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Plasma circulating microRNA-944 and microRNA-3662 as potential histologic type-specific early lung cancer biomarkers TOMASZ POWROZEK, PAWEŁ KRAWCZYK, DARIUSZ M. KOWALSKI, KINGA WINIARCZYK, MARTA OLSZYNA-SEREMENTA, and JANUSZ MILANOWSKI LUBLIN AND WARSAW, POLAND
Altered expression of microRNAs (miRNAs) is associated with the development and invasion of cancers by regulating post-transcriptionally gene function. Possibility of detection of miRNA expression in patients’ plasma or serum makes them valuable biomarkers of different neoplasms. In the present study, we investigated the potential role of miR-944 and miR-3662 expression analysis as novel lung cancer biomarkers and their lung tumor specificity in plasma samples of 90 patients with lung cancer and 85 healthy individuals using quantitative reverse transcription–polymerase chain reaction analysis. Expression of miR-944 and miR-3662 was upregulated in patients with lung cancer in comparison with healthy individuals. Receiver operating curve analysis has presented diagnostic power of analysis of both miRNA expressions for detection of patients with I and II stages of non–small cell lung cancer with area under the curve (AUC) of 0.881. Moreover, miR-944 has shown diagnostic accuracy for operable squamous cell carcinoma detection (AUC 5 0.982), whereas miR-3662 has shown the diagnostic accuracy for operable adenocarcinoma (AUC 5 0.926). Higher stage of lung cancer correlated with higher miRNA expressions. Our results suggest that the profile of studied miRNAs could be further evaluated and considered as potential lung cancer biomarkers. (Translational Research 2015;-:1–9)
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INTRODUCTION
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From the Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Lublin, Poland; Department of Lung and Chest Tumors, Oncology Centre, Institute M. Sklodowska-Curie in Warsaw, Warsaw, Poland. Submitted for publication March 15, 2015; revision submitted May 20, 2015; accepted for publication May 22, 2015. Reprint requests: Tomasz Powrozek, Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin, Poland; e-mail: tomaszpowrozek@gmail. com. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.05.009
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MicroRNAs as tumor biomarkers. Despite the availability of next-generation drugs and molecularly targeted therapies, lung cancer still remains a leading cause of cancer-related deaths worldwide.1 It is caused especially by late diagnosis of cancer, when tumor is detected in locally advanced or metastatic stages. Consequently, lung cancer patients are disqualified from surgical treatment and lose the possibility of complete recovery. Although, new molecular factors such as DNA methylation, DNA hydroxymethylation, or noncoding RNA molecules are considered as early lung cancer biomarkers, there are no established
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AT A GLANCE COMMENTARY zek T, et al. Powro Background
Possibility of detection of microRNA (miRNA) expression in patients’ blood samples make them valuable biomarkers of neoplasms. We investigated the role of miR-944 and miR-3662 expression analysis as potential histologic type-specific lung cancer biomarkers. The expression of both miRNAs has not been evaluated in blood of healthy individuals and patients with cancer before. Translational Significance
Receiver operating curve analysis has presented the diagnostic accuracy of studied miRNAs for the detection of patients with I–II stages of non– small cell lung cancer. Moreover, miR-944 has shown the diagnostic accuracy for detection of operable squamous cell carcinoma (area under the curve 0.982), whereas miR-3662 has shown the diagnostic accuracy for operable adenocarcinoma (area under the curve 0.926). These miRNAs may be considered as lung cancer biomarkers and may be involved in the development of particular histologic subtypes of non–small cell lung cancer.
criteria for their detection. Moreover, most of the studied biomarkers have limited diagnostic power and they are characterized by low sensitivity and low organ specificity. Thus, a novel approach to identify the molecular factors responsible for lung cancer development and progression should be achieved through the detection of tissue-specific agents. Moreover, development of sensitive, simple, and noninvasive (examination of blood serum or plasma) molecular methods becomes necessary.2-4 MicroRNA (miRNA) molecules seem to meet previously mentioned criteria. Several miRNAs are considered as neoplasm biomarkers. In cancers, the specific miRNAs have a regulatory function of tumor suppressor genes or oncogenes.5 Individual miRNAs are responsible for regulating the expression of various genes. Therefore, miRNAs profiling in human tumors present a diagnostic challenge. However, individual genes may be targeted by different miRNAs and numerous miRNA sequences are known.6 Thus, the role of miRNAs in human malignancies is still unknown and most studies have focused on their profiling in tumor tissues. Recognition of novel tumor-derived miRNAs
in human serum or plasma as diagnostic or prognostic biomarkers seems to be justified.7,8 Further selection of tumor-specific circulating molecules may improve early diagnosis of lung cancer. miRNA profiling could also expand the possibility of prevention of lung cancer, improving specificity of low-dose computed tomography (CT) (the differential diagnosis of small lung nodules).9,10 A few studies have reported potential clinical and diagnostic significance of circulating miRNAs in patients with lung cancer. These molecules include miRNA-17 (miR-17), miR-21, miR-93, miR-192, miR-429, and miR-1247.11-13 In the present study, we evaluated the potential role of miR-944 and miR-3662 as novel lung cancer biomarkers and its histologic subtype determinants. Criteria of testing. Gene
miR-944
and
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selection
for
coding miR-944 is located in the intron of TP63 gene, which encodes tumor protein 63 (p63). TP63 is significantly overexpressed in squamous types of cancer (in contrast to adenocarcinomas [ACs]), such as lung squamous cell carcinoma (SCC). Therefore, TP63 is considered as a squamous differentiation marker, and the expression of miR-944 may correlate with TP63 expression.14,15 Moreover, the miR-944 may target an mRNA of SOCS (suppressor of cytokine signaling) family tumor suppressor genes, which are expressed in epithelial cells of lung, and thus it can promote tumor growth, proliferation, and squamous differentiation.16 Therefore, localization and potential function of miR-944 proves its potential mechanism of acting in tumorigenesis and make it a promising marker of early squamous differentiation of lung tissue. The role of miR-3662 in human tumors is still unclear. However, on the basis of available miRNA tools (Target Scan Human 6.2 and miRDB target predictor) and mRNA-protein tissue expression database (Human Protein Atlas) as well as literature data, we assumed the potential role of miR-3662 in lung tumorigenesis and developing of lung AC.17-19 Firstly, we carefully selected suppressor genes that may be conservatively targeted by miR-3662. Subsequently, on the basis of mRNA-protein expression database, we evaluated the expression of selected genes in healthy lung tissue especially in pneumocytes, in which AC transformation is most typical. We selected few genes (PTAR1, NPR3, and SEPT10), which are cancer-related and the proteins encoded by these genes are involved in cell metabolism and structure and additionally SIX3 gene, which expression is a promising prognostic AC marker.20 Interestingly, these genes were highly or medium expressed in healthy pneumocytes compared with lung AC samples, where their expression was low. miR-3662 may be involved in this process and adenomatous
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differentiation. Moreover, miR-3662 targets CLDN gene family and TIMP3 gene, which were found downregulated in lung AC compared with SCC and healthy lung tissue and are investigated as markers of AC differentiation.21,22 These findings allow to consider the potential role of miR-3662 in lung cancer development and its AC differentiation. These molecular agents have not been described in literature data in patients with both non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Moreover, their expression in plasma of healthy individuals is unknown. This is a first study investigating the role of miR-3662 in human solid tumors. MATERIAL AND METHODS
We enrolled 90 patients with different pathologic diagnosis of lung cancer (60 patients with NSCLC and 30 with SCLC). Lung cancer group consisted of 56 males and 34 females with median age of 65 years. Among patients with NSCLC, 40 patients were in operable stages IA–IIIA and in 20 patients the locally advanced (IIIB) or advanced (IV) tumor was diagnosed, whereas in 25 patients with SCLC, 8 patients were in stage IIIA, 13 patients were in locally advanced stage IIIB, and 9 patients were in advanced stage IV. All patients’ plasma samples were collected before surgery, chemotherapy, and/or radiotherapy. Plasma samples were collected in the period between October 2013 and January 2015 at the Department of Pneumonology, Oncology and Allergology, Medical University of Lublin and at the Department of Lung and Chest Tumors Oncology Centre, Institute M. Sklodowska-Curie in Warsaw. Clinicopathologic characteristics of patients with lung cancer are shown in Table I. Moreover, we obtained plasma samples from 85 healthy volunteers without reported respiratory disease symptoms and without lung disorders in x-ray examination of the chest. Control group consisted of 51 males and 34 females with median age of 57 years. Whole blood (5 mL) was collected into tubes covered with EDTA-K2 and stored at room temperature for 30 minutes, and then immediately centrifuged at 1200 3 g for 12 minutes. About 2 mL of plasma was obtained from each blood sample. To avoid frequent refreezing of plasma, the collected samples were divided into smaller volumes (200 mL) in cryovials and stored at 280 C until used. Patients
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Quantitative reverse transcription–polymerase chain reaction analysis. Total RNA was extracted from Q7 Q8
200 mL of plasma using miRNeasy serum/plasma kit (Qiagen) according to the manufacturer’s protocol. Quantitative reverse transcription–polymerase chain
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Table I. Characteristics of studied lung cancer group Gender Male Female Age Median and standard deviation (y) Pathomorphologic diagnosis Adenocarcinoma Squamous cell carcinoma SCLC Disease stage of NSCLC patients Operable tumor IA, IB IIA, IIB IIIA Inoperable tumor IIIB IV Disease stage of SCLC patients Limited disease IIIA Extensive disease IIIB IV
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11 18 11 12 8
8 13 9
Abbreviations: NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.
reaction was conducted by TaqMan miRNA reverse transcription kit with miRNA specific RT primers (Applied Biosystems) in a total reaction volume of Q9 15 mL using TPersonal Thermocycler (Biometra, Germany) in accordance with the manufacturer’s instruction of complementary DNA (cDNA) synthesis. Q10 Then, miRNA cDNA was quantified using TaqMan Universal Master Mix II with UNG and TaqMan-specific primer probes (Applied Biosystems) against studied miRNAs in a total of 20 mL of reaction volume in Eco Illumina real-time polymerase chain reaction device (Illumina). To evaluate the quantity of miRNAs cDNA Q11 the following conditions were used: UNG activation, 2 minutes at 50 C; polymerase activation, 10 minutes at 95 C; preamplification, 10 cycles of 15 seconds at 95 C and 3 minutes at 60 C; and amplification, 35 cycles of 10 seconds at 95 C and 1 minute at 60 C. Ct Q12 values were generated and data were analyzed using Eco Study version 4.0 (Illumina). The studied miRNA expression level was normalized relative to U6 small nuclear RNA expression (internal control) using the 22DDCt analysis. Both relative expression level Q13 and fold change analysis were used to compare the expression of studied plasma miRNAs between studied groups. Statistical analysis. Statistical analysis and graphs were performed using Statistica version 12 software (Statsoft). The t test was used to compare the expression Q14
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Fig 1. Differences in the expressions of microRNA (miR)-944 and miR-3662 between patients with lung cancer and healthy individuals. Fold change for miR-944 was 20.54 and for miR-3662 was 19.45.
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of studied miRNAs between studied groups and between groups of patients with different tumor stages and tumor pathology. Receiver operating curves (ROCs) using MedCalc version 12 (MedCalc software, Belgium) were generated to assess the diagnostic accuracy of studied miRNAs. On the basis of ROC analysis the test sensitivity and specificity were assessed. The Fisher exact test was used to compare aberrant miRNA expressions with clinicopathologic data of patients with lung cancer. P values ,0.05 were considered statistically significant. The study was approved by the local Ethical Committee of Medical University of Lublin (KE-0254/156/ 2012).
RESULTS
On the basis of quantitative reverse transcription– polymerase chain reaction analysis, we evaluated the expression of studied miRNAs in plasma samples both in patients with lung cancer and in healthy individuals. Our findings seem to confirm results available in literature data concerning both miRNA overexpressions and their role in tumorigenesis as well as developing of particular histologic subtypes of lung cancer. We observed significantly higher expressions of miR-944 and miR-3662 in the plasma of patients with lung cancer compared with their expression in healthy individuals (P , 0.0001 for miR-944 and miR-3662, Fig 1). We found that the expression of the studied miRNAs was significantly higher in patients with both SCLC (P 5 0.003) and NCLC (P , 0.0001) compared with healthy volunteers.
Our second goal was comparison of the miRNA expressions between patients with early or late stages of lung cancer and healthy individuals. Significantly higher expression of studied miRNAs was observed in patients with early and late stages of lung cancer compared with healthy control (P , 0.0001 and P , 0.0001, respectively). Although, the expression of miR-944 and miR-3662 in advanced stages (IIIB and IV) of lung cancer was slightly higher than the expression of these molecules in patients with early stages (I–IIIA, P 5 0.178 and P 5 0.105, respectively). Patients with operable stages of NSCLC showed a lower expression of miR-944 and miR-3662 in comparison with patients inoperable stages of NSCLC (P 5 0.0033 and P 5 0.0278, respectively). However, patients with IIIA stage of SCLC showed a slightly lower expression of miR-944 and miR-3662 in comparison with patients with IIIB or IV stages of SCLC (P 5 0.436 and P 5 0.353, respectively). We also compared the expression of miRNAs among patients matched according to the NSCLC types and stages. The expression of miR-944 was insignificantly higher in patients with operable SCC than in patients with operable AC (P 5 0.059). Likewise, there were no significant differences in miR-3662 expressions between operable SCC and operable AC (P 5 0.451). We also observed no significant differences between expressions of miR-944 and miR-3662 in advanced SCC and advanced AC (P 5 0.791 and P 5 0.945, respectively). Interestingly, we observed a significantly higher miR944 expression in patients with IIIA NSCLC compared with patients with IIIA SCLC (P 5 0.0014). There was no such difference in miR-3662 (P 5 0.920). Moreover, studied miRNAs have shown a similar expression in
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Fig 2. Differences in miRNA expressions. (A) Comparison of miRNA expressions in patients with different stages of NSCLC and SCLC and in healthy individuals. (B) Expression of miR-944 and miR-3662 in early stages of NSLC and in healthy control. miRNA, microRNA; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.
patients with extensive SCLC and advanced NSCLC (P 5 0.310 and P 5 0.606). Differences in the expressions of miRNAs between groups of patients with different stages of lung cancer are shown in Fig 2, A and B. Expressions of studied miRNAs both for patients with lung cancer and healthy individuals’ group were plotted using ROC analysis to assess cutoff values for distinguishing patients with lung cancer from healthy volunteers. The ROC analysis has shown that the optimal cutoff point for plasma level of miR-944 had sensitivity of 81.5% and specificity of 90.5% (Fig 3, A), whereas for miR-3662 had sensitivity of 72.3% and specificity of 93.5% (Fig 3, B) for distinguishing patients with lung cancer (all tumor stages) from healthy controls with an area under the curve (AUC) of 0.914 (P , 0.0001; 95% confidence interval [CI], 0.839–
0.962) and AUC of 0.898 (P , 0.0001; 95% CI, 0.820–0.951), respectively. The combination analysis Q16 of expression of miR-944 and miR-3662 has assessed optimal cutoff for distinguishing lung cancer group from healthy individuals with sensitivity of 81.5% and specificity of 92% and AUC of 0.909 (P , 0.0001; 95% CI, 0.830–0.959) (Fig 3, C). We investigated ROC accuracy for our biomarkers used for distinguishing patients with I–IIIA stages of NSCLC from healthy individuals. The diagnostic accuracy of studied miRNA expressions was assessed as follows: for miR-944, sensitivity of 80% and specificity of 90.3% with an AUC of 0.900 (P , 0.0001; 95% CI, 0.802–0.960; Fig 4, A) and for miR-3662, 60% of sensitivity and 93.5% of specificity with an AUC of 0.845 (P , 0.0001; 95% CI, 0.735–0.922; Fig 4, B). The combination analysis of expression of these 2 miRNAs
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Fig 3. ROC analysis with AUC for plasma miRNAs level in patients with lung cancer. (A) ROC for miR-944, (B) ROC for miR-3662, and (C) combination analysis and diagnostic accuracy for studied miRNAs for distinguishing lung cancer patients from healthy individuals. AUC, area under the curve; miRNA, microRNA; ROC, receiver operating curve.
improved the test efficiency for distinguishing patients with operable NSCLC from healthy controls. The sensitivity of such a test was 91.7%, whereas the specificity was 85.7% and an AUC was 0.881 (P , 0.0001; 95% CI, 0.714–0.969; Fig 4, C). Moreover, we performed ROC analysis of miR-944 and miR-3662 expressions for patients with operable SCC and AC separately. For miR-944, we observed high diagnostic power for diagnosis of early stages SCC with sensitivity of 85.7% and specificity of 90.3% and an AUC of 0.929 (P , 0.0001; 95% CI, 0.823–0.982; Fig 4, D), whereas the analysis of miR3662 has shown high sensitivity for SCC detection (100%) but low specificity (56%). We observed diagnostic power for analysis of miR-3662 expression for detection of early stages of AC with sensitivity of 82.4%, specificity of 93.5%, and an AUC of 0.926 (P , 0.0001; 95% CI, 0.812–0.982; Fig 4, E). Diagnostic accuracy for the examination of miR-944 expression for early stages of AC was insignificant, with sensitivity of 70% and specificity of 80.3%. DISCUSSION
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Several studies have demonstrated that examination of certain profile of miRNA expression may be useful in early diagnosis of asymptomatic lung cancer. Additionally, the miRNA signature may explain the nature of small pulmonary nodules detected in prophylactic CT and identify high-risk individuals for prophylactic programs conducted in neoplasms diseases.8-10,23 On the basis of the large Bio-COSMOS trail study, a potential use of diagnostic miRNA signature was proposed to improve results of low-dose spiral CT (LD-CT) to qual-
ification of small lung nodules to invasive diagnosis. In the near future, biomarkers will be able to distinguish between LD-CT small benign nodules and truly malignant disease and may improve lung cancer screening in smokers’ population.24-26 However, in some developing countries the access for prophylactic LD-CT is restricted. Therefore, patients with lung cancer are rarely diagnosed in I and even II stage of the disease.27,28 Only a few studies have focused on the diagnostic role of miRNA profiling in patients with operable tumors and improvement in lung cancer diagnosis before the presence of symptomatic disease.11-13,29 Different studies have shown an aberrant expression of circulating miRNAs between patients with lung cancer and healthy individuals. Qi et al have found increased expressions of miR-17, miR-21, and miR-192 in patients with early stage of lung cancer compared with healthy individuals. Authors considered these miRNAs as early lung cancer biomarkers. However, they did not perform ROC analysis and test accuracy was not presented. Moreover, in their study there was no comparative analysis of miRNA expressions in early and late stages of lung cancer.11 In another study, Foss et al suggested that increased levels of serum miR-1254 and miR-574–5p were characteristic for patients with lung cancer with sensitivity of 73% and specificity of 71%.13 Zhu et al have found usefulness of detection of serum miR-429 decreased level (sensitivity of 50% and specificity of 95.8%, AUC 5 0.727) and upregulation of miR-93c (sensitivity of 94.4% and specificity of 41.7%, AUC 5 0.723) for distinguishing patients with stage I of NSCLC from healthy controls.12 In our study, we have shown a high diagnostic accuracy of combination analysis of
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Fig 4. ROCs for analysis of plasma circulating miRNA expressions in patients with NSCLC and their diagnostic accuracy: (A and B) ROCs for examination of miR-944 and miR-3662 expressions for early NSCLC stages, (C) ROC curves for combination analysis of both miRNA expressions for distinguishing early stages of NSCLC from healthy control, (D) ROCs for analysis of miR-944 expression for distinguishing early stages of SCC from healthy control, and (E) ROCs for analysis of miR-3662 expression for distinguishing early stages of AC from healthy control. Diagnostic power of analysis is shown as an AUC calculation and sensitivity and specificity of each ROCs. AC, adenocarcinoma; AUC, area under the curve; miRNA, microRNA; NSCLC, non–small cell lung cancer; ROC, receiver operating curve; SCC, squamous cell carcinoma.
miR-944 and miR-3662 expressions for early stage lung cancers (I–II) with sensitivity of 91.7% and specificity of 85.7% for distinguishing these patients from healthy people. On contrary to other studies, we recruited only a few patients with stage I of NSCLC. However, we compared these patients with larger cohort of healthy people, obtaining greater reliability and veracity of our test. We have proved that studied miRNAs may be considered as one of the potential tumor lung tissue-specific markers. We observed a gradual increase in both miRNA expressions through the tumor stages. It may suggest that plasma levels of miR-944 and miR-3662 increased simultaneously with enlarging of tumor size and its histologic differentiation. Interestingly, we found that investigated miRNAs may be also considered
as specific biomarkers for NSCLC types. The high diagnostic accuracy of analysis of miR-944 expression for SCC (stages I–II) diagnosis was reported. Our results are in accordance with Ma et al study. Authors in experimental study with next-generation sequencing found in paraffin-embedded SCC lung tissues, that miR-944 is one of few miRNAs, which showed a distinctive expression between AC and SCC with high sensitivity and specificity. Moreover, they investigated that overexpression of miR-944 may affect cell growth and proliferation by targeting SOCS4 tumor suppressor gene.16 Examination of miR-3662 level has shown high utility in AC (stages I–II) diagnosis. miR-3662 targeting genes highly expressed in healthy lung tissue (PTAR1, NPR3, and SEPT10) and CLDN-family genes, TIMP3 and SIX3 genes. It may lead to their knockdown and
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hence promotes cells proliferation for AC differentiation. Truly, in Nakamura et al21 and Wu et al22 studies these genes were found downexpressed in AC lung tissues compared with SCC and are considered as AC differentiation markers. Although, mechanisms leading to their silencing are still unknown, the function of miR-3662 in this process is possible. Several others miRNAs are also considered as tumor histologyspecific markers. On the basis of miRNA analysis in tissue samples of AC stage I tumors, Edmonds and Eischen30 have considered the miR-34b, miR-133b, and miR-1247 as AC-specific miRNAs. In other study, Tan et al investigated an SCC miRNA signature for distinguishing SCC from normal lung tissue. Expression analysis of miR-210, miR-182, miR-486–5p, miR-30a, and miR-140–3p had an accuracy of 96.2%.31 We generally focused on usefulness of miRNA examination in early tumor detection, based on data that patients with stages I–IIIA of NSCLC can benefit from surgical resection, which leads to survival prolongation. However, a few patients with SCLC were enrolled to the present study. SCLC patients characterize poor prognosis, lack of symptoms in early stages, and rapid tumor growth.32 The miRNA profile of such tumor is unknown and limited studies focused on its development.33,34 In the present study, we have shown a limited accuracy of analysis of miRNA expression for patients with stage IIIA SCLC. Instead, we detected a high expression of miR-944 and miR-3662 in advanced SCLC. Numerous molecular alternations and genetic disorders, which may be involved in SCLC development, make it one of the most complex human tumor.32,35 We noticed a significantly dynamic increase in both miRNA expressions in advanced compared with earlier stage of SCLC (IIIB and IV vs IIIA stages). Low expression of studied miRNAs in early stages of SCLC may exclude their role in developing of this lung cancer histologic type. Therefore, abnormalities of studied miRNA expression are not specific for patients with early SCLC. However, these miRNAs may be progression-related markers of SCLC. Therefore, these biomarkers may be considered as noninvasive markers of advanced SCLC, because their upregulation in plasma may proceed the disease symptoms. CONCLUSIONS
Our research is a first study investigating the plasma expression of miR-944 and miR-3662 in patients with neoplasms and in healthy individuals. Moreover, this is a first study that described an miR-3662 expression in patients with solid tumors. We have shown that examination of these 2 miRNAs may be considered as a
potential tool for lung cancer early diagnosis and for noninvasive diagnosis of lung cancer late stages. We also provided evidence that studied miRNAs may be involved in biogenesis of lung cancer with its histologic-type specific differentiation. We assume that simultaneous analysis of plasma expression of several miRNAs may improve patient qualification to surgical treatment and could rationalize programs using prophylactic LD-CT. We realize that our study has some limitations and more patients with lung cancer in early stages should be enrolled. Other possible mechanisms of studied miRNAs in the development of lung cancer should be also investigated in future. Nevertheless, proposed miRNAs might be considered in lung cancer screening, what should be proven on large cohort of patients in prospective clinical trials.
ACKNOWLEDGMENTS
Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare. This study was sponsored by a Polpharma Scientific Foundation. Q20 All authors have read the journal’s authorship agreement.
REFERENCES
1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008;359:1267–380. 2. Nikolaidis G, Raji OY, Markopoulou S, et al. DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer. Cancer Res 2012;72:5692–701. 3. Tang D, Shen Y, Wang M, et al. Identification of plasma microRNAs as novel noninvasive biomarkers for early detection of lung cancer. Eur J Cancer Prev 2013;22:540–8. 4. Olkhov-Mitsel E, Bapat B. Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers. Cancer Med 2012;1:237–60. 5. Zhang B, Pan X, Cobb GP, et al. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302:1–12. 6. MacFarlane LA, Murphy PR. MicroRNA: biogenesis, function and role in cancer. Curr Genomics 2010;11:537–61. 7. Lan H, Lu H, Wang X, Jin H. MicroRNAs as potential biomarkers in cancer: opportunities and challenges. Biomed Res Int 2015; 2015:125094. 8. Mitchelli PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513–8. 9. Sozzi G, Boeri M, Verri C, et al. Clinical utility of plasma-based miRNA signature classifier within computed tomography lung cancer screening: a correlative MILD trial study. J Clin Oncol 2014;32:768–73. 10. Boeri M, Verri C, Conte D, et al. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A 2011;108:3713–8.
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11. Qi Z, Yang DY, Cao J. Increased micro-RNA 17, 21, and 192 gene expression improve early diagnosis in non-small cell lung cancer. Med Oncol 2014;31:195. 12. Zhu W, He J, Chen D, et al. Expression of miR-29c, miR-93, and miR-429 as potential biomarkers for detection of early stage nonsmall lung cancer. PLoS One 2014;9:e87780. 13. Foss KM, Sima C, Ugolini D, et al. miR-1254 and miR-574–5p: serum-based microRNA biomarkers for early-stage non-small cell lung cancer. J Thorac Oncol 2011;6:482–8. 14. Xie H, Lee L, Scicluna P, et al. Novel functions and targets of miR-944 in human cervical cancer cells. Int J Cancer 2015;136: E230–41. 15. Au NH, Gown AM, Cheang M, et al. P63 expression in lung carcinoma: a tissue microarray study of 408 cases. Appl Immunhistochem Mol Morphol 2004;12:240–7. 16. Ma J, Mannoor K, Gao L, et al. Characterization of microRNA transcriptome in lung cancer by next-generation deep sequencing. Mol Oncol 2014;8:1208–19. 17. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15–20. 18. Wong N, Wang X. MiRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res 2015;43:D146–52. 19. Uhlen M, Fagerberg L, Hallstrom BM, et al. Tissue-based map of the human proteome. Science 2015;347:6220. 20. Mo ML, Okamoto J, Chen Z, et al. Down-regulation of SIX3 is associated with clinical outcome in lung adenocarcinoma. PLoS One 2013;8:e71816. 21. Nakamura N, Kobayashi K, Nakamoto M, et al. Identification of tumor markers and differentiation markers for molecular diagnosis of lung adenocarcinoma. Oncogene 2006;25:4245–55. 22. Wu M, Tu T, Huang Y, et al. Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3 as a novel lung cancer-related gene. Cancer 2013;13:44.
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23. Bianchi F, Nicassio F, Marzi M, et al. A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer. EMBO Mol Med 2011;3:495–503. 24. Bianchi F, Nicassio F, Veronessi G, di Fiore PP. Circulating microRNAs: next-generation biomarkers for early lung cancer detection. Ecancermedicalscience 2012;6:246. 25. Veronessi G, Bellomi M, Mulshine JL, et al. Lung cancer screening with low-dose computed tomography: a non-invasive diagnostic protocol baseline lung cancer nodules. Lung Cancer 2008;61:340–9. 26. Bach PB, Jett JR, Pastorino U, et al. Computed tomography screening and lung cancer outcomes. JAMA 2007;297:953–61. 27. Ellis PM, Vandermeer R. Delays in the diagnosis of lung cancer. J Thoracic Dis 2011;3:183–8. 28. Burki TK. Late detection of lung cancer. Lancet Oncol 2014;15: e590. 29. Deng Y, Chen H, Liu H, et al. Circulating microRNA profiling for early detection of non-small cell lung cancer. J Clin Oncol 2014; 32 (suppl; abstract e22051). Q22 30. Edmonds MD, Eischen CM. Differences in miRNA expression in early stage lung adenocarcinomas that did and did not relapse. PLoS One 2014;9:e101802. 31. Tan X, Qin W, Zhang L, et al. A 5-microRNA signature for lung squamous cell carcinoma diagnosis and hsa-miR-31 for prognosis. Clin Cancer Res 2011;17:6802–11. 32. Clark R, Ihde CD. Small-cell lung cancer: treatment progress and prospects. Oncology 1998;12:647–62. 33. Lee JH, Voortman J, Dingemans AC. MicroRNA expression and clinical outcome of small cell lung cancer. PLoS One 2011;6: e21300. 34. Miko E, Czimmerer Z, Csanky E, et al. Differentially expressed microRNAs in small cell lung cancer. Exp Lung Res 2009;35: 646–64. 35. Wistuba II, Gazdar AF, Minna JD. Molecular genetics of small cell lung carcinoma. Semin Oncol 2001;28:3–13.
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Plasma circulating microRNA-944 and microRNA-3662 as potential histologic type-specific early lung cancer biomarkers TOMASZ POWROZEK, PAWEŁ KRAWCZYK, DARIUSZ M. KOWALSKI, KINGA WINIARCZYK, MARTA OLSZYNA-SEREMENTA, and JANUSZ MILANOWSKI LUBLIN AND WARSAW, POLAND
Altered expression of microRNAs (miRNAs) is associated with the development and invasion of cancers by regulating post-transcriptionally gene function. Possibility of detection of miRNA expression in patients’ plasma or serum makes them valuable biomarkers of different neoplasms. In the present study, we investigated the potential role of miR-944 and miR-3662 expression analysis as novel lung cancer biomarkers and their lung tumor specificity in plasma samples of 90 patients with lung cancer and 85 healthy individuals using quantitative reverse transcription–polymerase chain reaction analysis. Expression of miR-944 and miR-3662 was upregulated in patients with lung cancer in comparison with healthy individuals. Receiver operating curve analysis has presented diagnostic power of analysis of both miRNA expressions for detection of patients with I and II stages of non–small cell lung cancer with area under the curve (AUC) of 0.881. Moreover, miR-944 has shown diagnostic accuracy for operable squamous cell carcinoma detection (AUC 5 0.982), whereas miR-3662 has shown the diagnostic accuracy for operable adenocarcinoma (AUC 5 0.926). Higher stage of lung cancer correlated with higher miRNA expressions. Our results suggest that the profile of studied miRNAs could be further evaluated and considered as potential lung cancer biomarkers. (Translational Research 2015;-:1–9)
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INTRODUCTION
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From the Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Lublin, Poland; Department of Lung and Chest Tumors, Oncology Centre, Institute M. Sklodowska-Curie in Warsaw, Warsaw, Poland. Submitted for publication March 15, 2015; revision submitted May 20, 2015; accepted for publication May 22, 2015. Reprint requests: Tomasz Powrozek, Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin, Poland; e-mail: tomaszpowrozek@gmail. com. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.05.009
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MicroRNAs as tumor biomarkers. Despite the availability of next-generation drugs and molecularly targeted therapies, lung cancer still remains a leading cause of cancer-related deaths worldwide.1 It is caused especially by late diagnosis of cancer, when tumor is detected in locally advanced or metastatic stages. Consequently, lung cancer patients are disqualified from surgical treatment and lose the possibility of complete recovery. Although, new molecular factors such as DNA methylation, DNA hydroxymethylation, or noncoding RNA molecules are considered as early lung cancer biomarkers, there are no established
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AT A GLANCE COMMENTARY zek T, et al. Powro Background
Possibility of detection of microRNA (miRNA) expression in patients’ blood samples make them valuable biomarkers of neoplasms. We investigated the role of miR-944 and miR-3662 expression analysis as potential histologic type-specific lung cancer biomarkers. The expression of both miRNAs has not been evaluated in blood of healthy individuals and patients with cancer before. Translational Significance
Receiver operating curve analysis has presented the diagnostic accuracy of studied miRNAs for the detection of patients with I–II stages of non– small cell lung cancer. Moreover, miR-944 has shown the diagnostic accuracy for detection of operable squamous cell carcinoma (area under the curve 0.982), whereas miR-3662 has shown the diagnostic accuracy for operable adenocarcinoma (area under the curve 0.926). These miRNAs may be considered as lung cancer biomarkers and may be involved in the development of particular histologic subtypes of non–small cell lung cancer.
criteria for their detection. Moreover, most of the studied biomarkers have limited diagnostic power and they are characterized by low sensitivity and low organ specificity. Thus, a novel approach to identify the molecular factors responsible for lung cancer development and progression should be achieved through the detection of tissue-specific agents. Moreover, development of sensitive, simple, and noninvasive (examination of blood serum or plasma) molecular methods becomes necessary.2-4 MicroRNA (miRNA) molecules seem to meet previously mentioned criteria. Several miRNAs are considered as neoplasm biomarkers. In cancers, the specific miRNAs have a regulatory function of tumor suppressor genes or oncogenes.5 Individual miRNAs are responsible for regulating the expression of various genes. Therefore, miRNAs profiling in human tumors present a diagnostic challenge. However, individual genes may be targeted by different miRNAs and numerous miRNA sequences are known.6 Thus, the role of miRNAs in human malignancies is still unknown and most studies have focused on their profiling in tumor tissues. Recognition of novel tumor-derived miRNAs
in human serum or plasma as diagnostic or prognostic biomarkers seems to be justified.7,8 Further selection of tumor-specific circulating molecules may improve early diagnosis of lung cancer. miRNA profiling could also expand the possibility of prevention of lung cancer, improving specificity of low-dose computed tomography (CT) (the differential diagnosis of small lung nodules).9,10 A few studies have reported potential clinical and diagnostic significance of circulating miRNAs in patients with lung cancer. These molecules include miRNA-17 (miR-17), miR-21, miR-93, miR-192, miR-429, and miR-1247.11-13 In the present study, we evaluated the potential role of miR-944 and miR-3662 as novel lung cancer biomarkers and its histologic subtype determinants. Criteria of testing. Gene
miR-944
and
miR-3662
selection
for
coding miR-944 is located in the intron of TP63 gene, which encodes tumor protein 63 (p63). TP63 is significantly overexpressed in squamous types of cancer (in contrast to adenocarcinomas [ACs]), such as lung squamous cell carcinoma (SCC). Therefore, TP63 is considered as a squamous differentiation marker, and the expression of miR-944 may correlate with TP63 expression.14,15 Moreover, the miR-944 may target an mRNA of SOCS (suppressor of cytokine signaling) family tumor suppressor genes, which are expressed in epithelial cells of lung, and thus it can promote tumor growth, proliferation, and squamous differentiation.16 Therefore, localization and potential function of miR-944 proves its potential mechanism of acting in tumorigenesis and make it a promising marker of early squamous differentiation of lung tissue. The role of miR-3662 in human tumors is still unclear. However, on the basis of available miRNA tools (Target Scan Human 6.2 and miRDB target predictor) and mRNA-protein tissue expression database (Human Protein Atlas) as well as literature data, we assumed the potential role of miR-3662 in lung tumorigenesis and developing of lung AC.17-19 Firstly, we carefully selected suppressor genes that may be conservatively targeted by miR-3662. Subsequently, on the basis of mRNA-protein expression database, we evaluated the expression of selected genes in healthy lung tissue especially in pneumocytes, in which AC transformation is most typical. We selected few genes (PTAR1, NPR3, and SEPT10), which are cancer-related and the proteins encoded by these genes are involved in cell metabolism and structure and additionally SIX3 gene, which expression is a promising prognostic AC marker.20 Interestingly, these genes were highly or medium expressed in healthy pneumocytes compared with lung AC samples, where their expression was low. miR-3662 may be involved in this process and adenomatous
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differentiation. Moreover, miR-3662 targets CLDN gene family and TIMP3 gene, which were found downregulated in lung AC compared with SCC and healthy lung tissue and are investigated as markers of AC differentiation.21,22 These findings allow to consider the potential role of miR-3662 in lung cancer development and its AC differentiation. These molecular agents have not been described in literature data in patients with both non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Moreover, their expression in plasma of healthy individuals is unknown. This is a first study investigating the role of miR-3662 in human solid tumors. MATERIAL AND METHODS
We enrolled 90 patients with different pathologic diagnosis of lung cancer (60 patients with NSCLC and 30 with SCLC). Lung cancer group consisted of 56 males and 34 females with median age of 65 years. Among patients with NSCLC, 40 patients were in operable stages IA–IIIA and in 20 patients the locally advanced (IIIB) or advanced (IV) tumor was diagnosed, whereas in 25 patients with SCLC, 8 patients were in stage IIIA, 13 patients were in locally advanced stage IIIB, and 9 patients were in advanced stage IV. All patients’ plasma samples were collected before surgery, chemotherapy, and/or radiotherapy. Plasma samples were collected in the period between October 2013 and January 2015 at the Department of Pneumonology, Oncology and Allergology, Medical University of Lublin and at the Department of Lung and Chest Tumors Oncology Centre, Institute M. Sklodowska-Curie in Warsaw. Clinicopathologic characteristics of patients with lung cancer are shown in Table I. Moreover, we obtained plasma samples from 85 healthy volunteers without reported respiratory disease symptoms and without lung disorders in x-ray examination of the chest. Control group consisted of 51 males and 34 females with median age of 57 years. Whole blood (5 mL) was collected into tubes covered with EDTA-K2 and stored at room temperature for 30 minutes, and then immediately centrifuged at 1200 3 g for 12 minutes. About 2 mL of plasma was obtained from each blood sample. To avoid frequent refreezing of plasma, the collected samples were divided into smaller volumes (200 mL) in cryovials and stored at 280 C until used. Patients
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Quantitative reverse transcription–polymerase chain reaction analysis. Total RNA was extracted from Q7 Q8
200 mL of plasma using miRNeasy serum/plasma kit (Qiagen) according to the manufacturer’s protocol. Quantitative reverse transcription–polymerase chain
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Table I. Characteristics of studied lung cancer group Gender Male Female Age Median and standard deviation (y) Pathomorphologic diagnosis Adenocarcinoma Squamous cell carcinoma SCLC Disease stage of NSCLC patients Operable tumor IA, IB IIA, IIB IIIA Inoperable tumor IIIB IV Disease stage of SCLC patients Limited disease IIIA Extensive disease IIIB IV
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11 18 11 12 8
8 13 9
Abbreviations: NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.
reaction was conducted by TaqMan miRNA reverse transcription kit with miRNA specific RT primers (Applied Biosystems) in a total reaction volume of Q9 15 mL using TPersonal Thermocycler (Biometra, Germany) in accordance with the manufacturer’s instruction of complementary DNA (cDNA) synthesis. Q10 Then, miRNA cDNA was quantified using TaqMan Universal Master Mix II with UNG and TaqMan-specific primer probes (Applied Biosystems) against studied miRNAs in a total of 20 mL of reaction volume in Eco Illumina real-time polymerase chain reaction device (Illumina). To evaluate the quantity of miRNAs cDNA Q11 the following conditions were used: UNG activation, 2 minutes at 50 C; polymerase activation, 10 minutes at 95 C; preamplification, 10 cycles of 15 seconds at 95 C and 3 minutes at 60 C; and amplification, 35 cycles of 10 seconds at 95 C and 1 minute at 60 C. Ct Q12 values were generated and data were analyzed using Eco Study version 4.0 (Illumina). The studied miRNA expression level was normalized relative to U6 small nuclear RNA expression (internal control) using the 22DDCt analysis. Both relative expression level Q13 and fold change analysis were used to compare the expression of studied plasma miRNAs between studied groups. Statistical analysis. Statistical analysis and graphs were performed using Statistica version 12 software (Statsoft). The t test was used to compare the expression Q14
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Fig 1. Differences in the expressions of microRNA (miR)-944 and miR-3662 between patients with lung cancer and healthy individuals. Fold change for miR-944 was 20.54 and for miR-3662 was 19.45.
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of studied miRNAs between studied groups and between groups of patients with different tumor stages and tumor pathology. Receiver operating curves (ROCs) using MedCalc version 12 (MedCalc software, Belgium) were generated to assess the diagnostic accuracy of studied miRNAs. On the basis of ROC analysis the test sensitivity and specificity were assessed. The Fisher exact test was used to compare aberrant miRNA expressions with clinicopathologic data of patients with lung cancer. P values ,0.05 were considered statistically significant. The study was approved by the local Ethical Committee of Medical University of Lublin (KE-0254/156/ 2012).
RESULTS
On the basis of quantitative reverse transcription– polymerase chain reaction analysis, we evaluated the expression of studied miRNAs in plasma samples both in patients with lung cancer and in healthy individuals. Our findings seem to confirm results available in literature data concerning both miRNA overexpressions and their role in tumorigenesis as well as developing of particular histologic subtypes of lung cancer. We observed significantly higher expressions of miR-944 and miR-3662 in the plasma of patients with lung cancer compared with their expression in healthy individuals (P , 0.0001 for miR-944 and miR-3662, Fig 1). We found that the expression of the studied miRNAs was significantly higher in patients with both SCLC (P 5 0.003) and NCLC (P , 0.0001) compared with healthy volunteers.
Our second goal was comparison of the miRNA expressions between patients with early or late stages of lung cancer and healthy individuals. Significantly higher expression of studied miRNAs was observed in patients with early and late stages of lung cancer compared with healthy control (P , 0.0001 and P , 0.0001, respectively). Although, the expression of miR-944 and miR-3662 in advanced stages (IIIB and IV) of lung cancer was slightly higher than the expression of these molecules in patients with early stages (I–IIIA, P 5 0.178 and P 5 0.105, respectively). Patients with operable stages of NSCLC showed a lower expression of miR-944 and miR-3662 in comparison with patients inoperable stages of NSCLC (P 5 0.0033 and P 5 0.0278, respectively). However, patients with IIIA stage of SCLC showed a slightly lower expression of miR-944 and miR-3662 in comparison with patients with IIIB or IV stages of SCLC (P 5 0.436 and P 5 0.353, respectively). We also compared the expression of miRNAs among patients matched according to the NSCLC types and stages. The expression of miR-944 was insignificantly higher in patients with operable SCC than in patients with operable AC (P 5 0.059). Likewise, there were no significant differences in miR-3662 expressions between operable SCC and operable AC (P 5 0.451). We also observed no significant differences between expressions of miR-944 and miR-3662 in advanced SCC and advanced AC (P 5 0.791 and P 5 0.945, respectively). Interestingly, we observed a significantly higher miR944 expression in patients with IIIA NSCLC compared with patients with IIIA SCLC (P 5 0.0014). There was no such difference in miR-3662 (P 5 0.920). Moreover, studied miRNAs have shown a similar expression in
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Fig 2. Differences in miRNA expressions. (A) Comparison of miRNA expressions in patients with different stages of NSCLC and SCLC and in healthy individuals. (B) Expression of miR-944 and miR-3662 in early stages of NSLC and in healthy control. miRNA, microRNA; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.
patients with extensive SCLC and advanced NSCLC (P 5 0.310 and P 5 0.606). Differences in the expressions of miRNAs between groups of patients with different stages of lung cancer are shown in Fig 2, A and B. Expressions of studied miRNAs both for patients with lung cancer and healthy individuals’ group were plotted using ROC analysis to assess cutoff values for distinguishing patients with lung cancer from healthy volunteers. The ROC analysis has shown that the optimal cutoff point for plasma level of miR-944 had sensitivity of 81.5% and specificity of 90.5% (Fig 3, A), whereas for miR-3662 had sensitivity of 72.3% and specificity of 93.5% (Fig 3, B) for distinguishing patients with lung cancer (all tumor stages) from healthy controls with an area under the curve (AUC) of 0.914 (P , 0.0001; 95% confidence interval [CI], 0.839–
0.962) and AUC of 0.898 (P , 0.0001; 95% CI, 0.820–0.951), respectively. The combination analysis Q16 of expression of miR-944 and miR-3662 has assessed optimal cutoff for distinguishing lung cancer group from healthy individuals with sensitivity of 81.5% and specificity of 92% and AUC of 0.909 (P , 0.0001; 95% CI, 0.830–0.959) (Fig 3, C). We investigated ROC accuracy for our biomarkers used for distinguishing patients with I–IIIA stages of NSCLC from healthy individuals. The diagnostic accuracy of studied miRNA expressions was assessed as follows: for miR-944, sensitivity of 80% and specificity of 90.3% with an AUC of 0.900 (P , 0.0001; 95% CI, 0.802–0.960; Fig 4, A) and for miR-3662, 60% of sensitivity and 93.5% of specificity with an AUC of 0.845 (P , 0.0001; 95% CI, 0.735–0.922; Fig 4, B). The combination analysis of expression of these 2 miRNAs
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Fig 3. ROC analysis with AUC for plasma miRNAs level in patients with lung cancer. (A) ROC for miR-944, (B) ROC for miR-3662, and (C) combination analysis and diagnostic accuracy for studied miRNAs for distinguishing lung cancer patients from healthy individuals. AUC, area under the curve; miRNA, microRNA; ROC, receiver operating curve.
improved the test efficiency for distinguishing patients with operable NSCLC from healthy controls. The sensitivity of such a test was 91.7%, whereas the specificity was 85.7% and an AUC was 0.881 (P , 0.0001; 95% CI, 0.714–0.969; Fig 4, C). Moreover, we performed ROC analysis of miR-944 and miR-3662 expressions for patients with operable SCC and AC separately. For miR-944, we observed high diagnostic power for diagnosis of early stages SCC with sensitivity of 85.7% and specificity of 90.3% and an AUC of 0.929 (P , 0.0001; 95% CI, 0.823–0.982; Fig 4, D), whereas the analysis of miR3662 has shown high sensitivity for SCC detection (100%) but low specificity (56%). We observed diagnostic power for analysis of miR-3662 expression for detection of early stages of AC with sensitivity of 82.4%, specificity of 93.5%, and an AUC of 0.926 (P , 0.0001; 95% CI, 0.812–0.982; Fig 4, E). Diagnostic accuracy for the examination of miR-944 expression for early stages of AC was insignificant, with sensitivity of 70% and specificity of 80.3%. DISCUSSION
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Several studies have demonstrated that examination of certain profile of miRNA expression may be useful in early diagnosis of asymptomatic lung cancer. Additionally, the miRNA signature may explain the nature of small pulmonary nodules detected in prophylactic CT and identify high-risk individuals for prophylactic programs conducted in neoplasms diseases.8-10,23 On the basis of the large Bio-COSMOS trail study, a potential use of diagnostic miRNA signature was proposed to improve results of low-dose spiral CT (LD-CT) to qual-
ification of small lung nodules to invasive diagnosis. In the near future, biomarkers will be able to distinguish between LD-CT small benign nodules and truly malignant disease and may improve lung cancer screening in smokers’ population.24-26 However, in some developing countries the access for prophylactic LD-CT is restricted. Therefore, patients with lung cancer are rarely diagnosed in I and even II stage of the disease.27,28 Only a few studies have focused on the diagnostic role of miRNA profiling in patients with operable tumors and improvement in lung cancer diagnosis before the presence of symptomatic disease.11-13,29 Different studies have shown an aberrant expression of circulating miRNAs between patients with lung cancer and healthy individuals. Qi et al have found increased expressions of miR-17, miR-21, and miR-192 in patients with early stage of lung cancer compared with healthy individuals. Authors considered these miRNAs as early lung cancer biomarkers. However, they did not perform ROC analysis and test accuracy was not presented. Moreover, in their study there was no comparative analysis of miRNA expressions in early and late stages of lung cancer.11 In another study, Foss et al suggested that increased levels of serum miR-1254 and miR-574–5p were characteristic for patients with lung cancer with sensitivity of 73% and specificity of 71%.13 Zhu et al have found usefulness of detection of serum miR-429 decreased level (sensitivity of 50% and specificity of 95.8%, AUC 5 0.727) and upregulation of miR-93c (sensitivity of 94.4% and specificity of 41.7%, AUC 5 0.723) for distinguishing patients with stage I of NSCLC from healthy controls.12 In our study, we have shown a high diagnostic accuracy of combination analysis of
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Fig 4. ROCs for analysis of plasma circulating miRNA expressions in patients with NSCLC and their diagnostic accuracy: (A and B) ROCs for examination of miR-944 and miR-3662 expressions for early NSCLC stages, (C) ROC curves for combination analysis of both miRNA expressions for distinguishing early stages of NSCLC from healthy control, (D) ROCs for analysis of miR-944 expression for distinguishing early stages of SCC from healthy control, and (E) ROCs for analysis of miR-3662 expression for distinguishing early stages of AC from healthy control. Diagnostic power of analysis is shown as an AUC calculation and sensitivity and specificity of each ROCs. AC, adenocarcinoma; AUC, area under the curve; miRNA, microRNA; NSCLC, non–small cell lung cancer; ROC, receiver operating curve; SCC, squamous cell carcinoma.
miR-944 and miR-3662 expressions for early stage lung cancers (I–II) with sensitivity of 91.7% and specificity of 85.7% for distinguishing these patients from healthy people. On contrary to other studies, we recruited only a few patients with stage I of NSCLC. However, we compared these patients with larger cohort of healthy people, obtaining greater reliability and veracity of our test. We have proved that studied miRNAs may be considered as one of the potential tumor lung tissue-specific markers. We observed a gradual increase in both miRNA expressions through the tumor stages. It may suggest that plasma levels of miR-944 and miR-3662 increased simultaneously with enlarging of tumor size and its histologic differentiation. Interestingly, we found that investigated miRNAs may be also considered
as specific biomarkers for NSCLC types. The high diagnostic accuracy of analysis of miR-944 expression for SCC (stages I–II) diagnosis was reported. Our results are in accordance with Ma et al study. Authors in experimental study with next-generation sequencing found in paraffin-embedded SCC lung tissues, that miR-944 is one of few miRNAs, which showed a distinctive expression between AC and SCC with high sensitivity and specificity. Moreover, they investigated that overexpression of miR-944 may affect cell growth and proliferation by targeting SOCS4 tumor suppressor gene.16 Examination of miR-3662 level has shown high utility in AC (stages I–II) diagnosis. miR-3662 targeting genes highly expressed in healthy lung tissue (PTAR1, NPR3, and SEPT10) and CLDN-family genes, TIMP3 and SIX3 genes. It may lead to their knockdown and
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hence promotes cells proliferation for AC differentiation. Truly, in Nakamura et al21 and Wu et al22 studies these genes were found downexpressed in AC lung tissues compared with SCC and are considered as AC differentiation markers. Although, mechanisms leading to their silencing are still unknown, the function of miR-3662 in this process is possible. Several others miRNAs are also considered as tumor histologyspecific markers. On the basis of miRNA analysis in tissue samples of AC stage I tumors, Edmonds and Eischen30 have considered the miR-34b, miR-133b, and miR-1247 as AC-specific miRNAs. In other study, Tan et al investigated an SCC miRNA signature for distinguishing SCC from normal lung tissue. Expression analysis of miR-210, miR-182, miR-486–5p, miR-30a, and miR-140–3p had an accuracy of 96.2%.31 We generally focused on usefulness of miRNA examination in early tumor detection, based on data that patients with stages I–IIIA of NSCLC can benefit from surgical resection, which leads to survival prolongation. However, a few patients with SCLC were enrolled to the present study. SCLC patients characterize poor prognosis, lack of symptoms in early stages, and rapid tumor growth.32 The miRNA profile of such tumor is unknown and limited studies focused on its development.33,34 In the present study, we have shown a limited accuracy of analysis of miRNA expression for patients with stage IIIA SCLC. Instead, we detected a high expression of miR-944 and miR-3662 in advanced SCLC. Numerous molecular alternations and genetic disorders, which may be involved in SCLC development, make it one of the most complex human tumor.32,35 We noticed a significantly dynamic increase in both miRNA expressions in advanced compared with earlier stage of SCLC (IIIB and IV vs IIIA stages). Low expression of studied miRNAs in early stages of SCLC may exclude their role in developing of this lung cancer histologic type. Therefore, abnormalities of studied miRNA expression are not specific for patients with early SCLC. However, these miRNAs may be progression-related markers of SCLC. Therefore, these biomarkers may be considered as noninvasive markers of advanced SCLC, because their upregulation in plasma may proceed the disease symptoms. CONCLUSIONS
Our research is a first study investigating the plasma expression of miR-944 and miR-3662 in patients with neoplasms and in healthy individuals. Moreover, this is a first study that described an miR-3662 expression in patients with solid tumors. We have shown that examination of these 2 miRNAs may be considered as a
potential tool for lung cancer early diagnosis and for noninvasive diagnosis of lung cancer late stages. We also provided evidence that studied miRNAs may be involved in biogenesis of lung cancer with its histologic-type specific differentiation. We assume that simultaneous analysis of plasma expression of several miRNAs may improve patient qualification to surgical treatment and could rationalize programs using prophylactic LD-CT. We realize that our study has some limitations and more patients with lung cancer in early stages should be enrolled. Other possible mechanisms of studied miRNAs in the development of lung cancer should be also investigated in future. Nevertheless, proposed miRNAs might be considered in lung cancer screening, what should be proven on large cohort of patients in prospective clinical trials.
ACKNOWLEDGMENTS
Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare. This study was sponsored by a Polpharma Scientific Foundation. Q20 All authors have read the journal’s authorship agreement.
REFERENCES
1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008;359:1267–380. 2. Nikolaidis G, Raji OY, Markopoulou S, et al. DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer. Cancer Res 2012;72:5692–701. 3. Tang D, Shen Y, Wang M, et al. Identification of plasma microRNAs as novel noninvasive biomarkers for early detection of lung cancer. Eur J Cancer Prev 2013;22:540–8. 4. Olkhov-Mitsel E, Bapat B. Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers. Cancer Med 2012;1:237–60. 5. Zhang B, Pan X, Cobb GP, et al. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302:1–12. 6. MacFarlane LA, Murphy PR. MicroRNA: biogenesis, function and role in cancer. Curr Genomics 2010;11:537–61. 7. Lan H, Lu H, Wang X, Jin H. MicroRNAs as potential biomarkers in cancer: opportunities and challenges. Biomed Res Int 2015; 2015:125094. 8. Mitchelli PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513–8. 9. Sozzi G, Boeri M, Verri C, et al. Clinical utility of plasma-based miRNA signature classifier within computed tomography lung cancer screening: a correlative MILD trial study. J Clin Oncol 2014;32:768–73. 10. Boeri M, Verri C, Conte D, et al. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A 2011;108:3713–8.
FLA 5.2.0 DTD TRSL917_proof 13 June 2015 2:45 pm ce
Q21
960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023
Translational Research Volume -, Number 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087
zek et al Powro
11. Qi Z, Yang DY, Cao J. Increased micro-RNA 17, 21, and 192 gene expression improve early diagnosis in non-small cell lung cancer. Med Oncol 2014;31:195. 12. Zhu W, He J, Chen D, et al. Expression of miR-29c, miR-93, and miR-429 as potential biomarkers for detection of early stage nonsmall lung cancer. PLoS One 2014;9:e87780. 13. Foss KM, Sima C, Ugolini D, et al. miR-1254 and miR-574–5p: serum-based microRNA biomarkers for early-stage non-small cell lung cancer. J Thorac Oncol 2011;6:482–8. 14. Xie H, Lee L, Scicluna P, et al. Novel functions and targets of miR-944 in human cervical cancer cells. Int J Cancer 2015;136: E230–41. 15. Au NH, Gown AM, Cheang M, et al. P63 expression in lung carcinoma: a tissue microarray study of 408 cases. Appl Immunhistochem Mol Morphol 2004;12:240–7. 16. Ma J, Mannoor K, Gao L, et al. Characterization of microRNA transcriptome in lung cancer by next-generation deep sequencing. Mol Oncol 2014;8:1208–19. 17. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15–20. 18. Wong N, Wang X. MiRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res 2015;43:D146–52. 19. Uhlen M, Fagerberg L, Hallstrom BM, et al. Tissue-based map of the human proteome. Science 2015;347:6220. 20. Mo ML, Okamoto J, Chen Z, et al. Down-regulation of SIX3 is associated with clinical outcome in lung adenocarcinoma. PLoS One 2013;8:e71816. 21. Nakamura N, Kobayashi K, Nakamoto M, et al. Identification of tumor markers and differentiation markers for molecular diagnosis of lung adenocarcinoma. Oncogene 2006;25:4245–55. 22. Wu M, Tu T, Huang Y, et al. Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3 as a novel lung cancer-related gene. Cancer 2013;13:44.
9
23. Bianchi F, Nicassio F, Marzi M, et al. A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer. EMBO Mol Med 2011;3:495–503. 24. Bianchi F, Nicassio F, Veronessi G, di Fiore PP. Circulating microRNAs: next-generation biomarkers for early lung cancer detection. Ecancermedicalscience 2012;6:246. 25. Veronessi G, Bellomi M, Mulshine JL, et al. Lung cancer screening with low-dose computed tomography: a non-invasive diagnostic protocol baseline lung cancer nodules. Lung Cancer 2008;61:340–9. 26. Bach PB, Jett JR, Pastorino U, et al. Computed tomography screening and lung cancer outcomes. JAMA 2007;297:953–61. 27. Ellis PM, Vandermeer R. Delays in the diagnosis of lung cancer. J Thoracic Dis 2011;3:183–8. 28. Burki TK. Late detection of lung cancer. Lancet Oncol 2014;15: e590. 29. Deng Y, Chen H, Liu H, et al. Circulating microRNA profiling for early detection of non-small cell lung cancer. J Clin Oncol 2014; 32 (suppl; abstract e22051). Q22 30. Edmonds MD, Eischen CM. Differences in miRNA expression in early stage lung adenocarcinomas that did and did not relapse. PLoS One 2014;9:e101802. 31. Tan X, Qin W, Zhang L, et al. A 5-microRNA signature for lung squamous cell carcinoma diagnosis and hsa-miR-31 for prognosis. Clin Cancer Res 2011;17:6802–11. 32. Clark R, Ihde CD. Small-cell lung cancer: treatment progress and prospects. Oncology 1998;12:647–62. 33. Lee JH, Voortman J, Dingemans AC. MicroRNA expression and clinical outcome of small cell lung cancer. PLoS One 2011;6: e21300. 34. Miko E, Czimmerer Z, Csanky E, et al. Differentially expressed microRNAs in small cell lung cancer. Exp Lung Res 2009;35: 646–64. 35. Wistuba II, Gazdar AF, Minna JD. Molecular genetics of small cell lung carcinoma. Semin Oncol 2001;28:3–13.
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