COMMENTARY Circulating microRNAs: novel biomarkers for early detection of colorectal cancer RAJAGOPAL N. ARAVALLI, and CLIFFORD J. STEER MINNEAPOLIS, MINN
Article on page XXX. Differential expression of circulating miRNAs according to severity of colorectal neoplasia Ho GY, Jung HJ, Schoen RE, Wang T, Lin J, Williams Z, Weissfeld JL, Park JY, Loudig O, and Suh Y
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olorectal cancer (CRC) is the third most common cancer in the United States,1 and about 1.2 million individuals are diagnosed each year with CRC worldwide.2 Although tumor resection is the most common curative treatment option for about two-thirds of patients with CRC, recurrence rates are very high (28%–50%) and pose a serious challenge to reducing mortality in these individuals.3 Five-year survival of patients with localized CRC is 90% after surgery.4 About 50% of patients with CRC develop metastasis, after which the 5-year survival rate drops to 60% in these individuals contributing to the increased rate of mortality.5 In the absence of a specific risk factor for this disease, prognosis is typically based on the extent of tumor at diagnosis. The detection of early stage disease and improved surveillance has reduced
From the Department of Radiology, University of Minnesota Medical School, Minneapolis, Minn; Department of Medicine, University of Minnesota Medical School, Minneapolis, Minn; Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minn. Reprint requests: Rajagopal N. Aravalli, Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455, and Clifford J. Steer, Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455; e-mail: arava001@ umn.edu or [email protected]. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.04.007
mortality rates, as shown recently in a 30-year followup study.6 Therefore, early detection is critical to effectively treating CRC, and novel biomarkers are urgently needed for this purpose. MicroRNAs (miRNAs) are a unique class of short noncoding RNA molecules (19–24 nucleotides long) that are implicated in numerous cancers.7 They exert their effects by regulating gene expression primarily at the post-transcriptional level, where they either induce mRNA degradation or translational inhibition. miRNAs are often dysregulated in CRC; and both overexpression and downregulation of specific miRNAs have been shown to be associated with CRC.8-10 Profiling of miRNAs in CRC cell lines and patient samples have shown that a large number of them were aberrantly expressed.8-11 Although it is not entirely clear how the dysregulation of miRNAs occurs in CRC, a number of potential mechanisms have been proposed, including epigenetic modifications and various cellular signaling cascades.11 In addition, putative targets of some of these miRNAs have been identified. Seminal discovery of circulating placenta-specific miRNAs in the maternal plasma12 has led to numerous studies on the identification of miRNAs in various body fluids, such as saliva, urine, and amniotic fluid.13,14 This subsequently resulted in the establishment of an miRNA database termed ‘‘miRandola’’ on circulating miRNAs found in various diseases.15 This database originally contained 581 miRNAs from 21 sample types and was expanded recently to include information on the function, diagnostic potential, and drug effects on cellular 1
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Table I. Circulating microRNAs identified in patients with colorectal cancer Sample type
Serum
Plasma
Stool
MicroRNA
Reference
miR-17-3p, miR-29a, miR-92a, miR-135b miR-20a, miR-130, miR-145, miR-216, miR-372 miR-21 miR-21 miR-21, miR-92a miR-21, miR-29a, miR-34a, miR-92a, miR-103 miR-106a, miR-107, miR-143, miR-146a miR-151-5p, miR-155, miR-191, miR-199a-3p miR-210, miR-221, miR-320a, miR-378, miR-382 miR-409-3p, miR-423-3p, miR-423-5p, miR-425 miR-484, miR-652, miR-720, let7d miR-27b, miR-106a, miR-130b, miR-148a miR-326, miR-484 miR-29a miR-21, miR-29a, miR-30b, miR-30c, miR-30d miR-146a, miR-486 miR-92, miR-17-3p miR-141 miR-200c miR-601, miR-760 let7a, miR-21, miR-23a, miR-150, miR-223 miR-1229, miR-1246 miR-15b, miR-19a, miR-19b, miR-29a, miR-335 miR-18a (only in adenoma patients) miR-15b, miR-17, miR-142-3p, miR-195, miR-331 miR-532, miR-532-3p, miR-652 miR-23a, miR-193a-3p, miR-338-5p miR-29a, miR-92a miR-92 miR-221 miR-21, miR-92a miR-135b miR-221, miR-18a
Faltejskova et al24 Zhang et al39 Wang and Zhang40 Kanaan et al41 Liu et al20
and circulating miRNAs.16 A number of studies have also reported the presence of miRNAs in high concentrations in body fluids and stool of patients with CRC.17-36 These miRNAs could potentially act as novel biomarkers for noninvasive diagnosis of CRC. Most of these miRNAs were identified using standard methods of microarray and real-time reverse transcription–polymerase chain reaction (RT-PCR) approaches. In this issue of Translational Research, Ho et al37 used a novel, cutting-edge technology of nextgeneration sequencing (NGS) to profile miRNAs in serum samples of 5 patients with colorectal neoplasia. These findings strongly support other reports that certain miRNAs are downregulated in CRC.22,38,39 NGS allows for the identification of novel miRNAs, including those with sequence changes and variants such as iso-miRs. In addition, downregulated or silenced miRNAs are interesting candidates, in that they could potentially function as tumor suppressor agents. Combinations of miRNAs (both upregulated and downregulated) and current diagnostic methods
Hofsli et al38
Kjersem et al42 Wang and Gu21 Ho et al37 Ng et al17 Cheng et al35 Toiyama et al30 Wang et al22 Ogata-Kawata43 ldez et al44 Gira Kanaan et al25 Yong et al29 Huang et al19 Ng et al17 Pu et al36 Wu et al32 Wu et al33 Yau et al34
have recently been shown to dramatically improve the detection of CRC.22 In that study, a combination of miR-92a, miR-29a, and miR-760 together with carcinoembryonic antigen (CEA) analysis significantly enhanced early detection of CRC. Among the so-called downregulated miRNAs reported in the literature as potential biomarkers for CRC are miR-601, miR-760, miR-106a, miR-143, miR-103, miR-199a-3p, miR151-5p, miR-107, miR-191, miR-423-3p, let7d, miR-409-3p, miR-652, miR-20a, miR-130, miR-145, miR-216, and miR-37222,38,39 (Table I). In their study, Ho et al37 have identified miR-30 family (miR-30b, miR-30C, and miR-30d), and miR-146a as putative candidate circulating biomarkers for the early detection of colorectal neoplasia. However, these miRNAs are not novel for CRC studies. MiR-30a was previously shown to function as a tumor suppressor by targeting the Akt/mTOR pathway,45 and insulin receptor substrate 246 in colon cancer cell lines and tissues. Interestingly, a polymorphism (rs2690164) in the miR-146a was found to be associated with susceptibility
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and prognosis in a study cohort of 399 patients with CRC.47 Although others have reported miR-21 and miR-29a as miRNAs that were significantly upregulated in CRC,20,21,24,28,38,40,41 Ho et al found that these circulating miRNAs were downregulated in CRC and advanced cases of adenoma. There was, however, no correlation with tissue samples and their respective levels of miRNAs. Specific miRNAs identified in tissue specimens of patients with CRC and body fluid samples would enhance their value as reliable biomarkers for noninvasive diagnosis. For instance, the upregulated expression of miR-21 secreted from CRC cells in vitro correlated with that found in tissue and serum sets, and mirrored its downregulated expression levels in serum after complete tumor resection.28 In addition, identification of high-risk patients among those with stage II/III CRC using circulating miRNAs would greatly benefit in selecting candidates for adjuvant therapy, which was shown to be effective in stage II48 and stage III patients.49 It has been shown that the circulating miRNAs are tissue-specific, stable in circulation, potentially derived from tumor tissues, and possibly correlate with tumor progression and recurrence, which may render them as highly sensitive biomarkers for CRC.30,50 Stability and circulation of miRNAs in body fluids was attributed to RNA-binding proteins51,52 or subcellular particles, such as microvesicles53,54 and exosomes.55 Recent studies have unraveled secretory mechanisms and incorporation of extracellular miRNAs into mammalian cells. Kosaka et al56 suggested that miRNAs are released through a ceramide-dependent secretory machinery, and the secreted miRNAs are transported between the cells. Zernecke et al57 suggested that apoptotic bodies may transfer miRNAs between cells. In that study, the authors showed that miR-126 was enriched in apoptotic bodies of endothelial cells during atherosclerosis that conveyed paracrine alarm signals to recipient vascular cells triggering the production of chemokine CXCL12. These studies, taken together with the finding that cancer cells communicate with other cells in the tumor microenvironment via exosomes,53,58 underscore that miRNAs are secreted and transported by cells in vivo. In one of the earliest studies on circulating miRNAs, Ng et al17 used RT-PCR with plasma and tissue samples from patients with CRC and found that 5 miRNAs were upregulated. Subsequent validation studies with 90 more patient plasma samples showed that miR-92 was significantly increased suggesting that this miRNA might be a potential biomarker for CRC. Other groups have also found that miR-92 could be a marker for the early detection of CRC.19,20,24 Similarly, a number of other miRNAs have been found to be either
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upregulated or downregulated during various stages of CRC (Table I) suggesting that these miRNAs have greater sensitivity and specificity than the use of single miRNAs. In contrast to these studies, Ho et al37 focused on miRNAs that were downregulated in CRC serum specimens to test whether they can act as biomarkers. They report on the differential expression of circulating miRNAs that are correlated with the severity of CRC. The novelty of the study was not for establishing the feasibility of using serum miRNAs to differentiate a spectrum of cancers, but rather for identifying miRNAs that were downregulated compared with controls using NGS. This case-control study examined the feasibility of using serum-based miRNAs to differentiate a spectrum of early to advanced colorectal neoplasia. Archived serum samples of 10 normal controls and 31 cases, including 10 with nonadvanced adenoma, 10 with advanced adenoma, and 11 with CRC, were profiled for circulating miRNAs using rapid sequencing technology. The authors used multiple linear regression, adjusting for age, gender, and smoking status, to compare 3 case groups to controls for levels of 175 miRNAs that met stringent criteria for sequencing analysis. Of the 175 miRNAs, 106 miRNAs were downregulated according to the severity of neoplasia and showed a significant and relative decrease in expression from controls to nonadvanced adenoma to advanced adenoma to CRC. Pairwise group comparisons showed that 39 and 80 miRNAs were differentially expressed in the advanced adenoma and cancer groups, compared with the controls, respectively. The study found that expression of many miRNAs in serum was inversely correlated with the severity of colorectal neoplasia, and differential miRNA profiles were apparent in preneoplastic cases with advanced lesions. Taken together, the results suggested that circulating miRNAs could serve as potential biomarkers for CRC screening. Although the number of downregulated miRNAs was somewhat unexpected, such a trend has been previously reported in colon cancer.17 Even aside from the potential concerns of the sequencing methods used to detect them, it remains to be seen as to how useful the measure of downregulated circulating miRNAs will be in assessing disease. That said, even the authors admitted that the study was not intended to identify miRNA markers that have clinical utility, but rather as a proof-of-concept to stimulate future translational research on the use of measuring circulating miRNAs for CRC screening. Currently, the fecal occult blood test and the measure of CEA in the serum are the most widely used screens for CRC. Fecal occult blood test has a sensitivity between 30% and 50%,59 and specificity for CRC and
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adenomas between 90% and 98%60, and therefore is a good screening modality for CRC. On the other hand, the sensitivity of CEA was found to be very low in some studies,61 whereas it was shown to be high in others.62 Because of these inconsistencies, CEA may not be a suitable biomarker for early stage detection of CRC. In contrast to CEA, certain miRNAs exhibit higher sensitivity and specificity that make them potentially useful as reliable biomarkers. In addition, serum miRNAs are resistant to RNase A digestion, and are stable under conditions, such as low pH and high pH, extended storage and freeze-thaw cycles, and high temperatures.63 Because of these distinct advantages, circulating miRNAs may be more suited for screening of CRC as they have significantly lower rates of producing false-positive and false-negative results than conventional diagnostic methods.64 However, studies on serum miRNAs are associated with high cost and long latency time. Although the study by Ho et al37 is aimed at identifying reliable biomarkers using NGS, it is not cost effective, which may limit screening of a wide range of patients, such as those with different age and health status, comorbidities, and lifestyle variations. Moreover, because of the small sample size, their results are not translatable unless the miRNAs identified in this study are validated in a large sample of patients from different geographical regions. This is critical because occurrence of miRNA polymorphisms and cancer risk vary in different populations.65,66 Despite these differences, a combination of biomarkers would certainly improve the early diagnosis, and only a small number of miRNAs might be sufficient to differentiate the various stages of CRC in a costeffective manner. The translational potential and importance of measuring circulating miRNAs for CRC screening will require additional studies that include (1) far larger samples sizes; (2) advanced precancerous adenomas; (3) identification and validation of promising miRNA markers when adjusting for confounding factors, such as inflammatory bowel disease or other inflammatory conditions, that could affect miRNA expression; (4) correlation of serum levels with those of tissues specific for colorectal tumors; (5) variation and latency of these promising miRNAs over time in serial blood samples; and (6) establishing the utility of quantitative RT-PCR in detecting miRNA markers in a target population for CRC screening. Given that most of the miRNAs in the study were downregulated in individuals with disease, it will be critical to pinpoint the markers that despite their levels in diseased individuals are less than normal can be detected reliably with high sensitivity by a clinical diagnostic technology.
The study by Ho et al37 provides additional support for the role of circulating miRNAs in a new frontier of diagnostics. More significantly, it underscores the importance of developing robust, sensitive, accurate, and reproducible methods for their identification and application to clinical disease. With such a small sample size, the study was not designed to identify and validate diagnostic or early detection miRNA markers for colorectal neoplasia, but rather a proof-of-concept to demonstrate differential expression of circulating miRNAs against severity of disease using NGS. Although research on circulating miRNAs is still relatively recent, stringent analytical standards for study design and statistics are prerequisites for understanding their role in health and disease. The identification of potential targets of these circulating miRNAs will almost invariably uncover, heretofore, unrecognized regulatory mechanisms of cell function. The effective diagnosis and surveillance of complex multifactorial disorders such as CRC mandates the discovery of easily accessible biomarkers, such as tissue-specific and circulating miRNAs. Most importantly, we now recognize the critical considerations required for their isolation and characterization using cutting edge technology that is accurate and reproducible. There is an urgent need to develop a CRC-screening test that is noninvasive, cost effective, and sensitive enough to detect both neoplastic and perhaps even more importantly, preneoplastic lesions. The findings by Ho et al37 that miRNAs were not only downregulated but also decreased in a linear fashion with the stage of CRC highlight the timely, important, and potentially translational nature of the study. REFERENCES
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29. Yong FL, Law CW, Wang CW. Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer. BMC Cancer 2013;13:280. 30. Toiyama Y, Hur K, Tanaka K, et al. Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer. Ann Surg 2014;259:735–43. 31. Fesler A, Jiang J, Zhai H, Ju J. Circulating microRNA testing for the early diagnosis and follow-up of colorectal cancer patients. Mol Diagn Ther 2014;18:303–8. 32. Wu CW, Ng SS, Dong YJ, et al. Detection of miR-92a and miR-21 in stool samples as potential screening biomarkers for colorectal cancer and polyps. Gut 2012;61:739–45. 33. Wu CW, Ng SC, Dong Y, et al. Identification of microRNA-135b in stool as a potential noninvasive biomarker for colorectal cancer and adenoma. Clin Cancer Res 2014;20:2994–3002. 34. Yau TO, Wu CW, Dong Y, et al. microRNA-221 and microRNA18a identification in stool as potential biomarkers for the noninvasive diagnosis of colorectal carcinoma. Br J Cancer 2014; 111:1765–71. 35. Cheng H, Zhang L, Cogdell DE, et al. Circulating plasma MiR141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis. PLoS One 2011;6:e17745. 36. Pu XX, Huang GL, Guo HQ, et al. Circulating miR-221 directly amplified from plasma is a potential diagnostic and prognostic marker of colorectal cancer and is correlated with p53 expression. J Gastroenterol Hepatol 2010;25:1674–80. 37. Ho GY, Jung JH, Schoen RE, et al. Differential expression of circulating miRNAs according to severity of colorectal neoplasia. Transl Res 2015 (in press). 38. Hofsli E, Sjursen W, Prestvik WS, et al. Identification of serum microRNA profiles in colon cancer. Br J Cancer 2013;108: 1712–9. 39. Zhang J, Zhang K, Bi M, Jiao X, Zhang D, Dong Q. Circulating microRNA expressions in colorectal cancer as predictors of response to chemotherapy. Anticancer Drugs 2014;25: 346–52. 40. Wang B, Zhang Q. The expression and clinical significance of circulating microRNA-21 in serum of five solid tumors. J Cancer Res Clin Oncol 2012;138:1659–66. 41. Kanaan Z, Rai SN, Eichenberger MR, et al. Plasma miR-21: a potential diagnostic marker of colorectal cancer. Ann Surg 2012;256:544–51. 42. Kjersem JB, Ikdahl T, Lingjaerde OC, Guren T, Tveit KM, Kure EH. Plasma microRNAs predicting clinical outcome in metastatic colorectal cancer patients receiving first-line oxaliplatinbased treatment. Mol Oncol 2014;8:59–67. 43. Ogata-Kawata H. Circulating exosomal microRNAs as biomarkers of colon cancer. PLoS One 2014;9:e92921. 44. Giraldez MD, Lozano JJ, Ramırez G, et al. Circulating microRNAs as biomarkers of colorectal cancer: results from a genome-wide profiling and validation study. Clin Gastroenterol Hepatol 2013;11:681–8. 45. Zhong M, Bian Z, Wu Z. miR-30a suppresses cell migration and invasion through downregulation of PIK3CD in colorectal carcinoma. Cell Physiol Biochem 2013;31:209–18. 46. Zhang Q, Tang Q, Qin D, et al. Role of microRNA 30a targeting insulin receptor substrate 2 in colorectal tumorigenesis. Mol Cell Biol 2015;35:988–1000. 47. Chae YS, Kim JG, Lee SJ, et al. A miR-146a polymorphism (rs2910164) predicts risk of and survival from colorectal cancer. Anticancer Res 2013;33:3233–9. 48. Figueredo A, Charette ML, Maroun J, Brouwers MC, Zuraw L. Adjuvant therapy for stage II colon cancer: a systematic review from the Cancer Care Ontario Program in evidence-based care’s
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58. Wolfers J, Lozier A, Raposo G, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL crosspriming. Nat Med 2001;7:297–303. 59. Graser A, Stieber P, Nagel D, et al. Comparison of CT colonography, colonoscopy, sigmoidoscopy and faecal occult blood tests for the detection of advanced adenoma in an average risk population. Gut 2009;58:241–8. 60. Duffy MJ, van Dalen A, Haglund C, et al. Tumour markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines for clinical use. Eur J Cancer 2007;43: 1348–60. 61. Eleftheriadis N, Papaloukas C, Pistevou-Gompaki K. Diagnostic value of serum tumor markers in asymptomatic individuals. J BUON 2009;14:707–10. 62. Wang YR, Yan JX1, Wang LN. The diagnostic value of serum carcino-embryonic antigen, alpha fetoprotein and carbohydrate antigen 19-9 for colorectal cancer. J Cancer Res Ther 2014;10: 307–9. 63. Ahmed FE, Vos PW, Jeffries C, et al. Differences in mRNA and microRNA microarray expression profiles in human colon adenocarcinoma HT-29 cells treated with either intensity-modulated radiation therapy (IMRT), or conventional radiation therapy (RT). Cancer Genomics Proteomics 2009;6:109–27. 64. Kosaka N, Iguchi H, Ochiya T. Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci 2010;101:2087–92. 65. Srivastava K, Srivastava A. Comprehensive review of genetic association studies and meta-analyses on miRNA polymorphisms and cancer risk. PLoS One 2012;7:e50966. 66. Xu Y, Gu L, Pan Y, et al. Different effects of three polymorphisms in microRNAs on cancer risk in Asian population: evidence from published literatures. PLoS One 2013;8:e65123.
Article on page XXX. Differential expression of circulating miRNAs according to severity of colorectal neoplasia Ho GY, Jung HJ, Schoen RE, Wang T, Lin J, Williams Z, Weissfeld JL, Park JY, Loudig O, and Suh Y
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olorectal cancer (CRC) is the third most common cancer in the United States,1 and about 1.2 million individuals are diagnosed each year with CRC worldwide.2 Although tumor resection is the most common curative treatment option for about two-thirds of patients with CRC, recurrence rates are very high (28%–50%) and pose a serious challenge to reducing mortality in these individuals.3 Five-year survival of patients with localized CRC is 90% after surgery.4 About 50% of patients with CRC develop metastasis, after which the 5-year survival rate drops to 60% in these individuals contributing to the increased rate of mortality.5 In the absence of a specific risk factor for this disease, prognosis is typically based on the extent of tumor at diagnosis. The detection of early stage disease and improved surveillance has reduced
From the Department of Radiology, University of Minnesota Medical School, Minneapolis, Minn; Department of Medicine, University of Minnesota Medical School, Minneapolis, Minn; Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minn. Reprint requests: Rajagopal N. Aravalli, Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455, and Clifford J. Steer, Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455; e-mail: arava001@ umn.edu or [email protected]. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.04.007
mortality rates, as shown recently in a 30-year followup study.6 Therefore, early detection is critical to effectively treating CRC, and novel biomarkers are urgently needed for this purpose. MicroRNAs (miRNAs) are a unique class of short noncoding RNA molecules (19–24 nucleotides long) that are implicated in numerous cancers.7 They exert their effects by regulating gene expression primarily at the post-transcriptional level, where they either induce mRNA degradation or translational inhibition. miRNAs are often dysregulated in CRC; and both overexpression and downregulation of specific miRNAs have been shown to be associated with CRC.8-10 Profiling of miRNAs in CRC cell lines and patient samples have shown that a large number of them were aberrantly expressed.8-11 Although it is not entirely clear how the dysregulation of miRNAs occurs in CRC, a number of potential mechanisms have been proposed, including epigenetic modifications and various cellular signaling cascades.11 In addition, putative targets of some of these miRNAs have been identified. Seminal discovery of circulating placenta-specific miRNAs in the maternal plasma12 has led to numerous studies on the identification of miRNAs in various body fluids, such as saliva, urine, and amniotic fluid.13,14 This subsequently resulted in the establishment of an miRNA database termed ‘‘miRandola’’ on circulating miRNAs found in various diseases.15 This database originally contained 581 miRNAs from 21 sample types and was expanded recently to include information on the function, diagnostic potential, and drug effects on cellular 1
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Table I. Circulating microRNAs identified in patients with colorectal cancer Sample type
Serum
Plasma
Stool
MicroRNA
Reference
miR-17-3p, miR-29a, miR-92a, miR-135b miR-20a, miR-130, miR-145, miR-216, miR-372 miR-21 miR-21 miR-21, miR-92a miR-21, miR-29a, miR-34a, miR-92a, miR-103 miR-106a, miR-107, miR-143, miR-146a miR-151-5p, miR-155, miR-191, miR-199a-3p miR-210, miR-221, miR-320a, miR-378, miR-382 miR-409-3p, miR-423-3p, miR-423-5p, miR-425 miR-484, miR-652, miR-720, let7d miR-27b, miR-106a, miR-130b, miR-148a miR-326, miR-484 miR-29a miR-21, miR-29a, miR-30b, miR-30c, miR-30d miR-146a, miR-486 miR-92, miR-17-3p miR-141 miR-200c miR-601, miR-760 let7a, miR-21, miR-23a, miR-150, miR-223 miR-1229, miR-1246 miR-15b, miR-19a, miR-19b, miR-29a, miR-335 miR-18a (only in adenoma patients) miR-15b, miR-17, miR-142-3p, miR-195, miR-331 miR-532, miR-532-3p, miR-652 miR-23a, miR-193a-3p, miR-338-5p miR-29a, miR-92a miR-92 miR-221 miR-21, miR-92a miR-135b miR-221, miR-18a
Faltejskova et al24 Zhang et al39 Wang and Zhang40 Kanaan et al41 Liu et al20
and circulating miRNAs.16 A number of studies have also reported the presence of miRNAs in high concentrations in body fluids and stool of patients with CRC.17-36 These miRNAs could potentially act as novel biomarkers for noninvasive diagnosis of CRC. Most of these miRNAs were identified using standard methods of microarray and real-time reverse transcription–polymerase chain reaction (RT-PCR) approaches. In this issue of Translational Research, Ho et al37 used a novel, cutting-edge technology of nextgeneration sequencing (NGS) to profile miRNAs in serum samples of 5 patients with colorectal neoplasia. These findings strongly support other reports that certain miRNAs are downregulated in CRC.22,38,39 NGS allows for the identification of novel miRNAs, including those with sequence changes and variants such as iso-miRs. In addition, downregulated or silenced miRNAs are interesting candidates, in that they could potentially function as tumor suppressor agents. Combinations of miRNAs (both upregulated and downregulated) and current diagnostic methods
Hofsli et al38
Kjersem et al42 Wang and Gu21 Ho et al37 Ng et al17 Cheng et al35 Toiyama et al30 Wang et al22 Ogata-Kawata43 ldez et al44 Gira Kanaan et al25 Yong et al29 Huang et al19 Ng et al17 Pu et al36 Wu et al32 Wu et al33 Yau et al34
have recently been shown to dramatically improve the detection of CRC.22 In that study, a combination of miR-92a, miR-29a, and miR-760 together with carcinoembryonic antigen (CEA) analysis significantly enhanced early detection of CRC. Among the so-called downregulated miRNAs reported in the literature as potential biomarkers for CRC are miR-601, miR-760, miR-106a, miR-143, miR-103, miR-199a-3p, miR151-5p, miR-107, miR-191, miR-423-3p, let7d, miR-409-3p, miR-652, miR-20a, miR-130, miR-145, miR-216, and miR-37222,38,39 (Table I). In their study, Ho et al37 have identified miR-30 family (miR-30b, miR-30C, and miR-30d), and miR-146a as putative candidate circulating biomarkers for the early detection of colorectal neoplasia. However, these miRNAs are not novel for CRC studies. MiR-30a was previously shown to function as a tumor suppressor by targeting the Akt/mTOR pathway,45 and insulin receptor substrate 246 in colon cancer cell lines and tissues. Interestingly, a polymorphism (rs2690164) in the miR-146a was found to be associated with susceptibility
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and prognosis in a study cohort of 399 patients with CRC.47 Although others have reported miR-21 and miR-29a as miRNAs that were significantly upregulated in CRC,20,21,24,28,38,40,41 Ho et al found that these circulating miRNAs were downregulated in CRC and advanced cases of adenoma. There was, however, no correlation with tissue samples and their respective levels of miRNAs. Specific miRNAs identified in tissue specimens of patients with CRC and body fluid samples would enhance their value as reliable biomarkers for noninvasive diagnosis. For instance, the upregulated expression of miR-21 secreted from CRC cells in vitro correlated with that found in tissue and serum sets, and mirrored its downregulated expression levels in serum after complete tumor resection.28 In addition, identification of high-risk patients among those with stage II/III CRC using circulating miRNAs would greatly benefit in selecting candidates for adjuvant therapy, which was shown to be effective in stage II48 and stage III patients.49 It has been shown that the circulating miRNAs are tissue-specific, stable in circulation, potentially derived from tumor tissues, and possibly correlate with tumor progression and recurrence, which may render them as highly sensitive biomarkers for CRC.30,50 Stability and circulation of miRNAs in body fluids was attributed to RNA-binding proteins51,52 or subcellular particles, such as microvesicles53,54 and exosomes.55 Recent studies have unraveled secretory mechanisms and incorporation of extracellular miRNAs into mammalian cells. Kosaka et al56 suggested that miRNAs are released through a ceramide-dependent secretory machinery, and the secreted miRNAs are transported between the cells. Zernecke et al57 suggested that apoptotic bodies may transfer miRNAs between cells. In that study, the authors showed that miR-126 was enriched in apoptotic bodies of endothelial cells during atherosclerosis that conveyed paracrine alarm signals to recipient vascular cells triggering the production of chemokine CXCL12. These studies, taken together with the finding that cancer cells communicate with other cells in the tumor microenvironment via exosomes,53,58 underscore that miRNAs are secreted and transported by cells in vivo. In one of the earliest studies on circulating miRNAs, Ng et al17 used RT-PCR with plasma and tissue samples from patients with CRC and found that 5 miRNAs were upregulated. Subsequent validation studies with 90 more patient plasma samples showed that miR-92 was significantly increased suggesting that this miRNA might be a potential biomarker for CRC. Other groups have also found that miR-92 could be a marker for the early detection of CRC.19,20,24 Similarly, a number of other miRNAs have been found to be either
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upregulated or downregulated during various stages of CRC (Table I) suggesting that these miRNAs have greater sensitivity and specificity than the use of single miRNAs. In contrast to these studies, Ho et al37 focused on miRNAs that were downregulated in CRC serum specimens to test whether they can act as biomarkers. They report on the differential expression of circulating miRNAs that are correlated with the severity of CRC. The novelty of the study was not for establishing the feasibility of using serum miRNAs to differentiate a spectrum of cancers, but rather for identifying miRNAs that were downregulated compared with controls using NGS. This case-control study examined the feasibility of using serum-based miRNAs to differentiate a spectrum of early to advanced colorectal neoplasia. Archived serum samples of 10 normal controls and 31 cases, including 10 with nonadvanced adenoma, 10 with advanced adenoma, and 11 with CRC, were profiled for circulating miRNAs using rapid sequencing technology. The authors used multiple linear regression, adjusting for age, gender, and smoking status, to compare 3 case groups to controls for levels of 175 miRNAs that met stringent criteria for sequencing analysis. Of the 175 miRNAs, 106 miRNAs were downregulated according to the severity of neoplasia and showed a significant and relative decrease in expression from controls to nonadvanced adenoma to advanced adenoma to CRC. Pairwise group comparisons showed that 39 and 80 miRNAs were differentially expressed in the advanced adenoma and cancer groups, compared with the controls, respectively. The study found that expression of many miRNAs in serum was inversely correlated with the severity of colorectal neoplasia, and differential miRNA profiles were apparent in preneoplastic cases with advanced lesions. Taken together, the results suggested that circulating miRNAs could serve as potential biomarkers for CRC screening. Although the number of downregulated miRNAs was somewhat unexpected, such a trend has been previously reported in colon cancer.17 Even aside from the potential concerns of the sequencing methods used to detect them, it remains to be seen as to how useful the measure of downregulated circulating miRNAs will be in assessing disease. That said, even the authors admitted that the study was not intended to identify miRNA markers that have clinical utility, but rather as a proof-of-concept to stimulate future translational research on the use of measuring circulating miRNAs for CRC screening. Currently, the fecal occult blood test and the measure of CEA in the serum are the most widely used screens for CRC. Fecal occult blood test has a sensitivity between 30% and 50%,59 and specificity for CRC and
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adenomas between 90% and 98%60, and therefore is a good screening modality for CRC. On the other hand, the sensitivity of CEA was found to be very low in some studies,61 whereas it was shown to be high in others.62 Because of these inconsistencies, CEA may not be a suitable biomarker for early stage detection of CRC. In contrast to CEA, certain miRNAs exhibit higher sensitivity and specificity that make them potentially useful as reliable biomarkers. In addition, serum miRNAs are resistant to RNase A digestion, and are stable under conditions, such as low pH and high pH, extended storage and freeze-thaw cycles, and high temperatures.63 Because of these distinct advantages, circulating miRNAs may be more suited for screening of CRC as they have significantly lower rates of producing false-positive and false-negative results than conventional diagnostic methods.64 However, studies on serum miRNAs are associated with high cost and long latency time. Although the study by Ho et al37 is aimed at identifying reliable biomarkers using NGS, it is not cost effective, which may limit screening of a wide range of patients, such as those with different age and health status, comorbidities, and lifestyle variations. Moreover, because of the small sample size, their results are not translatable unless the miRNAs identified in this study are validated in a large sample of patients from different geographical regions. This is critical because occurrence of miRNA polymorphisms and cancer risk vary in different populations.65,66 Despite these differences, a combination of biomarkers would certainly improve the early diagnosis, and only a small number of miRNAs might be sufficient to differentiate the various stages of CRC in a costeffective manner. The translational potential and importance of measuring circulating miRNAs for CRC screening will require additional studies that include (1) far larger samples sizes; (2) advanced precancerous adenomas; (3) identification and validation of promising miRNA markers when adjusting for confounding factors, such as inflammatory bowel disease or other inflammatory conditions, that could affect miRNA expression; (4) correlation of serum levels with those of tissues specific for colorectal tumors; (5) variation and latency of these promising miRNAs over time in serial blood samples; and (6) establishing the utility of quantitative RT-PCR in detecting miRNA markers in a target population for CRC screening. Given that most of the miRNAs in the study were downregulated in individuals with disease, it will be critical to pinpoint the markers that despite their levels in diseased individuals are less than normal can be detected reliably with high sensitivity by a clinical diagnostic technology.
The study by Ho et al37 provides additional support for the role of circulating miRNAs in a new frontier of diagnostics. More significantly, it underscores the importance of developing robust, sensitive, accurate, and reproducible methods for their identification and application to clinical disease. With such a small sample size, the study was not designed to identify and validate diagnostic or early detection miRNA markers for colorectal neoplasia, but rather a proof-of-concept to demonstrate differential expression of circulating miRNAs against severity of disease using NGS. Although research on circulating miRNAs is still relatively recent, stringent analytical standards for study design and statistics are prerequisites for understanding their role in health and disease. The identification of potential targets of these circulating miRNAs will almost invariably uncover, heretofore, unrecognized regulatory mechanisms of cell function. The effective diagnosis and surveillance of complex multifactorial disorders such as CRC mandates the discovery of easily accessible biomarkers, such as tissue-specific and circulating miRNAs. Most importantly, we now recognize the critical considerations required for their isolation and characterization using cutting edge technology that is accurate and reproducible. There is an urgent need to develop a CRC-screening test that is noninvasive, cost effective, and sensitive enough to detect both neoplastic and perhaps even more importantly, preneoplastic lesions. The findings by Ho et al37 that miRNAs were not only downregulated but also decreased in a linear fashion with the stage of CRC highlight the timely, important, and potentially translational nature of the study. REFERENCES
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