkers

One of the current challenges facing clinicians diagnosing malignancies is the lack of robust biomarkers for early detection and monitoring of disease progression. Over the last 5 years, the appeal of using circulating microRNAs (miRNAs) from sources such as blood, saliva, plasma and other bodily fluids has gathered much interest. miRNAs are small noncoding RNAs of approximately 22 nucleotides in length and are important controllers of gene expression [1–3] . They are processed from much larger RNA precursors through a series of enzymatic steps to form a small double-stranded RNA structure [4] . It is this small RNA, which can be detected in the blood of patients. The first realization that miRNAs could be found in serum was made by Chim et al. [5] who detected stable plasma miRNAs that could distinguish between women still carrying and those that had already given birth. They identified that miR-141, miR-149, miR299–5p and miR-135b increased in concentration throughout pregnancy. This revelation opened up a new class of serum markers for pregnancy monitoring. Similarly, Lawrie et al. published the existence of miRNAs in

10.2217/BMM.14.102 © Nahm Tran and Samantha Khoury

Samantha Khoury1,2 & Nham Tran*,2,3 1 School of Medical & Molecular Biosciences, Faculty of Science, University of Technology, Sydney, NSW, Australia 2 Centre for Health Technologies, Faculty of Engineering & Information Technology University of Technology, Sydney, NSW, Australia 3 The Sydney Head & Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, Sydney, NSW, Australia *Author for correspondence: nham.tran@ uts.edu.au

the serum of patients with diffuse large B-cell lymphoma. This study identified miR-155, miR-21 and miR-210 as being overexpressed in patients with lymphoma when compared with healthy controls [6] . Further evidence of serum miRNAs as cancer markers was provided by Mitchell et al. whereby serum miR-141 was rapidly released into the blood stream and could distinguish healthy individuals from patients with advanced prostate cancer [7] . Since these original reports, multiple studies in a wide range of malignancies have shown unique miRNA biomarkers in the plasma or serum of cancer patients. The potential of miRNAs to act as both diagnostic and prognostic cancer biomarkers relies heavily on their robust nature and high stability in bodily fluids. It has been shown that serum miRNAs can remain stable after exposure to very high or low pH levels, extended storage, boiling temperatures and up to ten freeze-thaw cycles [8] . Furthermore, miRNAs were detected in the serum of archived breast cancer samples stored for over a decade [9] . This stability was further exemplified when miRNAs could be detected in unrefrigerated dried serum blots after a period of 5 months

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The stability is due to several factors, which protects them from both cellular and environmental degradation. First, miRNAs can be packaged in microvesicles such as microparticles exosomes, and apoptotic bodies [11,12] . Second, most miRNAs are bound to RNA binding proteins such as AGO2 [13] , or lipoprotein complexes such as HDLs [14] . Given all these characteristics and the fact that circulating miRNAs can be collected from blood, they have the potential to be developed into next-generation b iomarkers for cancer screening.

The origin of circulating miRNAs Extracellular miRNAs present in human bodily fluids are remarkably stable and are protected from endogenous RNase activity [15] . Currently, there are several models that can explain the stability of the circulating miRNAs. They can be released from cells in membrane-bound vesicles, which prevents degradation from serum RNase. These vesicles include exosomes which are 50–90 nm [16] and other membrane-bound particles such as microparticles which are much larger at 1 μm [17] . It is generally known that cells release exosomes which harbor miRNAs and other bioactive compounds [12,18] . These exosomes transfer the RNA contents to modulate biological activity in the recipient cell [19–21] . It is now accepted that exosomes transport information in the form of RNA and this RNA has an effect on cell activity [22] . Not all miRNAs found in circulation are derived from transporting vesicles. It was found that a significant portion of circulating miRNAs in human plasma and serum was associated with protein complexes rather than with vesicles [13] . These miRNAs were bound to and stabilized by AGO2 and thus were protected from degradation by serum RNases. Other RNA-binding proteins such as HDL [14] and NPM1 [23] were shown to also bind to miRNAs in circulation. Currently, there is no evidence that AGO2 or NPM1associated miRNAs are actively released from cancer cells. The last source of circulating miRNAs is from leakage from damaged cells due to chronic inflammation, tissue injury and cell death, or from cells with a short half-life [24,25] . The role of circulating miRNAs in cancer The interaction of miRNAs with cellular messenger RNAs (mRNA) is well documented [4,26] and miRNA expression is strongly deregulated in almost all human malignancies [27,28] . The majority of miRNAs target the 3′ untranslated region (UTR) of the mRNA. Regulation of the target RNA is dependent on the base pairing between the miRNA and its target. Perfect binding induces cleavage of the target RNA, whereas

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imperfect base pairing elicits polydeadenylation or translational repression. miRNAs can modulate the development and spread of a tumor by playing multiple roles as either oncogenes or tumor suppressors depending on the cancer type [29] . Aberrant regulation of miRNA expression alters important events leading to cancer, including differentiation [30] , proliferation [31] , apoptosis, migration, invasion and metastasis [32] and chemotherapy resistance [33–35] . More recently, miRNAs have been shown to have a paracrine effect on tumor growth. It was found that miR-21 and 29a excreted by tumor cell exosomes are attracted and bind to TLR8 from surrounding immune cells. They activate these receptors in a paracrine loop which releases cytokines that increases cell proliferation and metastatic potential of the cell [36,37] . Through this association, these secreted miRNAs are not only regulators of the tumor microenvironment but are important in the growth and spread of the tumor. Aside from their functional roles in tumorigenesis, these circulating miRNAs represent a less invasive alternative for diagnostic testing. Given the lack of robust clinical markers for many cancers, circulating miRNAs may have important implications for patient management as they can potentially predict tumor aggressiveness, metastasis and disease outcome [38] . This extensive review will highlight our current understanding of miRNAs as diagnostic biomarkers in some of the world’s most common malignancies. Colorectal cancer Though rare in underdeveloped countries, colorectal cancer (CRC) is among the leading causes of cancerrelated morbidity and mortality in western countries [39] . Evidence from national awareness programs [40] indicates that 60% of CRCs may be preventable through a combination of early screening procedures and lifestyle modifications. Clinically, there are no guidelines for dietary or lifestyle changes that can assist in primary prevention of CRC. Research is now supporting the development of secondary prevention methods which aim to detect and remove lesions at an early or premalignant stage. Currently, the following methods exist for patients: widespread screening by regular fecal occult blood testing is a method that can reduce CRC mortality by 15–20% and does increase the proportion of early cancers detected [41–44] . In the United States, annual fecal occult blood testing is recommended after the age of 50 years. However, these tests currently lack sensitivity and specificity; colonoscopy does remain the gold standard but requires expertise, is expensive and carries risks. Furthermore, most countries lack the resources to offer this form of screening; flexible sigmoidoscopy is an alternative option and

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Circulating microRNAs

has been shown to reduce overall colorectal cancer mortality by approximately 28% [45–47] . Screening by molecular genetic analysis is an exciting prospect but there are no assays currently available. In order to develop a more effective method of secondary prevention, several studies have explored the potential of circulating miRNAs as low-invasive diagnostic biomarkers. These studies, discussed below, explore circulating miRNA levels across serum and plasma of CRC patients. The first report to describe circulating miRNA levels in CRC patients utilized Solexa Sequencing [8] . The study showed that CRC patients had a different serum miRNA profile when compared with healthy subjects. In total, 69 miRNAs were detected in colorectal cancer serum. These colorectal cancer patients shared a large number of serum miRNAs such as miR-134, miR-146a, miR-221, miR-222 and miR23a with lung cancer patients. Fourteen of the 69 serum miRNAs were unique to CRC and included miR-485–5p, miR-361–3p, miR-326, miR-487b. These findings provided the first evidence to show that serum miRNAs could potentially contain a diagnostic fingerprint for CRC disease. There have been numerous studies since this report and for this review we will focus on studies with larger patient numbers. The Ng et al. study showed that miR-17–3p and miR-92 were significantly upregulated in 180 patients and 50 controls. The plasma levels of these two markers were significantly reduced after surgery but only miR-92 could differentiate CRC from gastric cancer and inflammatory bowel disease [48] . Huang et al. identified 12 miRNAs (miR-134, miR146a, miR-17–3p, miR-181d, miR-191, miR-221, miR222, miR-223, miR-225, miR-229, miR-320 and miR92a) that had elevated expression in 100 patients with CRC and 37 colorectal adenomas when compared with 59 healthy patients. [49] . Similarly, miR-92 could discriminate between advanced colorectal adenomas and CRC, suggesting differential diagnosis using miRNAs was possible [49] . In a study to discover metastatic CRC makers, miR-141 was measured in an independent cohort of 156 plasma samples [50] . It was significantly associated with stage 4 colon cancer and metastases to the liver. It was suggested that plasma miR-141 might represent a biomarker that could complement CEA in the accurate detection of colon cancer with distant metastasis. The study by Giraldez et al. profiled 123 patients with sporadic colorectal neoplasia (63 with CRC and 60 with advanced adenomas) [51] . Six miRNAs (miR-18a, miR-19a, miR-19b, miR-15b, miR-29a and miR-335) were upregulated in patients with CRC and only miR18a was observed to be upregulated in patients with advanced adenomas. Vega et al. also identified elevated


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serum levels of both miR-18a and miR-29a in CRC patients [52] . In an attempt to discover more common miRNA markers, Luo et al. reanalyzed seven published miRNAs in a cohort of 80 CRC patients, 50 advanced adenomas and 194 neoplasm-free controls [53] . Surprisingly, not one of the published miRNAs showed any difference between the adenoma patients and neoplasm-free controls. However, the study did find nine miRNAs (miR-18a, miR-20a, miR-21, miR-29a, miR92a, miR-106b, miR-133a, miR-143, miR-145), which were differentially expressed in CRC patients when compared with control. The use of multiple classifiers is another approach for disease diagnosis. By using a Pearson’s test, positive correlations were observed in the tissue and blood levels for miR-193a-3p, miR-23a and miR-338–5p. This triple miRNA classifier was shown to be a strong candidate biomarker for the early detection of CRC [54] . The levels of serum miR-21 alone have the diagnostic power to discern advanced adenomas from primary CRCs [55] . The caveat for miR-21 is specificity with other cancers also overexpressing this miRNA. Recently, several studies have combined miR-21 with other miRNAs to increase the power of detection. The Lui study utilized over 330 serum samples to identify miR-92a as a co-marker with miR-21 for the early detection of CRC [56] . Recurrence of CRC is a major factor in the overall survival and it is well established that early recurrence of CRC is frequent within the first year of curative resection surgery [57] . The Menendez et al. study indicated that miR-21 can act as an independent predictor of recurrence and survival [58] . Several other studies showed that miR-15a, mir-103, miR-148a, miR-320a, miR-451, miR-596 [59] , miR-378 [60] and miR-29a/c [61] could be used to further predict recurrence in early-stage CRCs. MiR-29c was then demonstrated to inhibit cancer cell proliferation, migration and tumor growth but not invasion [62] . These studies provide strong evidence that circulating miRNAs have potential as biomarkers in CRC. In serum and plasma, it has been shown that the expression of certain miRNAs is indicative of the presence of CRC, the stage of CRC and early data suggest that they can also distinguish between a good or poor prognosis. Breast cancer Cancer of the breast is the leading malignant tumor in females. It is the second leading cause of cancer death among woman worldwide and the US National Cancer Institute estimates that the lifetime risk of a woman in the USA developing breast cancer is one in eight. The cancer may not produce obvious symptoms until relatively late, by which time it may have spread

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Review Khoury & Tran to other organs. As such, there has been a worldwide focus to identify breast cancers at an early stage. These initiatives involve women performing self-examination for the presence of a hard, immovable lump and then screening by a mammography. Despite these prevention strategies, all patients must undergo a biopsy or fine needle aspiration for a positive diagnosis. There have been two approaches to analyze circulating miRNA expression in breast cancer: validating single miRNA probes using qRT-PCR [9,63–71] or a genomewide approach such as oligonucleotide arrays or next-generation sequencing [72–76] . In order to use serum miRNAs as biomarkers, Cookson et al. examined the fundamental question, do circulating miRNA profiles actually resemble those of miRNAs within the tumors [77] . The study analyzed plasma and tissue from ten patients before and after resection of the tumor. In total, they detected 210 miRNAs and subsequently normalized their results to mean miRNA expression. It was found that overexpressed miRNAs in the serum matched those in the tumors, suggesting that circulating miRNAs are representative of the solid tumor. One of the first genome-wide studies to examine miRNA plasma expression was performed by Zhao et al. [76] . This study compared the plasma miRNA profiles of 20 early-stage breast cancer patients and 20 healthy controls. This analysis indicated that 15% of the miRNA population was differentially expressed with miR-425* and miR-302b highly expressed in early-stage breast cancer. Studies have also been extended to utilize next-generation sequencing extensively in cancer tissue and cell lines [78–80] . These studies have so far indicated that circulating miRNAs such as miR-21 could separate normal breast tissue samples from the most invasive ductal carcinoma in situ and invasive carcinomas. The stability of these miRNAs as biomarkers was highlighted by Zhu et al. who measured differential miRNA expression in 10-year-old archival human serum specimens [9] . In 13 patients with breast cancer and eight healthy patients, they detected miR-16, miR-145 and miR-155 and found them to have similar expression levels. Of interest, women in this group with progesterone receptor (PR) positive tumors had higher miR-155 expression than tumors that were negative for these receptors. These results suggested that serum miRNAs were detectable in archived serum samples and miR-155 may be differentially expressed in relation to the PR status of the patient. Subsequent to this report, Heneghan et al. [63] used qRT-PCR and quantified levels of seven candidate miRNAs in tissue and blood specimens of 148 patients with breast cancer and 44 age-matched, healthy con-

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trols. Increased levels of miR-195 and let-7a were reflective of the presence of a tumor. These two miRNAs were then assessed at a 2-week postoperative period and both were found to be decreased in expression. This study also correlated the miRNA expression with specific clinicopathological variables such as nodal status and estrogen receptor status. These findings suggested that miRNAs can be exploited to detect early pathological signs associated with breast cancer and monitor disease progression. Individual miRNAs such as miR-155 have been strongly studied in breast cancer tissue [81] . To further clarify its expression and application for diagnosis, Sun et al. [82] looked into serum levels of miR-155 in a group of 103 breast cancer patients and 55 controls. Using qRT-PCR, they demonstrated that miR-155 had significantly increased levels in the cancer sera. Furthermore, they collected 29 sera samples from after surgery and after four cycles of chemotherapy to track the effects of clinical treatment on serum miR155. Surprisingly, a decrease in miR-155 was found; whereas the concentrations of CA15–3, CEA and tissue polypeptide-specific antigen did not change. These findings suggested that patients who responded well to therapy or had stable disease after therapy exhibited low levels of serum miR-155. This trend of high-to-low miR-155 levels after surgery and chemotherapy raises the possibility of using this marker as an indicator for treatment response. Using plasma not serum, Lui et al. showed that miR-155 was overexpressed more than twofold when compared with normal tissue whereas let-7b, miR381, miR-10b, miR-125a-5p, miR-335, miR-205 and miR-145 were downregulated [83] . This analysis was then extended to archived serum, which indicated low expression of miR-205 and an overexpression of miR-155. The study found no significant correlation between miR-205 and clinical parameters. In contrast, miR-155 showed a strong correlation to clinical stage, ki-67 staining and a negative correlation with p53 status. In a more recent study, Wang et al. [84] examined miR-182 levels in the serum of 46 breast cancer patients and 58 controls using qRT-PCR. The results showed that serum miR-182 levels were significantly higher in cancer patients. Interestingly, the serum levels of miR182 in estrogen receptor (ER) positive patients were considerably lower compared with those in the ERnegative patients. Similarly, serum miR-182 levels in the progesterone receptor (PR) positive patients were lower when compared with those in the PR-negative patients. The current study suggested miR-182 might be a valuable biomarker for the diagnosis of breast cancer.



A study by Kim et al. [85] prepared modified zwitterionic sulfobetaine-conjugated immunobeads using CD83 as a candidate protein marker for breast cancer derived microvesicles. The zwitterionic immunobeads are very efficient at isolating tumor derived-mirovesicles TMVs from clinical plasma samples by suppressing nonspecific protein binding than conventional immunobeads. They were able to distinguish earlystage breast cancer from benign patients using this modified protocol in comparison with conventional immunobead methods. Furthermore, the expression levels of six miRNAs from tumor-derived microvesicles were significantly overexpressed in the microvesicles from the CD83-positive cohort. Their results show an alternative approach to comparing the miRNA expression profile between total plasma and tumor-derived microvesicles. The use of combination markers may be a better approach than using a single miRNA. To address the use of multiple miRNA markers, Ng et al. [86] conducted TaqMan-based miRNA profiling on breast cancer tumors (n = 5), adjacent nontumors (n = 5), plasma from breast cancer patients (n = 260) and plasma from matched healthy controls (n = 170). Their results showed eight miRNAs were elevated (miR16, miR-21, miR-27a, miR-150, miR-191, miR-200c, miR-210, miR-451) and miR-145 which was reduced in both the plasma and tumor tissue of breast cancer patients. In the validation cohort, miR-16, miR-21 and miR-451, were significantly decreased after surgery. They focused on using the combination of miR145 and miR-451 to discriminate breast cancer from healthy controls. A panel of serum markers has also been investigated by Mar Aguilar et al. [87] using a Mexican population cohort. MiRNA profiling using qRT-PCR on serum from patients (n = 61) found that miR-10b, miR-21, miR-125b, miR-145, miR-155 miR-191 and miR-382 had different expression patterns compared with healthy controls. ROC curve analysis also revealed that miR-145, miR-155 and miR-382 showed better sensitivity and specificity in combination with each other rather than as individual markers. In a cohort of Chinese breast cancer patients, Chen et al. [88] compared paired serum and tumor miRNA profiles using microarrays or locked nucleic acid realtime PCR panels. Statistically significant serum miRNAs were validated in an independent set of serum samples from patients (n = 164) and healthy controls (n = 123). The most significant, differentially expressed miRNAs were miR-21, miR-10b and miR145. Only seven miRNAs were elevated both in tumors and serum, suggesting that miRNAs may be selectively released into the serum. The study suggested miR-1,


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miR-92a, miR-133a and miR-133b as the strongest diagnostic markers. Cuk et al. [89] using qRT-PCR arrays, investigated miRNA expression extracted from the plasma of earlystage breast cancer patients samples taken at the time of initial diagnosis. Four miRNAs (miR-148b, miR376c, miR-409–3p and miR-801) were shown to be significantly overexpressed in the early-stage plasma. ROC curve analysis showed that the combination of miR-148b, miR-409–3p and miR-801 had an equal discriminatory power between patients and healthy controls. They concluded that multi-blood-based miRNA tests can improve early detection, and be developed into a prescreening tool in younger women. A follow-up study by Cuk et al. [90] described the overexpression of a cluster of miRNAs from the 14q32 chromosome region. Patients with benign breast tumors had high levels of miR-148b, miR-652 and miR-801 levels in their plasma. Furthermore, an analysis of samples stratified by stage demonstrated that miR-127–3p, miR-148b, miR-409–3p, miR-652 and miR-801 can differentiate stage I or stage II breast cancer from healthy controls making them attractive candidates for early detection. Analysis of miRNA levels in tissue showed that miR-127–3p, miR-376a and miR-652 are present at lower levels in malignant breast cancer tissue in comparison with benign breast tissue. An interesting study by Zeng et al. [91] compared the biomarker potential of miR-30a to conventional circulating tumor markers CA153 and CEA. This was significantly downregulated in cancer patients than healthy controls. In comparison, the levels of CEA and CA153 were all significantly higher in preoperative cancer plasma compared with the control. Only the level of CA153 decreased in postoperative plasma samples from breast cancer patients. ROC analysis showed the sensitivity and specificity of miR-30a for diagnosis at 74.0 and 65.6%, respectively, whereas the sensitivities of CEA and CA153 were 12.0 and 14.0%, respectively. Interestingly, the status of ER and triplenegative breast cancer was significantly associated with miR-30a. These findings suggested that the combination of a single miRNA maker with conventional protein markers CA153 and CEA has the potential to significantly improve diagnosis. To determine if serum miRNAs were altered in metastatic breast cancer, Roth et al. demonstrated that miR-10b, miR-34a and miR-155 could distinguish primary and metastatic breast cancer patients from healthy controls [65] . In addition, miR-155 appeared to be the only single marker to distinguish primary breast cancer from the healthy controls. Several studies have linked specific miRNAs to lymph node status. The expression of both miR-10b


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Review Khoury & Tran and miR-373 was measured in a cohort of ductal carcinoma patients with metastasis, ductal carcinoma patients without metastasis and healthy female donors. The levels of these miRNAs were roughly fourfold higher in the patients with lymph node metastasis. This is similar to another study where the combination of the two plasma miRNAs increases the sensitivity and specificity for detecting lymph node positive status in breast cancer patients [92] . Other studies have expanded the list of potential miRNAs as possible markers for metastasis. Si et al. tested miR-106b, miR-125b, miR-17, miR-185, miR21, miR-558, miR-625, miR-665, miR-92a and miR93 and only the levels of miR-92a were significantly lower in tissue and serum samples of breast cancer patients, in contrast, miR-21 was higher in tissue and serum. It was further shown that low miR-92a and high miR-21 levels were associated with tumor size and a positive lymph node status [93] . In one of the larger studies (n = 269 individuals), miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375 and miR-801 were elevated relative to the controls. Interestingly, miR-200b which is involved in the EMT process and may promote cell invasion was identified as the strongest marker for identifying circulating tumor cells which is an established prognostic indicator for metastatic breast cancer [94] . Eichelser et al. [95] analyzed serum samples from 120 patients with primary breast cancer of three different subgroups taken postsurgery and before chemotherapy, 32 patients with overt metastasis and 40 healthy women. Using qRT-PCR, they measured the relative concentrations of six miRNAs (miR-10b, miR-17, miR-34a, miR-93, miR-155 and miR-373) known to be relevant for tumor development and progression. The concentrations of miR-34a, miR93 and miR-373 were significantly different between M0 stage patients and healthy women, whereas miR17 and miR-155 were differently expressed between M0 and M1 patients. Increased concentrations of miR-373 were associated with negative HER2 status of the primary tumor. Deregulated concentrations of miR-17 and miR-34a were detected in patients with progesterone/estrogen receptor-positive and -negative status, respectively. These findings indicate that serum levels of deregulated miRNAs may be linked to a particular subtype of cancers that favor progression and metastatic spread. Prostate cancer Prostate cancer is the most commonly diagnosed cancer in men in the western world. Early diagnosis is critical in achieving a beneficial response to treatment. Current diagnosis for this disease involves the highly

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invasive digital rectal examination and then followed by diagnosis with a needle biopsy of the prostate gland. To date, there is no effective early screening test for the early diagnosis of prostate cancer. A blood test for PSA, an antigen produced by prostate cells is available but there are limitations to this assay [96–98] . Mitchell et al. examined a panel of miRNAs in the blood of advanced prostate cancer and showed that miR-141 was highly elevated in the cancer serum. Moreover, miR-141 demonstrated a high correlation with serum PSA levels and could identify individuals with advanced prostate cancer with 60% sensitivity and 100% specificity [7] . Subsequent studies have supported this observation with additional miRNAs that were altered in the serum of patients with prostate cancer [99] . In one study, 36 early-stage prostate cancer patients (prior to a prostatectomy) were compared with a cohort of healthy men. This study indicated that serum miR-93, miR-106a and miR-24 were overexpressed with further supporting studies suggesting that miR-21 and miR-221 were significantly higher in early-stage or localized prostate cancer compared with controls [100] . The potential use of serum miRNAs as discriminators for local disease or benign prostatic hyperplasia was then highlighted by Mahn et al. [101] . This study suggested that miR-26a, miR-195 and let-7i were increased in the serum of men with localized prostate cancer. Bryant et al. in 2012 using a large cohort of 78 patients suggested that miR-107 was overexpressed in only prostate cancer when compared with healthy controls [102] . In a similar size study, Chen et al. compared miRNA serum signatures between patients with prostate cancer and patients diagnosed with benign enlargement of the prostate. They identified two significantly upregulated miRNAs (miR-622 and miR1285) which could discern the two diseased states [103] . One of the major challenges in prostate cancer treatment is the ability to predict disease progression. In one recent study, circulating miRNAs were measured in serum of patients who had experienced rapid biochemical recurrence (BCR) or no recurrence following a radical prostatectomy. It was shown that miR-141, miR-146b-3p and miR-194 were elevated in patients who experienced BCR. In addition, miR-146b-3p and miR-194 were also associated with disease progression. The latter, being robustly elevated in metastatic tissue, and its expression in primary tumors was associated with a poor prognosis. This study suggests that circulating miRNAs, measured at the time of a radical prostatectomy, could determine intermediate risk of prostate cancers [104] . The Shen et al. study then investigated plasma miRNAs associated with the development and pro-



gression of prostate cancer in 82 patients [105] . By measuring miRNA copy number, they found significant elevated levels of miR-20a in patients with stage 3 compared with stage 2 or lower. The expression levels for miR-20a and miR-21 were significantly increased in patients with high prostate risk assessment scores (CAPRA). Increased miR-21 and miR-145 expression was observed for patients with intermediate or high risk classified by D’Amico, compared with patients with low risk scores. Expression of miR-21 and miR221 differentiated patients with intermediate risk from those with low-risk CAPRA scores. MiR-20a, miR-21, miR-145 and miR-221 could also distinguish high versus low risk in prostate cancer patients. Nguyen et al. [106] examined circulating miRNAs associated with the progression of hormone-sensitive primary tumors to metastatic castration-resistant prostate cancer (CRPC) after androgen deprivation therapy. Expression levels of miRNAs in serum from 28 patients of low-risk localized disease, 30 of highrisk localized disease and 26 of metastatic CRPC was assessed using genome-wide miRNA Arrays and qRTPCR. The data indicated that miR-375, miR-378* and miR-141 were significantly overexpressed in serum of CRPC patients compared with serum from low-risk localized patients, while miR-409–3p was s ignificantly downregulated. To further support the application of miRNAs as possible CRPC biomarkers, Watahiki et al. profiled plasma samples from patients with localized prostate cancer (n = 25) or mCRPC (n = 25) [107] . The study identified 63 miRNAs that were overexpressed in mCRPC compared with localized prostate cancer. Correlation analysis revealed three groups of specific miRNAs. The first comprised miR-141, miR-375 and miR-200c, the second included miR-151–3p, miR423–3p, miR-126, miR-152 and miR-21. The third group made of miR-16 and miR-205, showed less correlation. While no miRNA alone differentiated localized prostate cancer and mCRPC, they found that combinations had greater sensitivity and specificity. A subsequent study by Cheng et al. [108] also looked at mCRPC. Out of the 365 miRNAs profiled, miR141, miR-200a, miR-200c, miR-210 and miR-375, all overexpressed in mCRPC. They also found that serum levels of miR-210, a miRNA associated with the hypoxia pathway, varied widely among mCRPC patients undergoing therapy. In another notable study, Lodes et al. [109] used an oligonucleotide microarray to profile miRNAs in serum from patients with stage 3 and 4 prostate cancer. This study unearthed 15 overexpressed miRNAs that were upregulated in serum from prostate cancer patients. It was further suggested that the miRNA


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expression patterns seen in serum were not identical to miRNA patterns measured from cancer cell lines. When the miRNA data from prostate cancer cell lines were compared with patient data, there is an upregulation of miRNAs but these miRNAs were of a different species. It was concluded that cell line data are not representative of serum expression and miRNAs found in the serum were speculated to be a product of tumor cell lysis or a product(s) released through active transport. These biomarker studies clearly suggest the potential of using miRNAs as biomarkers for the diagnosis and prognosis of prostate cancer. For this field to move forward and deliver a clinical biomarker, most of these studies require better aged-matched controls. Moreover, it is likely that miRNA expression is sensitive and profoundly altered with aging and also hormonal state of this disease. Lung cancer There are two major types of lung cancer; small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) [110] , the latter being much more prevalent. Despite advances made in surgery, radiation therapy and chemotherapy, the 5-year survival for lung cancer remains roughly 16% [110] . Similar to the other malignancies, early detection for lung cancer is the key to overall long-term survival. The study by Hu et al. utilized Solexa sequencing to examine 60 serum samples from NSCLC patients. A biomarker suite of miR-1, miR-30d, miR-486 and miR-499 was shown to be significantly associated with long-term survival in lung cancer [111] . A similar finding was also reported by Yu et al. who used qRTPCR to show that in 112 NSCLC patients, low levels of miR-221 and let-7a, and high miR-137, miR-372 and miR-182* can predict overall and disease-free survival outcomes [112] . This miRNA signature is an independent predictor of the cancer relapse and survival of NSCLC patients. In a microarray study (serum of 11 patients with NSCLC and 11 controls), miR-1254 and miR-574–5p were shown to be significantly overexpressed in the serum of patients with early-stage NSCLC [113] . In support of these initial discoveries, a recent study showed that miR-17–5p expression levels in serum were significantly increased in patients with lung cancer compared with healthy individuals [114] . Survival analysis showed that low serum miR-17–5p expression was correlated to the survival of patients with lung cancer. Two additional miRNAs (miR-361–3p and miR-625*) also identified using microarray profiling were observed to be downregulated in serum of lung cancer patients. In the most recent study, high miR125b expression in lung cancer serum was correlated


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Review Khoury & Tran to poorer prognosis when compared with patients with low expressing miR-125b [115] . Yuxia et al. [115] further tested miR-125b as a biomarker for stage stratification and prognosis in NSCLC. With a cohort of 193 patients representing different stages of NSCLC, blood samples were collected before surgery and therapy. Serum miR-125b was consistently expressed in the nontumor group and significantly associated with NSCLC stage. Furthermore, patients with high miR-125b expression displayed a significantly poorer prognosis compared with patients with low expression. Multivariate analysis indicated that high miR-125b expression was an independent prognostic factor for survival. The results suggest that serum miR-125b may represent a novel biomarker in NSCLC patients and that high miR125b expression is an independent prognostic factor for survival. Several other miRNAs have been suggested as potential indicators for outcomes. MiR-21, miR-10a and miR-30e-5p were elevated in NSCLC plasma when compared with healthy donors. High expression of miR-21 was associated with disease-free intervals. In contrast, low expression of miR-10a was associated with poor disease-free intervals and high expression of miR-30e-5p was linked with shorter overall survival [116] . Another potential prognostic biomarker is miR-375, with low expression in sera associated with poorer overall survival rates [117] . Interestingly, the study by Zhang et al. [118] analyzed plasma miRNA levels in combination with mutations in the EGFR. These mutations in EGFR are often observed in female patients with adenocarcinomas and no prior history of smoking. The study showed that miR-122 and miR195 in the plasma were also associated with overall survival in the patients, especially in those with advanced stage and EGFR mutation. As in the case with all cancer, early detection is vital for early intervention to improve survival rates. Toward this end, the study by Zhang [119] using published miRNA microarray data of primary lung cancer identified 15 miRNAs most frequently overexpressed in lung cancer tissues. The levels of these miRNAs were then determined by qRT-PCR in the plasma of 74 lung cancer patients and 68 age-matched cancer-free controls. The results indicated that miR-155, miR-197 and miR-182 in the plasma of lung cancer including stage I patients were significantly higher when compared with controls. The combination of these three miRNAs yielded 81.33% sensitivity and 86.76% specificity in identifying lung cancer patients. The levels of miR155 and miR-197 were higher in the plasma from lung cancer patients with metastasis than in those without and were significantly decreased in responsive patients

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during chemotherapy. These results indicate that miR155, miR-197 and miR-182 can be potential biomarkers for the early detection of lung cancer. In another study to discover early detection biomarkers, Heegaard et al. [120] measured miRNA levels in paired serum and plasma patients from 220 patients with early-stage NSCLC and 220 matched controls. This study demonstrated that miR-146b, miR-221, let7a, miR-155, miR-17–5p, miR-27a and miR-106a were significantly reduced in the serum of NSCLC cases, while miR-29c was significantly increased. In contrast, there were no significant differences in the plasma of patients compared with controls. Overall, expression levels in serum did not correlate with levels in plasma. Furthermore, low plasma let-7b levels were modestly associated with mortality in patients and that low serum expression of miR-223 was linked to mortality in stage IA/B patients. In a similar study by Kaduthanam et al. [121] , miR142–3p and miR-29b, were confirmed to be increased in sera of early-stage adenocarcinoma patients suffering from recurrence within 24 months. Elevated miRNA levels were exclusively observed in the group of high-risk patients diagnosed for operable adenocarcinoma compared with benign diagnosis or advanced tumor disease. The differentiation between pulmonary adenocarcinoma patients with low and high risk for recurrence was improved by accounting for both miR-142–3p levels and tumor stage. The study concluded that serum miR-142–3p was associated with a high risk of recurrence in early-stage lung adenocarcinoma patients and can be a putative serum marker for risk assessment. Further studies to determine serum miRNAs in lung adenocarcinoma were performed by Rani et al. [122] and Patnaik et al. [123] . Using a large testing cohort of 180 serum samples, the group of miR-30c-1*, miR-616*, miR-146b-3p, miR-566, miR550, miR-939, miR-190b, miR-630, miR-942 and miR-1284 were significantly dsyregulated in cancer sera only. Classification analysis on the latter three miRNAs suggested they would be the best classifiers for the identification of lung adenocarcinomas. Serum miRNAs can also discriminate different forms of lung diseases. Tomasetti et al. [124] determine the diagnostic potential of miR-126 in the serum of malignant mesothelioma and NSCLC patients using absolute qRT-PCR. The levels of miR-126 could significantly differentiate malignant mesothelioma patients from healthy controls and NSCLC from malignant mesothelioma, but could not discriminate NSCLC patients from control subjects. Furthermore, a Kaplan–Meier analysis indicated that low levels of serum miR-126 in mesothelioma patients were strongly associated with poor prognosis.



In another study, Akbas et al. [125] profiled serum from patients with chronic obstructive pulmonary disease (COPD) a disease that is characterized by lower airway inflammation. The levels of 72 serum miRNAs were measured by qRT-PCR in 20 COPD patients and 12 control patients. This study found that miR-20a, miR-28–3p, miR-34c-5p and miR-100 were significantly dysregulated with miR-7 overexpressed. This is one of the first studies to screen serum miRNAs as potential biomarkers for COPD. Wang et al. [125] then analyzed miRNA expression using 184 malignant pleural effusions from NSCLC cases. Thirty-three miRNAs were found to be altered by twofold in malignant effusions between longersurvival and shorter-survival groups, and the levels of miR-93, miR-100, miR-134, miR-151 and miR-345 were significantly associated with overall survival. Further studies by Han et al. [126] identified miRNAs differentially expressed between benign pleural effusion samples and adenocarcinoma-associated malignant pleural effusion (LA-MPE). Microarray profiling showed that miR-198 was significantly downregulated in LA-MPE compared with benign pleural effusion samples. The diagnostic power of miR-198 was comparable with that of CEA, a lung biomarker, but better than that of CYFRA 21–1 a second lung marker. The present study suggests that serum miR-198 may have diagnostic potential for differentiating LA-MPE from benign pleural effusion. Other lung diseases such as tuberculosis and pneumonia could also be differentiated from lung cancer. In the study by Foss et al., qPCR levels of miR-21, miR155, miR-182 and miR-197 were measured in serum from patients with lung cancer (n = 65), pulmonary tuberculosis (n = 29), pneumonia (n = 29) and transudate (n = 16) compared with matched healthy controls (n = 37) [127] . Serum levels of miR-21, miR-155 and miR-197 were significantly elevated in the patients with lung cancer and pneumonia whereas miR-182 and miR-197 levels were increased only in patients with lung cancer and tuberculosis. ROC analysis revealed that miR-182, miR-155 and miR-197 work together well as a suite of diagnostic miRNAs for d iscrimination between the diseases. Another powerful approach to identify miRNA biomarkers is to examine expression in pre- and post-tissue or serum. Both the Aushev et al. [128] and the Le et al. [129] measured serum miRNA expression between preoperative and postoperative lung carcinoma patients. The group of miR-21, miR-24, miR-205, miR-19a, miR-19b, miR-30b and miR-20a was decreased after surgery. In addition, high expressions of miR-21 and miR-30d in preoperative sera were independently correlated with shorter overall survival in lung cancer


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patients. In contrast to a decrease post surgery, miR361–3p and miR-625* demonstrated increased levels postoperatively. This finding may suggest that miR361–3p and miR-625* have a protective influence on the development of NSCLC [130] . Gastric cancer Although the incidence of gastric cancer in countries has fallen markedly in recent years, it remains the leading cause of cancer death worldwide. Mortality rates are high in China, Japan and parts of South America, however it is less common in the United Kingdom and almost uncommon in America. Of interest is that Japan is one of the few countries where patients are diagnosed often at an early stage. This directly correlates to the widespread and heavy practice of endoscopic screening across the country. Currently, there are no laboratory markers of sufficient accuracy for the diagnosis of gastric cancer. Upper gastrointestinal endoscopy is the diagnosis of choice and is performed on patients who present with symptoms such as weight loss, anemia, hematamesis or melena, dysphagia or the presence of a palpable abdominal mass. Inevitably, multiple biopsies will follow that are taken from the different parts of the cancer or primary ulcer in order to improve the diagnostic evaluation. Extensive imaging is then required for accurate staging and assessment of resectability [131–133] . Currently, this field requires further development in order to improve the prognosis for gastric cancer patients whose outlook at the moment is very poor. Sadly, less than 10% of all patients survive 5 years. The best hope for their improved survival lies in greater detection of tumors at an earlier stage. Surprisingly, there have only been very few studies on circulating miRNA expression in gastric cancer, predominantly using Asian male population aged between 46 and 66. These studies first appeared in 2010 and assessed the expression of circulating miRNAs across different sample types; blood, plasma and serum [134,135] . A separate study on plasma samples showed that miR-17–5p, miR-21, miR-106a and miR106b were significantly upregulated during gastric cancer and decreased postoperatively [136] . In support of these initial findings, Zhou et al. [137] detected miR106a and miR-17 in gastric cancer patients (n = 90). In preoperative and postoperative groups, miR-106a and miR-17 levels were expressed higher than the controls with areas under the ROC curve suggesting a strong correlation with the number of cancer cells. Given that miR-21 is already considered a biomarker for other cancers, several studies examined the utility of this miR-21 and the miR-17 cluster in gastric cancers. Zheng et al. [138] , investigated miR-21 in peripheral


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Review Khoury & Tran blood taken from 53 preoperative patients with gastric cancer and 20 healthy volunteers. They showed that miR-21 was significantly higher in the blood of gastric cancer patients with levels associated with tumor node metastasis stage, tumor size and tissue categories. Komatsu et al. further reported that miR-21, miR17–5p, miR-106a and miR-106b were highly expressed in gastric cancer tissue (n = 69) and plasma [139] . Postoperative high levels of both miR-21 and miR-106a were associated with poor survival rates and slightly higher rates of vascular invasion. Multivariate analysis indicated that high miR-21 in plasma was an independent prognostic factor. Kim et al. [140] then investigated the expression of miR-21 in lymph node positive patients. Serum levels of miR-21, miR-27a, miR-106b, miR-146a, miR-148a and miR-223 were significantly increased in lymph node positive when compared with lymph node negative patients. These findings suggest that serum miRNAs may be used to predict the presence of lymph node metastasis in gastric cancers. In contrast to the high levels of miR-21 seen in these studies, the absence or low expression of miR-21 was noted in the Ma et al. study. They showed that miR-21 was significantly reduced in postoperative plasma when compared with preoperative samples. Interestingly, this difference was very pronounced in a subgroup of patients without any family disease h istory. The ability to forecast disease progression would impact greatly in the treatment of patients with gastric cancer. The study by Chen et al. [141] investigated if specific plasma miRNAs could be associated with distant metastasis. Their results identified that miR-192 levels were significantly higher in patients with a distal metastasis. Concomitantly, plasma miR-122 levels were low in the same patients. However, elevated levels of plasma miR-122 were correlated to increased survival rates. The Zhang et al. [118] used distal gastric adenocarcinomas to show that miR-375 serum levels were significantly reduced. As a biomarker, miR-375 yielded an ROC area under the curve of 0.835 with a specificity and sensitivity of 80 and 85%. Interestingly, the expression of miR-375 was underexpressed both in distal gastric adenocarcinoma tissues and serum of patients with distal gastric adenocarcinoma. The ability to predict relapse, which can be up to 50–60% in some patients will dramatically improve survival rates. Tsai et al. [142] reported that miR-196a was significantly increased in gastric tumors. In addition, increased levels of serum miR-196a were associated with gastric cancer relapse. Functional studies, demonstrated that overexpression of miR-196a promoted the epithelial–mesenchymal transition (EMT) and migration/invasion of these cells. Previous studies have suggested that miR-200 is an important player

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in the EMT process. Toward this end, the expression of miR-200a, miR-200b, miR-200c and miR-141 was measured in the blood of gastric cancer samples. It was shown that miR-200c levels were significantly higher in cancer sera and levels correlated with the number of lymph node metastases and a poor overall survival [143] . In an attempt to identify very early biomarkers, Li et al. assessed the expression of miR-199a-3p in early gastric cancer [144] . The expression of miR-199a-3p in the plasma of early gastric cancer patients (n = 50) was significantly higher when compared with healthy controls and patients with gastric precancerous diseases. In a more recent study, Tsujiura et al. [145] suggested that miR-18a (part of the miR-17–92 cluster) could be a early plasma biomarker in patients with gastric cancer. Plasma levels of miR-18a were significantly higher in gastric cancer patients than in healthy controls. Additionally, both miR-199a-3p and miR-18a were reduced in postoperative samples compared with preoperative samples. Supporting the miRNA biomarkers found earlier by Liu et al., a study by Wang et al. that looked at multiple types of cancers, also noted that miR-20b, miR-20a, miR-17, miR-106a, miR-18a and miR-21 were upregulated in gastric cancers [146] . Furthermore, they found that their concentrations were significantly associated with the differentiation status and TNM stages. Apart from being able to track tumor progression, a Kaplan–Meier curve analysis revealed that high expression levels were significantly correlated with poor overall survival. Additional studies have used next-generation Solexa sequencing [147] and the BioMark™ 96.96 Dynamic Array [148] to further identify novel markers. These studies demonstrated that miR-1, miR-20a, miR-27a, miR-34 and miR-423–5p serum levels were higher in gastric cancers and these levels could discern the specific tumor stage. In contrast to these elevated levels, plasma miR-195–5p was significantly downregulated from a panel of 740 miRNA tested [148] . Five miRNAs (miR-16, miR-25, miR-92a, miR-451 and miR486–5p) showed consistently elevated levels in plasma of the gastric cancer patients as compared with controls, and were identified to be potential markers for gastric noncardia adenocarcinoma. Conclusion & future perspective This review has assessed the potential of using circulating miRNAs as biomarkers in common malignancies (prostate, breast, lung, gastric and colorectal cancers). There is a diverse range of circulating miRNAs, which occur in each of these malignancies. To provide a future perspective of the biomarker landscape, we summarized these serum miRNAs for each cancer under diagnostic or prognostic potential (Table 1) .



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Table 1. Circulating miRNAs as potential diagnostic and prognostic biomarkers in the five most common malignancies. Biomarkers

Differential diagnosis

Prognostic

Breast cancer Cancer vs healthy

Early-stage cancers

Benign

Invasive ductal PR + vs PR -ve Metastatic carcinoma in situ marker vs invasive carcinomas

Survival

miR-16

miR-425*

miR-148b

miR-21

miR-155

miR-141

miR-10b

miR-145

miR-302b

miR-652

miR-10b

Low miR-182 in PR+

miR-106b

miR-34a

miR-155

miR-148b

miR-801

miR-373

miR-125b

miR-155

miR-195

miR-376c

Low miR-17

miR-17

Let-7a

miR-409-3p

Low miR-34a

miR-185

miR-182

miR-801

miR-21

miR-21

miR-127-3p

miR-558

miR-27a

miR-148b

miR-625

miR-150

miR-409-3p

miR-665

miR-191

miR-652

miR-92a

miR-200c

miR-801

miR-93

miR-210

miR-200a

miR-382

miR-200b

miR-10b

miR-200c

miR-125b

miR-203

miR-1

miR-210

miR-92a

miR-375

miR-133a

miR-801

miR-133b

miR-30a

M0 vs M1

miR-34a

miR-93

miR-373

miR-17

miR-155

Lung cancer NSCLC biomarkers Cancer vs healthy

COPD

Malignant pleural effusions vs NSCLC

Malignant Pneumonia vs Poor mesothelioma vs tuberculosis vs NSCLC NSCLC

NSCLC long-term Survival

miR-155

Low Levels

Low levels

miR-126

miR-182

miR-125b

miR-21

miR-197

miR-20a

miR-155

Low miR-10a

miR-1

miR-182

miR-28-3p

miR-198

miR-197

High miR30e-5p

miR-30d

This summary list of the individual miRNAs from the studies reviewed. The miRNAs shown have been categorized according to their diagnostic and prognostic potential and then classified into specific disease states. Adenoma: Advanced adenoma; Ca: Cancer; COPD: Chronic obstructive pulmonary disease; CRPC: Castration-resistant prostate cancer; NSCLC: Non-small-cell lung cancer; PR+: Progesterone receptor positive; PR-ve: Progesterone receptor negative.



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Table 1. Circulating miRNAs as potential diagnostic and prognostic biomarkers in the five most common malignancies (cont.). Biomarkers


Prognostic

Lung cancer (cont.) NSCLC biomarkers Cancer vs healthy

COPD

Malignant pleural effusions vs NSCLC

Malignant Pneumonia vs Poor mesothelioma vs tuberculosis NSCLC vs NSCLC

NSCLC long-term survival

miR-1254

miR-34c-5p

miR-93

miR-486

miR-574-5p

miR-100

miR-100

Low miR-375

miR-499

miR-29c

miR-134

miR-223

miR-122

miR-142-3p

High levels

miR-151

Let-7b

miR-195

miR-29b

miR-345

miR-142-3p

miR-30c-1*

miR-7

miR-29b

Low levels miR-221

miR-616*

miR-146b-3p

Low miR-126

Let-7a

miR-566

miR-21

miR-17-5p

miR-550

miR-30d

miR-939

High levels

miR-190b

miR-630

miR-137

miR-942

miR-372

miR-1284

miR-182*

Low levels

miR-146b

miR-221

Let-7a

miR-155

miR-27a

miR-106a

Cancer vs healthy

Early-stage cancers (localized)

Benign

Progression and high-risk patients

CRPC versus Poor prognosis localized lowand high-risk disease

miR-141

miR-26a

miR-622

miR-20a

miR-375

miR-141

miR-93

miR-195

miR-1285

miR-21

miR-378*

miR-146b-3p

miR-106a

Let-7i

miR-221

miR-141

miR-194

miR-24

miR-145

miR-200c

miR-107

miR-151-3p

Prostate cancer


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Prognostic

Prostate cancer (cont.) Cancer vs healthy

Early-stage cancers (localized)

Benign

Progression and high-risk patients

CRPC versus localized low and high-risk disease

Poor prognosis

miR-107 (cont.)

miR-423-3p

miR-126

miR-152

mi-21

miR-16

miR-205

miR-200a

miR-210

Cancer vs healthy

Predictors of recurrence and survival in earlystage cancers

Adv. Diff between GCa and adenomas inflammatory bowel disease vs primary CRCs

Metastatic marker

miR-485–5p

miR-21

miR-92a

miR-92

miR-141

mir-361-3p

miR-15a

miR-18a

miR-326

miR-103

miR-21

miR-487b

miR-148a

miR-17-3p

miR-320a

miR-92

miR-451

miR-134

miR-596

miR-146a

miR-378

miR-181s

miR-29c

miR-191

miR-221

miR-222

miR-223

miR-225

miR-229

miR-320

miR-18a

miR-20a

miR-21

miR-29a

Colorectal cancer




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Prognostic

Colorectal cancer (cont.) Cancer vs healthy

Predictors of recurrence and survival in earlystage cancers

Adv. Diff between GCa and adenomas inflammatory bowel disease vs primary CRCs

Metastatic marker

miR-106b

miR-133a

miR-143

miR-145

miR-193a-3p

miR-23a miR-338-5p

Gastric cancer Cancer vs healthy

Lymph node positive vs negative

Metastasis

Relapse

miR-17-5p

miR-21

miR-21

miR-196a

miR-21

miR-27a

miR-106a

miR-200c

miR-106a

miR-106b

miR-192

miR-106b

miR-146a

miR-17

miR-148a

miR-196a

miR-223

miR-199a-3p

miR-200c

miR-18a

miR-20b

miR-20a

miR-1

miR-27a

miR-34

miR-423-5p

Low miR-375

1


These combined studies have identified 127 serum miRNAs which are dysregulated from normal patients. Surprisingly, the majority of these serum miRNAs were unique to their origin with only 17% or 22 miRNAs common between these five cancers. The most frequent and common being miR-21, followed by miR-17 and miR-155. We suggest these are the main serum oncomiRs present in most cancers. In this review, they are nearly always expressed at ele-

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vated levels in the serum of cancer patients when compared with a normal population. The combination of serum miR-21 and miR-155 is also seen in pancreatic [149] and cervical cancers [150] . Although these miRNAs may be elevated in cancers, they also represent other physiological conditions. Both these miRNAs are elevated in immune responses [151] and bacterial infections [152] . A critical evaluation by Haider et al. identified miR-21, miR-16, miR-146a, miR-155, miR-



126 and miR-223 as biomarkers for nine or more distinct diseases [153] . Thus, one of the major considerations when using miRNAs as distinct cancer serum biomarkers is their presence and concentrations in other diseases. Most of the studies in prostate, lung and breast cancers have been focused on finding serum miRNAs that can predict disease outcomes. In contrast, gastric and colorectal studies are centered on discriminating cancer from healthy patients. There are very few serum miRNA biomarkers that can predict metastasis or relapse in either gastric or colorectal cancers. miR141 is suggested to be a metastatic maker for colorectal cancers, but it is also expressed in prostate cancer. We also found 7 common serum miRNAs between gastric and colorectal cancers. In comparison, circulating miR-199a-3p, miR-20b, mir-34, miR-423–5p and miR-192 were unique to gastric cancers. Given the significant increased risk of developing lung cancer after breast cancer diagnosis [154] , there were 28 serum miRNAs in common, the most frequent being miR-155, then, miR-21, and Let-7a/b. Interestingly, we found that miR-182 was common in both cancers but only miR-182* was specific to lung cancer. From the reviewed studies, we also noted that prostate, breast, lung and colorectal cancers might have 11, 15, 29 and 21 unique miRNA in circulation, respectively. It is not unexpected that some cancer serum miRNAs are common in other physiological conditions, but their exact concentrations in these conditions may differ greatly. It is plausible that miR-21 for example is detected at a certain concentration range and this is indicative of a malignant phenotype. In comparison, a nonneoplastic phenotype may have a concentration outside this range. The majority of studies reviewed use qRT-PCR to calculated fold change relative to a normal cohort to evaluate expression levels. This approach does not provide an accurate measure of miRNAs in serum. Furthermore, these fold changes for the same miRNA can vary from study to study and is dependent on the reproducibility of the assay from each study. This relative fold calculation is entirely dependent on normalizing the data to an internal calibrator (another miRNA), which does not change. In large cohort studies, these internal calibrators may fluctuate from an individual-by-individual basis and introduce bias in calculating the fold change. We put forward the recommendation of using an absolute qRT-PCR assay to measure the exact concentrations of serum miRNAs. A synthetic oligonucleotide is used in a qRT-PCR to generate a standard concentration curve, which is used to calculate the exact molar concentration of miRNAs. By using


Review

this approach, we can assess the exact molar amounts of these common serum miRNAs in malignant and nonneoplastic conditions. It would provide insights to determine if the concentration range for a single miRNA can d iscriminate between these different physiological states. The review also demonstrated a lack of consistent miRNA expression in the same type of malignancy. This is mainly attributed to different methods of collecting the patient material, storage environment, isolation of total RNA and the different endpoints for assessing miRNA levels. Detection methodology ranges from qRT-PCR, oligonucleotide arrays and RNA sequencing platforms. All of which rely on different technologies and the levels of miRNAs measured using each platform may vary greatly. Cross platform comparisons can be done but we suggest that qRT-PCR is the standard to assess individual miRNA expression. There is also the concern of hemolysis in the blood prior to processing these samples. Several studies have shown that hemolysis of the blood during collection may release miRNAs from white and red blood cells [155,156] . This hemolysis would contaminate the overall miRNA population and result in a false biomarker(s) not truly representative of the disease. The majority of these studies do not perform a hemolysis assay to assess the degree of hemolysis. In our lab, all serum samples are screened for hemolysis by measuring free hemoglobin using the Drabkin assay. A standard hemoglobin concentration curve is generated and used to determine the level of free hemoglobin in serum samples. Serums with a concentration of hemoglobin greater than 2 mg/ml are considered to be hemolyzed [157] and excluded from further testing. Other approaches to measure hemolysis include the Harboe assay [158] and measuring at 414 nm [159] . Given the ease of collecting blood with routine testing done by pathology laboratories and major hospitals, circulating miRNAs could be used as a frontline screening program for large populations. It could be implemented into screening programs with positive results being further tested using routine pathology tests. Circulating miRNAs are not only restricted to the blood but can be found in other body fluids such as urine, saliva, tears, breast milk and sweat [160] . Although the majority of studies have used serum or plasma, some studies have now utilized saliva to screen for potential cancer biomarkers [160,161] . In summary, circulating miRNAs are indicative of various cancers, some of these are unique but several share commonality in malignancies such as prostate, breast, lung, gastric and colorectal cancers. As yet, there are no miR-


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Review Khoury & Tran NAs either circulating or derived from solid tissue, which have approval for clinical use. One of the major obstacles impeding this progress is the inconsistency of expression patterns between studies. If the field can agree on uniform guidelines for sample collection, processing with QC for hemolysis and employ a robust detection platform, this would allow for the reliable and reproducible detection of circulating miRNAs in any disease system. Open access This work is licensed under the Creative Commons Attribution-NonCommercial 3.0 Unported License. To view a copy

of this license, visit http://creativecommons.org/licenses/bync-nd/3.0/

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary The origin of circulating miRNAs • These are small non-coding RNAs of 22 nucleotides that can be found in most bodily fluids such as blood. The most studied are miRNAs and their main function is gene regulation. • These miRNAs can be released into the circulation via membrane-bound vesicles such as exosomes, bound to protein complexes or accidental leakage from damaged cells.

The role of circulating miRNAs • miRNAs can modulate the development and spread of a tumor by playing multiple roles as either oncogenes or tumor suppressors. • Their expression in the blood is altered in different diseases such as cancer. • The presence of these specific circulating miRNAs can then be utilized to diagnose different cancer stages and predict disease outcomes in cancer patients.

Circulating miRNAs in prostate, breast, lung, gastric and colorectal cancers • From 127 miRNAs, only 22 were common between the five cancers with miR-21, miR-17 and miR-155 being the most frequent. • These three circulating miRNAs were universally elevated in cancer when compared with normal patients and may represent the key miRNAs involved in cancer. • miR-141 may be a metastatic maker for colorectal cancers, but is also found in prostate cancer. • Circulating miR-199a-3p, miR-20b, mir-34, miR-423-5p and miR-192 were unique to gastric cancers. • Breast and lung cancer share 28 common serum miRNAs, the most frequent being miR-155, then, miR-21 and Let-7a/b. • Prostate, breast, lung and colorectal cancers have 11, 15, 29 and 21 unique miRNAs in circulation, respectively. • The expression of these circulating miRNAs may have the potential to act as both diagnostic and prognostic tools.

References

146

1

Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 294(5543), 853–858 (2001).

2

Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundantxclass of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294(5543), 858–862 (2001).

3

Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 294(5543), 862–864 (2001).

4

Tran N, Hutvagner G. Biogenesis and the regulation of the maturation of miRNAs. Essays Biochem. 54(1), 17–28 (2013).

5

Chim SS, Shing TK, Hung EC et al. Detection and characterization of placental microRNAs in maternal plasma. Clin. Chem. 54(3), 482–490 (2008).


6

Lawrie CH, Gal S, Dunlop HM et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br. J. Haematol. 141(5), 672–675 (2008).

7

Mitchell PS, Parkin RK, Kroh EM et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl Acad. Sci. USA 105(30), 10513–10518 (2008).

8

Chen X, Ba Y, Ma L et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18(10), 997–1006 (2008).

9

Zhu W, Qin W, Atasoy U, Sauter ER. Circulating microRNAs in breast cancer and healthy subjects. BMC Res. Notes 2, 89 (2009).

10

Patnaik SK, Mallick R, Yendamuri S. Detection of microRNAs in dried serum blots. Anal. Biochem. 407(1), 147–149 (2010).



11

12

13

14

Zernecke A, Bidzhekov K, Noels H et al. Delivery of microRNA-126 by apoptotic bodies induces CXCL12dependent vascular protection. Sci. Signal. 2(100), ra81 (2009). Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9(6), 654–659 (2007). Arroyo JD, Chevillet JR, Kroh EM et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc. Natl Acad. Sci. USA 108(12), 5003–5008 (2011). Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat. Cell Biol. 13(4), 423–433 (2011).

15

Fleischhacker M, Schmidt B. Circulating nucleic acids (CNAs) and cancer – a survey. Biochim. Biophys. Acta 1775(1), 181–232 (2007).

16

Fevrier B, Raposo G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 16(4), 415–421 (2004).

17

Hunter MP, Ismail N, Zhang X et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS ONE 3(11), e3694 (2008).

18

Skog J, Wurdinger T, Van Rijn S et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10(12), 1470–1476 (2008).

19

Ismail N, Wang Y, Dakhlallah D et al. Macrophage microvesicles induce macrophage differentiation and miR223 transfer. Blood 121(6), 984–995 (2013).

20

Van Balkom BW, De Jong OG, Smits M et al. Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells. Blood 121(19), 3997–4006, S3991–S3915 (2013).

21

Mittelbrunn M, Gutierrez-Vazquez C, Villarroya-Beltri C et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011).

22

Xu L, Yang BF, Ai J. MicroRNA transport: a new way in cell communication. J. Cell Physiol. 228(8), 1713–1719 (2013).

23

Wang K, Zhang S, Weber J, Baxter D, Galas DJ. Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res. 38(20), 7248–7259 (2010).

24

Chen X, Ba Y, Ma L et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18(10), 997–1006 (2008).

25

Mitchell PS, Parkin RK, Kroh EM et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl Acad. Sci. USA 105(30), 10513–10518 (2008).

26

Hutvagner G, Zamore PD. RNAi: nature abhors a doublestrand. Curr. Opin. Genet. Dev. 12(2), 225–232 (2002).


27

Lu J, Getz G, Miska EA et al. MicroRNA expression profiles classify human cancers. Nature 435(7043), 834–838 (2005).

28

Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 10(10), 704–714 (2009).

29

Esquela-Kerscher A, Slack FJ. Oncomirs – microRNAs with a role in cancer. Nat. Rev. Cancer 6(4), 259–269 (2006).

30

Pelosi A, Careccia S, Lulli V et al. miRNA let-7c promotes granulocytic differentiation in acute myeloid leukemia. Oncogene 32(31), 3648–3654 (2013).

31

Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat. Genet. 39(5), 673–677 (2007).

32

Nicoloso MS, Spizzo R, Shimizu M, Rossi S, Calin GA. MicroRNAs – the micro steering wheel of tumour metastases. Nat. Rev. Cancer 9(4), 293–302 (2009).

33

Zhang L, Pickard K, Jenei V et al. miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance. Cancer Res. 73(21), 6435–6447 (2013).

34

Feng DD, Zhang H, Zhang P et al. Down-regulated miR331–5p and miR-27a are associated with chemotherapy resistance and relapse in leukemia. J. Cell Mol. Med. 15(10), 2164–2175 (2010).

35

Liang Z, Wu H, Xia J et al. Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1. Biochem. Pharmacol. 79(6), 817–824 (2009).

36

Fabbri M, Paone A, Calore F et al. MicroRNAs bind to Tolllike receptors to induce prometastatic inflammatory response. Proc. Natl Acad. Sci. USA 109(31), E2110–E2116 (2012).

37

Fabbri M, Paone A, Calore F, Galli R, Croce CM. A new role for microRNAs, as ligands of Toll-like receptors. RNA Biol. 10(2), 169–174 (2013).

38

Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 18(3), 350–359 (2008).

39

Kamangar F, DoRes. GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J. Clin. Oncol. 24(14), 2137–2150 (2006).

40

He J. Screening for colorectal cancer. Adv. Surg. 45, 31–44 (2011).

41

Hardcastle JD, Chamberlain JO, Robinson MHE et al. Randomised controlled trial of faecal-occult-blood screening for colorectal cancer. Lancet 348(9040), 1472–1477 (1996).

42

Kronborg O, Fenger C, Olsen J, JØrgensen OD, SØndergaard O. Randomised study of screening for colorectal cancer with faecal-occult-blood test. Lancet 348(9040), 1467–1471 (1996).

43

Lindholm E, Brevinge H, Haglind E. Survival benefit in a randomized clinical trial of faecal occult blood screening for colorectal cancer. Br. J. Surg. 95(8), 1029–1036 (2008).

44

Mandel JS, Church TR, Ederer F, Bond JH. Colorectal cancer mortality: effectiveness of biennial screening for fecal occult blood. J. Natl Cancer Inst. 91(5), 434–437 (1999).


Review

147

Review Khoury & Tran 45

Atkin WS, Edwards R, Kralj-Hans I et al. Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 375(9726), 1624–1633

61

Kuo T-Y, Hsi E, Yang IP, Tsai P-C, Wang J-Y, Juo S-HH. Computational analysis of mRNA expression profiles identifies microRNA-29a/c as predictor of colorectal cancer early recurrence. PLoS ONE 7(2), e31587 (2012).

46

Schoen RE, Pinsky PF, Weissfeld JL et al. Colorectalcancer incidence and mortality with screening flexible sigmoidoscopy. N. Engl. J. Med. 366(25), 2345–2357 (2012).

62

Matsuo M, Nakada C, Tsukamoto Y et al. MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2. Mol. Cancer 12, 15 (2013).

47

Segnan N, Armaroli P, Bonelli L et al. Once-only sigmoidoscopy in colorectal cancer screening: follow-up findings of the Italian randomized controlled trial – SCORE. J. Natl Cancer Inst. 103(17), 1310–1322 (2011).

63

Heneghan HM, Miller N, Lowery AJ, Sweeney KJ, Newell J, Kerin MJ. Circulating microRNAs as novel minimally invasive biomarkers for breast cancer. Ann. Surg. 251(3), 499–505 (2010).

48

Ng EK, Chong WW, Jin H et al. Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut 58(10), 1375–1381 (2009).

64

49

Huang Z, Huang D, Ni S, Peng Z, Sheng W, Du X. Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int. J. Cancer 127(1), 118–126 (2009).

Heneghan HM, Miller N, Kelly R, Newell J, Kerin MJ. Systemic miRNA-195 differentiates breast cancer from other malignancies and is a potential biomarker for detecting noninvasive and early stage disease. Oncologist 15(7), 673–682 (2010).

65

Roth C, Rack B, Muller V, Janni W, Pantel K, Schwarzenbach H. Circulating microRNAs as blood-based markers for patients with primary and metastatic breast cancer. Breast Cancer Res. 12(6), R90 (2010).

66

Wang F, Zheng Z, Guo J, Ding X. Correlation and quantitation of microRNA aberrant expression in tissues and sera from patients with breast tumor. Gynecol. Oncol. 119(3), 586–593 (2010).

67

Appaiah H, Goswami C, Mina L et al. Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients. Breast Cancer Res. 13(5), R86 (2011).

68

Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE, Hoon DSB. Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin. Chem. 57(1), 84–91 (2011).

69

Wu Q, Lu Z, Li H, Lu J, Guo L, Ge Q. Next-generation sequencing of microRNAs for breast cancer detection. J. Biomed. Biotechnol. 2011, 597145 (2011).

70

Van Schooneveld E, Wouters M, Van Der Auwera I et al. Expression profiling of cancerous and normal breast tissues identifies microRNAs that are differentially expressed in serum from patients with (metastatic) breast cancer and healthy volunteers. Breast Cancer Res. 14(1), R34 (2012).

71

Schwarzenbach H, Milde-Langosch K, Steinbach B, Müller V, Pantel K. Diagnostic potential of PTEN-targeting miR214 in the blood of breast cancer patients. Breast Cancer Res. Treat. 134(3), 933–941 (2012).

50

51

52

53

54

148

Cheng H, Zhang L, Cogdell DE et al. Circulating plasma MiR-141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis. PLoS ONE 6(3), e17745 (2011). Giráldez MD, Lozano JJ, Ramírez G et al. Circulating microRNAs as biomarkers of colorectal cancer: results from a genome-wide profiling and validation study. Clin. Gastroenterol. Hepatol. 11(6), 681–688.e683 (2013). Anna Brunet Vega CP, Irene M, Anna F et al. Eugeni Saigí MicroRNA expression profile in stage III colorectal cancer: circulating miR-18a and miR-29a as promising biomarkers. Oncol. Rep. 30(1), 320–326 (2013). Luo X, Stock C, Burwinkel B, Brenner H. Identification and evaluation of plasma microRNAs for early detection of colorectal cancer. PLoS ONE 8(5), e62880 (2013). Yong FL, Law CW, Wang CW. Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR338–5p for early detection of colorectal cancer. BMC Cancer 13(1), 280 (2013).

55

Toiyama Y, Takahashi M, Hur K et al. Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer. J. Natl Cancer Inst. 105(12), 849–859 (2013).

56

Liu G-H, Zhou Z-G, Chen R et al. Serum miR-21 and miR-92a as biomarkers in the diagnosis and prognosis of colorectal cancer. Tumor Biol. 34(4), 2175–2181 (2013).

57

Kobayashi H, Mochizuki H, Sugihara K et al. Characteristics of recurrence and surveillance tools after curative resection for colorectal cancer: a multicenter study. Surgery 141(1), 67–75 (2007).

72

Sieuwerts A, Mostert B, Bolt-De Vries J et al. mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients. Clin. Cancer Res. 17, 3600–3618 (2011).

58

Menéndez P, Padilla D, Villarejo P et al. Prognostic implications of serum microRNA-21 in colorectal cancer. J. Surg. Oncol. 108(6), 369–373 (2013).

73

59

Shivapurkar N, Weiner LM, Marshall JL et al. Recurrence of early stage colon cancer predicted by expression pattern of circulating microRNAs. PLoS ONE 9(1), e84686 (2014).

Schrauder MG, Strick R, Schulz-Wendtland R et al. Circulating micro-RNAs as potential blood-based markers for early stage breast cancer detection. PLoS ONE 7(1), e29770 (2012).

74

60

Zanutto S, Pizzamiglio S, Ghilotti M et al. Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer. Br J. Cancer 110(4), 1001–1007 (2014).

Hu Z, Dong J, Wang L-E et al. Serum microRNA profiling and breast cancer risk: the use of miR-484/191 as endogenous controls. Carcinogenesis 33(4), 828–834 (2012).

75

Wu Q, Wang C, Lu Z, Guo L, Ge Q. Analysis of serum genome-wide microRNAs for breast cancer detection. Clin. Chim. Acta 413(13–14), 1058–1065 (2012).




76

77

78

Zhao H, Shen J, Medico L, Wang D, Ambrosone CB, Liu S. A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer. PLoS ONE 5(10), e13735 (2010). Cookson V, Bentley M, Hogan B et al. Circulating microRNA profiles reflect the presence of breast tumours but not the profiles of microRNAs within the tumours. Cell. Oncol. 35(4), 301–308 (2012). Ryu S, Joshi N, McDonnell K et al. Discovery of novel human breast cancer microRNAs from deep sequencing data by analysis of pri-microRNA secondary structures. PLoS ONE 6(2), e16403 (2011).

Tumor Biol. 34(1), 455–462 (2013). 93

Si H, Sun X, Chen Y et al. Circulating microRNA-92a and microRNA-21 as novel minimally invasive biomarkers for primary breast cancer. J. Cancer Res. Clin. Oncol. 139(2), 223–229 (2013).

94

Madhavan D, Zucknick M, Wallwiener M et al. Circulating miRNAs as surrogate markers for circulating tumor cells and prognostic markers in metastatic breast cancer. Clin. Cancer Res. 18(21), 5972–5982 (2012).

95

Eichelser C, Flesch-Janys D, Chang-Claude J, Pantel K, Schwarzenbach H. Deregulated serum concentrations of circulating cell–free microRNAs miR-17, miR-34a, miR-155, and miR-373 in human breast cancer development and progression. Clin. Chem. 59(10), 1489–1496 (2013).

79

Volinia S, Galasso M, Sana ME et al. Breast cancer signatures for invasiveness and prognosis defined by deep sequencing of microRNA. Proc. Natl Acad. Sci. USA 109(8), 3024–3029 (2012).

96

80

Farazi TA, Horlings HM, Ten Hoeve JJ et al. MicroRNA sequence and expression analysis in breast tumors by deep sequencing. Cancer Res. 71(13), 4443–4453 (2011).

Haythorn MR, Ablin RJ. Prostate-specific antigen testing across the spectrum of prostate cancer. Biomark. Med. 5(4), 515–526 (2011).

97

81

Jiang S, Zhang H-W, Lu M-H et al. MicroRNA-155 functions as an OncomiR in breast cancer by targeting the suppressor of cytokine signaling 1 gene. Cancer Res. 70(8), 3119–3127 (2010).

Canto EI, Slawin KM. Early management of prostate cancer. How to respond to an elevated PSA? Annu. Rev. Med. 53(1), 355–368 (2002).

98

Gulati R, Gore JL, Etzioni R. Comparative effectiveness of alternative prostate-specific antigen-based prostate cancer screening strategies model estimates of potential benefits and harms. Ann. Intern. Med. 158(3), 145–153 (2013). Moltzahn F, Olshen AB, Baehner L et al. Microfluidic-based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in the sera of prostate cancer patients. Cancer Res. 71(2), 550–560 (2011).

82

Sun Y, Wang M, Lin G et al. Serum microRNA-155 as a potential biomarker to track disease in breast cancer. PLoS ONE 7(10), e47003 (2012).

83

Liu J, Mao Q, Liu Y, Hao X, Zhang S, Zhang J. Analysis of miR-205 and miR-155 expression in the blood of breast cancer patients. Chin. J. Cancer Res. 25(1), 46–54 (2012).

99

84

Wang Py GH, Li Bf, Lv Cl et al. Higher expression of circulating miR-182 as a novel biomarker for breast cancer. Oncol. Lett. 6(6), 1681–1686 (2013).

100 Yaman Agaoglu F, Kovancilar M, Dizdar Y et al.

85

86

87

88

89

90

91

92

Kim G, Yong Y, Kang HJ et al. Zwitterionic polymercoated immunobeads for blood-based cancer diagnostics. Biomaterials 35(1), 294–303 (2014). Ng EKO, Li R, Shin VY et al. Circulating microRNAs as specific biomarkers for breast cancer detection. PLoS ONE 8(1), e53141 (2013). Mar-Aguilar F, Mendoza-Ramirez JA, Malagón-Santiago I et al. Serum circulating microRNA profiling for identification of potential breast cancer biomarkers. Dis. Markers 34(3), 163–169 (2013).

Investigation of miR-21, miR-141, and miR-221 in blood circulation of patients with prostate cancer. Tumor Biol. 32(3), 583–588 (2011). 101 Mahn R, Heukamp LC, Rogenhofer S, Von Ruecker A,

Müller SC, Ellinger J. Circulating microRNAs (miRNA) in serum of patients with prostate cancer. Urology 77(5), 1265. e1269–1265.e1216 (2011). 102 Bryant RJ, Pawlowski T, Catto JWF et al. Changes in

circulating microRNA levels associated with prostate cancer. Br J. Cancer 106(4), 768–774 (2012). 103 Chen Z-H, Zhang G-L, Li H-R et al. A panel of five

circulating microRNAs as potential biomarkers for prostate cancer. Prostate 72(13), 1443–1452 (2012).

Chan M, Liaw CS, Ji SM et al. Identification of circulating microRNA signatures for breast cancer detection. Clin. Cancer Res. 19(16), 4477–4487 (2013).

104 Selth LA, Townley SL, Bert AG et al. Circulating

Cuk K, Zucknick M, Heil J et al. Circulating microRNAs in plasma as early detection markers for breast cancer. Int. J. Cancer 132(7), 1602–1612 (2013).

105 Shen J, Hruby GW, McKiernan JM et al. Dysregulation of

Cuk K, Zucknick M, Madhavan D et al. Plasma microRNA panel for minimally invasive detection of breast cancer. PLoS ONE 8(10), e76729 (2013).

106 Nguyen HCN, Xie W, Yang M et al. Expression differences

Zeng R-C, Zhang W, Yan X-Q et al. Down-regulation of miRNA-30a in human plasma is a novel marker for breast cancer. Med. Oncol. 30(1), 1–8 (2013). Chen W, Cai F, Zhang B, Barekati Z, Zhong X. The level of circulating miRNA-10b and miRNA-373 in detecting lymph node metastasis of breast cancer: potential biomarkers.


Review

microRNAs predict biochemical recurrence in prostate cancer patients. Br J. Cancer 109(3), 641–650 (2013). circulating microRNAs and prediction of aggressive prostate cancer. Prostate 72(13), 1469–1477 (2012). of circulating microRNAs in metastatic castration resistant prostate cancer and low-risk, localized prostate cancer. Prostate 73(4), 346–354 (2013). 107 Watahiki A, MacFarlane R, Gleave M et al. Plasma miRNAs

as biomarkers to identify patients with castration-resistant metastatic prostate cancer. Int. J. Mol. Sci. 14(4), 7757–7770 (2013).


149

Review Khoury & Tran 108 Cheng HH, Mitchell PS, Kroh EM et al. Circulating

microRNA profiling identifies a subset of metastatic prostate cancer patients with evidence of cancer-associated hypoxia. PLoS ONE 8(7), e69239 (2013). 109 Lodes MJ, Caraballo M, Suciu D, Munro S, Kumar A,

Anderson B. Detection of cancer with serum miRNAs on an oligonucleotide microarray. PLoS ONE 4(7), e6229 (2009). 110 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer

Statistics, 2009. CA. Cancer J. Clin. 59(4), 225–249 (2009). 111 Hu Z, Chen X, Zhao Y et al. Serum microRNA signatures

identified in a genome-wide serum microRNA expression profiling predict survival of non–small-cell lung cancer. J. Clin. Oncol. 28(10), 1721–1726 (2010). 112 Yu S-L, Chen H-Y, Chang G-C et al. MicroRNA signature

predicts survival and relapse in lung cancer. Cancer Cell 13(1), 48–57 (2008). 113 Foss KM, Sima C, Ugolini D, Neri M, Allen KE, Weiss

GJ. miR-1254 and miR-574–5p: serum-based microRNA biomarkers for early-stage non-small cell lung cancer. J. Thorac. Oncol. 6(3), 482–488 (2011). 114 Chen Q, Si Q, Xiao S et al. Prognostic significance of serum

miR-17–5p in lung cancer. Med. Oncol. 30(1), 1–6 (2012). 115 Yuxia M, Zhennan T, Wei Z. Circulating miR-125b is a

novel biomarker for screening non-small-cell lung cancer and predicts poor prognosis. J. Cancer Res. Clin. Oncol. 138(12), 2045–2050 (2012). 116 Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou

E. Clinical evaluation of microRNA expression profiling in non small cell lung cancer. Lung Cancer (Amsterdam, The Netherlands) 81(3), 388–396 (2013). 117 Yu H, Jiang L, Sun C et al. Decreased circulating miR-375:

a potential biomarker for patients with non-small-cell lung cancer. Gene 534(1), 60–65 (2014). 118 Zhang W-H, Gui J-H, Wang C-Z et al. The identification

of miR-375 as a potential biomarker in distal gastric adenocarcinoma. Oncol. Res. 20(4), 139–147 (2012). 119 Zheng D HS, Wang Y, Gu LQ, Perry MC, Freter CE, Wang

MX.. Plasma microRNAs as novel biomarkers for early detection of lung cancer. Int. J. Clin. Exp. Pathol. 4(6), 575–586 (2011). 120 Heegaard NHH, Schetter AJ, Welsh JA, Yoneda M, Bowman

ED, Harris CC. Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer. Int. J. Cancer 130(6), 1378–1386 (2012). 121 Kaduthanam S, Gade S, Meister M et al. Serum miR-

142–3p is associated with early relapse in operable lung adenocarcinoma patients. Lung Cancer (Amsterdam, The Netherlands) 80(2), 223–227 (2013). 122 Rani S, Gately K, Crown J, O’Byrne K, O’Driscoll L. Global

analysis of serum microRNAs as potential biomarkers for lung adenocarcinoma. Cancer Biol. Ther. 14(12), 1104–1112 (2013). 123 Patnaik SK, Yendamuri S, Kannisto E, Kucharczuk JC,

Singhal S, Vachani A. MicroRNA expression profiles of whole blood in lung adenocarcinoma. PLoS ONE 7(9), e46045 (2012).

150


124 Tomasetti M, Staffolani S, Nocchi L et al. Clinical

significance of circulating miR-126 quantification in malignant mesothelioma patients. Clin. Biochem. 45(7–8), 575–581 (2012). 125 Akbas F, Coskunpinar E, Aynacı E, Müsteri Oltulu Y, Yildiz

P. Analysis of serum micro-RNAs as potential biomarker in chronic obstructive pulmonary disease. Exp. Lung Res. 38(6), 286–294 (2012). 126 Han H-S, Yun J, Lim S-N et al. Downregulation of cell-free

miR-198 as a diagnostic biomarker for lung adenocarcinomaassociated malignant pleural effusion. Int. J. Cancer 133(3), 645–652 (2013). 127 Abd-El-Fattah A, Sadik N, Shaker O, Aboulftouh M.

Differential microRNAs expression in serum of patients with lung cancer, pulmonary tuberculosis, and pneumonia. Cell Biochem. Biophys. 67(3), 875–884 (2013). 128 Aushev VN, Zborovskaya IB, Laktionov KK et al.

Comparisons of microRNA patterns in plasma before and after tumor removal reveal new biomarkers of lung squamous cell carcinoma. PLoS ONE 8(10), e78649 (2013). 129 Le H-B, Zhu W-Y, Chen D-D et al. Evaluation of dynamic

change of serum miR-21 and miR-24 in pre- and postoperative lung carcinoma patients. Med. Oncol. 29(5), 3190–3197 (2012). 130 Roth C, Stückrath I, Pantel K, Izbicki JR, Tachezy M,

Schwarzenbach H. Low levels of cell-free circulating miR-361–3p and miR-625* as blood-based markers for discriminating malignant from benign lung tumors. PLoS ONE 7(6), e38248 (2012). 131 Pan Z, Pang L, Ding B et al. Gastric cancer staging with

dual energy spectral CT imaging. PLoS ONE 8(2), e53651 (2013). 132 Burbidge S, Mahady K, Naik K. The role of CT and

staging laparoscopy in the staging of gastric cancer. Clin. Radiol. 68(3), 251–255 (2013). 133 Gong J, Kang W, Zhu J, Xu J. CT and MR imaging

of gastrointestinal stromal tumor of stomach: a pictorial review. Quant. Imaging Med. Surg. 2(4), 274–279 (2012). 134 Liu R, Zhang C, Hu Z et al. A five-microRNA signature

identified from genome-wide serum microRNA expression profiling serves as a fingerprint for gastric cancer diagnosis. Eur. J. Cancer 47(5), 784–791 (2010). 135 Okubo M, Tahara T, Shibata T et al. Association between

common genetic variants in pre-microRNAs and gastric cancer risk in Japanese population. Helicobacter 15(6), 524–531 (2010). 136 Tsujiura M, Ichikawa D, Komatsu S et al. Circulating

microRNAs in plasma of patients with gastric cancers. Br. J. Cancer 102(7), 1174–1179 (2010). 137 Zhou H, Guo J-M, Lou Y-R et al. Detection of circulating

tumor cells in peripheral blood from patients with gastric cancer using microRNA as a marker. J. Mol. Med. 88(7), 709–717 (2010). 138 Zheng Y, Cui L, Sun W et al. MicroRNA-21 is a new marker

of circulating tumor cells in gastric cancer patients. Cancer Biomark. 10(2), 71–77 (2011).



139 Komatsu S, Ichikawa D, Tsujiura M et al. Prognostic impact

of circulating miR-21 in the plasma of patients with gastric carcinoma. Anticancer Res. 33(1), 271–276 (2013). 140 Kim SY, Jeon TY, Choi CI et al. Validation of circulating

miRNA biomarkers for predicting lymph node metastasis in gastric cancer. J. Mol. Diagn. 15(5), 661–669 (2013). 141 Chen Q, Ge X, Zhang Y et al. Plasma miR-122 and miR-

192 as potential novel biomarkers for the early detection of distant metastasis of gastric cancer. Oncol. Rep. 31(4), 1863–1870 (2014). 142 Tsai Kw LY, Wu Cw, Hu Ly et al. Aberrant expression

of miR-196a in gastric cancers and correlation with recurrence. Genes Chromosomes Cancer 51(4), 394–401 (2012). 143 Valladares-Ayerbes M, Reboredo M, Medina-Villaamil V

et al. Circulating miR-200c as a diagnostic and prognostic biomarker for gastric cancer. J. Transl. Med. 10(1), 186 (2012). 144 Li C, Li JF, Cai Q et al. MiRNA-199a-3p: a potential

circulating diagnostic biomarker for early gastric cancer. J. Surg. Oncol. 108(2), 89–92 (2013). 145 Tsujiura M, Komatsu S, Ichikawa D et al. Circulating miR-18a

in plasma contributes to cancer detection and monitoring in patients with gastric cancer. Gastric Cancer doi:10.1007/ s10120-014-0363-1 (2014) (Epub ahead of print). 146 Wang B, Zhang Q. The expression and clinical

significance of circulating microRNA-21 in serum of five solid tumors. J. Cancer Res. Clin. Oncol. 138(10), 1659–1666 (2012). 147 Liu R, Zhang C, Hu Z et al. A five-microRNA signature

identified from genome-wide serum microRNA expression profiling serves as a fingerprint for gastric cancer diagnosis. Eur. J. Cancer 47(5), 784–791 (2011). 148 Gorur A, Balci Fidanci S, Dogruer Unal N et al.

Determination of plasma microRNA for early detection of gastric cancer. Mol. Biol. Rep. 40(3), 2091–2096 (2013). 149 Wang J, Chen J, Chang P et al. MicroRNAs in plasma of

pancreatic ductal adenocarcinoma patients as novel bloodbased biomarkers of disease. Cancer Prev. Res. (Phila PA) 2(9), 807–813 (2009).


Review

150 Wang X, Tang S, Le SY et al. Aberrant expression of

oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS ONE 3(7), e2557 (2008). 151 Tili E, Michaille JJ, Croce CM. MicroRNAs play a central

role in molecular dysfunctions linking inflammation with cancer. Immunol. Rev. 253(1), 167–184 (2013). 152 Staedel C, Darfeuille F. MicroRNAs and bacterial infection.

Cell. Microbiol. 15(9), 1496–1507 (2013). 153 Haider BA, Baras AS, McCall MN, Hertel JA, Cornish

TC, Halushka MK. A critical evaluation of microRNA biomarkers in non-neoplastic disease. PLoS ONE 9(2), e89565 (2014). 154 Prochazka M, Granath F, Ekbom A, Shields PG, Hall P.

Lung cancer risks in women with previous breast cancer. Eur J. Cancer 38(11), 1520–1525 (2002). 155 Kirschner MB, Kao SC, Edelman JJ et al. Haemolysis during

sample preparation alters microRNA content of plasma. PLoS ONE 6(9), e24145 (2011). 156 Pritchard CC, Kroh E, Wood B et al. Blood cell origin of

circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prev Res. (Phila) 5(3), 492–497 (2012). 157 Sawant RB, Jathar SK, Rajadhyaksha SB, Kadam PT.

Red cell hemolysis during processing and storage. Asian J. Transfus. Sci. 1(2), 47–51 (2007). 158 Noe DA, Weedn V, Bell WR. Direct spectrophotometry of

serum hemoglobin: an Allen correction compared with a three-wavelength polychromatic analysis. Clin. Chem. 30(5), 627–630 (1984). 159 Kirschner MB, Van Zandwijk N, Reid G. Cell-free

microRNAs: potential biomarkers in need of standardized reporting. Front. Genet. 4, 56 (2013). 160 Weber JA, Baxter DH, Zhang S et al. The microRNA

spectrum in 12 body fluids. Clin. Chem. 56(11), 1733–1741 (2010). 161 Park NJ, Zhou H, Elashoff D et al. Salivary

microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin. Cancer Res. 15(17), 5473–5477 (2009).


151