G Model
ARTICLE IN PRESS
LR-5384; No. of Pages 6
Leukemia Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
MicroRNA-194-5p could serve as a diagnostic and prognostic biomarker in myelodysplastic syndromes Ji-Seon Choi a , Myung-Hyun Nam a , Soo-Young Yoon a , Seong-Ho Kang b,∗ a b
Department of Laboratory Medicine, Korea University College of Medicine, Seoul, South Korea Department of Laboratory Medicine, Chosun University College of Medicine, Gwangju, South Korea
a r t i c l e
i n f o
Article history: Received 15 January 2015 Received in revised form 26 March 2015 Accepted 19 April 2015 Available online xxx Keywords: MDS microRNA Deregulation Diagnosis Prognosis
a b s t r a c t Trisomy 8 and trisomy 1q are the most frequent chromosomal abnormalities in Korean patients with myelodysplastic syndrome (MDS). MicroRNA (miRNA) deregulation is involved in the development of hematological malignancies, including MDS, and cancer-associated genomic regions are known to encode miRNAs. The aim of the present study was to investigate the involvement of miRNAs encoded by chromosomes 8 and 1q in MDS. For this, the expression of nine miRNAs encoded by chromosome 8 (miR-30b-5p, miR-30d-5p, miR-101-3p, miR-124-3p, miR-151a-5p, miR-320a, miR-486-5p, miR-596, and miR-8755p) and three miRNAs encoded by chromosome 1q (miR-29c-3p, miR-194-5p, and miR-214-3p) was compared between 65 MDS patients and 11 controls. We found a significant upregulation of miR-1945p (5.1-fold, P = 0.002) and miR-320a (2.94-fold, P = 0.016) in MDS patients compared with controls. The patients with low miR-194-5p expression showed a significantly decreased overall survival (P = 0.049). The areas under the miR-194-5p and miR-320a ROC curves were 0.797 (P = 0.002) and 0.729 (P = 0.016), respectively. Although these findings need to be validated in a larger patient population, our results indicate that miR-194-5p is a candidate diagnostic biomarker for MDS and that low miR-194-5p expression could be associated with poor overall survival for MDS patients. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The myelodysplastic syndromes (MDS) are characterized by pancytopenia of peripheral blood, dysplasia in one or more myeloid cell lines, ineffective hematopoiesis of hematopoietic stem cells, and increased risk of development of acute myeloid leukemia. Dysplasia in one or more myeloid cell lines is a prerequisite for the diagnosis of MDS and diagnosis of MDS was difficult when dysplasia is not definite; however, a presumptive diagnosis can be made if dysplasia is not definite but certain cytogenetic abnormalities are present [1]. The most frequent cytogenetic abnormalities in Korean MDS patients are +8 and 1q+ observed in 19.5–34.5% and 15.2–17.2%, respectively, of MDS cases [2,3]. Previous studies have suggested that +8 is the most frequent cytogenetic abnormality found in MDS patients from Asian countries and that the proportion of 1q+ is specifically increased in MDS patients
∗ Corresponding author at: Department of Laboratory Medicine, Chosun University College of Medicine, 365, Pilmun-daero, Dong-gu, Gwangju 501-717, South Korea. Tel.: +82 62 220 3272; fax: +82 62 232 2063. E-mail address: [email protected] (S.-H. Kang).
from Korea compared to those from Western countries and Japan [2–5]. MicroRNAs (miRNAs) are non-coding RNA species of approximately 22 nucleotides that bind 3 -untranslated regions (UTR) of target mRNAs to repress their translation and stability. MiRNAs are involved in cell proliferation, development, and differentiation [6,7]. Approximately half of miRNA-encoding genes are frequently located at the fragile sites and regions of copy-number alterations associated with cancer [8]. More than 70% of miRNAs are located at the genomic regions carrying leukemia-associated copy-number alterations in MDS and acute myeloid leukemia cell lines [9]. These studies suggest that miRNAs located at the regions of genomic alterations can be deregulated in MDS patients. For instance, miR-145 and miR-146a located at 5q have been reported to be downregulated in patients with 5q-syndrome and to be involved in the mediation of the 5q-syndrome phenotype [10]. Similarly, miRNAs located at chromosomes 8 and 1q, which most frequently carry abnormalities detected in Korean MDS patients, could also be deregulated. Currently, MDS diagnostics is based on the dysplasia of one or more myeloid cell lines, the presence of ring sideroblasts, and blast counts. Conventional cytogenetics and fluorescence in situ hybridization (FISH) can assist the diagnosis of MDS. However,
http://dx.doi.org/10.1016/j.leukres.2015.04.013 0145-2126/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Choi J-S, et al. MicroRNA-194-5p could serve as a diagnostic and prognostic biomarker in myelodysplastic syndromes. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.04.013
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ARTICLE IN PRESS J.-S. Choi et al. / Leukemia Research xxx (2015) xxx–xxx
2
these methods have limitations: cytogenetics requires long cell culturing (>2 days) and FISH needs multiple expensive probes to detect frequent aneuploidy. In contrast, the detection of miRNAs using real-time PCR is fast and simple, and may be successfully used for MDS diagnostics. Thus, miRNAs located at chromosomes 8 and 1q could be the biomarkers specific to Korean MDS patients, as +8 and +1q are the most frequent cytogenetic abnormalities in this population. The aim of the present study was to investigate whether the expression of miRNAs located at chromosomes 8 and 1q could be used as diagnostic as well as outcome prediction biomarkers for MDS in Korean patients. 2. Material and methods 2.1. Patients The present study included 65 MDS patients and 11 controls. MDS diagnosis was based on the WHO classification of MDS [11]. Controls exhibited no evidence of cancer, and had normocellular bone marrow with no signs of hematologic disease. Clinical and laboratory data (karyotyping, complete blood cell counts, and analysis of bone marrow blasts) were obtained from patients’ medical records. This research was approved by the Korea University Anam Hospital Institutional Review Board and Korea University Ansan Hospital Institutional Review Board. 2.2. MiRNA extraction and quantification MiRNA was extracted from archived unstained bone marrow aspirate slides of the participants using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction, and used to evaluate the relative expression of nine miRNAs encoded by chromosome 8 (miR-30b-5p, miR-30d-5p, miR-1013p, miR-124-3p, miR-151a-5p, miR-320a, miR-486-5p, miR-596, and miR-875-5p) and three miRNAs encoded by chromosome 1q (miR-29c-3p, miR-194-5p, and miR214-3p). Multiplex reverse transcription was performed using the primer pool of the TaqMan MicroRNA Assay (Applied Biosystems, Carlsbad, CA, USA) and the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instruction. Because the amount of total RNA extracted from each sample was less than 350 ng, the synthesized cDNA was pre-amplified prior to performing real-time PCR using the primer pool and TaqMan probe of the TaqMan MicroRNA Assay and TaqMan PreAmp Master Mix (Applied Biosystems) according to the manufacturer’s instruction. MiRNA expression was evaluated by real-time PCR using the TaqMan MicroRNA Assay and a StepOne Real-Time PCR system (Applied Biosystems) according to the manufacturer’s instruction. The relative expression of each miRNA was quantified using the 2−Ct method [12]. To compare miRNA expression between MDS patients and controls, 2–Ct of each miRNA was calculated, where Ct = CtmiRNA – Ctendogenous control and Ct is the threshold cycle defined as the number of cycles at which the fluorescent signal exceeds the threshold. RNU48 expression was used as an endogenous control. 2.3. Statistics The relative expression of each miRNA was compared between MDS patients and controls using the non-parametric Mann Whitney test. The Revised International Prognostic Scoring System (IPSS-R) for myelodysplasia score was used as previously described [13] and the IPSS-R score was calculated using an IPSS-R calculator tool: (http://www.ipss-com, or http://mds-foundation.org/calcaulator/index.php). Overall survival was determined as the time from initial diagnosis to death and analyzed by the Kaplan–Meier method. SPSS version 21.0 (SPSS, Chicago, IL, USA) was used for the statistical analysis. The data are expressed as the mean ± statistical significance (SD).
3. Results 3.1. Patients’ characteristics Patients’ clinical and hematological parameters are presented in Table 1. The study included 35 male and 30 female MDS patients with a median age of 68 years (range, 21–88 years). MDS diagnosis based on the WHO criteria included 26 cases (34.2%) of refractory cytopenia with multilineage dysplasia (RCMD), 12 cases (15.8%) of myelodysplastic syndrome-unclassified (MDS-U), 10 cases (13.2%) of refractory anemia with excess blasts-2 (RAEB-2), nine cases (11.8%) of RAEB-1, seven cases (9.2%) of refractory anemia with unilineage dysplasia (RCUD), and one case (1.3%) of refractory anemia with ring sideroblasts (RARS). Of the 65 MDS patients, 28 patients
Table 1 Patients’ characteristics. Descriptive statistics Total patients Median age, years (minimum–maximum)
65 68 (21–88)
Age 61
6 (9.2%) 14 (21.5%) 45 (69.2%)
Gender Male Female
35 (53.8%) 30 (46.2%)
Marrow blasts (%) ≤2 >2–10
35 (53.8%) 13 (20.0%) 10 (15.4%) 7 (10.8%)
Hemoglobin (g/dL)
ARTICLE IN PRESS
LR-5384; No. of Pages 6
Leukemia Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
MicroRNA-194-5p could serve as a diagnostic and prognostic biomarker in myelodysplastic syndromes Ji-Seon Choi a , Myung-Hyun Nam a , Soo-Young Yoon a , Seong-Ho Kang b,∗ a b
Department of Laboratory Medicine, Korea University College of Medicine, Seoul, South Korea Department of Laboratory Medicine, Chosun University College of Medicine, Gwangju, South Korea
a r t i c l e
i n f o
Article history: Received 15 January 2015 Received in revised form 26 March 2015 Accepted 19 April 2015 Available online xxx Keywords: MDS microRNA Deregulation Diagnosis Prognosis
a b s t r a c t Trisomy 8 and trisomy 1q are the most frequent chromosomal abnormalities in Korean patients with myelodysplastic syndrome (MDS). MicroRNA (miRNA) deregulation is involved in the development of hematological malignancies, including MDS, and cancer-associated genomic regions are known to encode miRNAs. The aim of the present study was to investigate the involvement of miRNAs encoded by chromosomes 8 and 1q in MDS. For this, the expression of nine miRNAs encoded by chromosome 8 (miR-30b-5p, miR-30d-5p, miR-101-3p, miR-124-3p, miR-151a-5p, miR-320a, miR-486-5p, miR-596, and miR-8755p) and three miRNAs encoded by chromosome 1q (miR-29c-3p, miR-194-5p, and miR-214-3p) was compared between 65 MDS patients and 11 controls. We found a significant upregulation of miR-1945p (5.1-fold, P = 0.002) and miR-320a (2.94-fold, P = 0.016) in MDS patients compared with controls. The patients with low miR-194-5p expression showed a significantly decreased overall survival (P = 0.049). The areas under the miR-194-5p and miR-320a ROC curves were 0.797 (P = 0.002) and 0.729 (P = 0.016), respectively. Although these findings need to be validated in a larger patient population, our results indicate that miR-194-5p is a candidate diagnostic biomarker for MDS and that low miR-194-5p expression could be associated with poor overall survival for MDS patients. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The myelodysplastic syndromes (MDS) are characterized by pancytopenia of peripheral blood, dysplasia in one or more myeloid cell lines, ineffective hematopoiesis of hematopoietic stem cells, and increased risk of development of acute myeloid leukemia. Dysplasia in one or more myeloid cell lines is a prerequisite for the diagnosis of MDS and diagnosis of MDS was difficult when dysplasia is not definite; however, a presumptive diagnosis can be made if dysplasia is not definite but certain cytogenetic abnormalities are present [1]. The most frequent cytogenetic abnormalities in Korean MDS patients are +8 and 1q+ observed in 19.5–34.5% and 15.2–17.2%, respectively, of MDS cases [2,3]. Previous studies have suggested that +8 is the most frequent cytogenetic abnormality found in MDS patients from Asian countries and that the proportion of 1q+ is specifically increased in MDS patients
∗ Corresponding author at: Department of Laboratory Medicine, Chosun University College of Medicine, 365, Pilmun-daero, Dong-gu, Gwangju 501-717, South Korea. Tel.: +82 62 220 3272; fax: +82 62 232 2063. E-mail address: [email protected] (S.-H. Kang).
from Korea compared to those from Western countries and Japan [2–5]. MicroRNAs (miRNAs) are non-coding RNA species of approximately 22 nucleotides that bind 3 -untranslated regions (UTR) of target mRNAs to repress their translation and stability. MiRNAs are involved in cell proliferation, development, and differentiation [6,7]. Approximately half of miRNA-encoding genes are frequently located at the fragile sites and regions of copy-number alterations associated with cancer [8]. More than 70% of miRNAs are located at the genomic regions carrying leukemia-associated copy-number alterations in MDS and acute myeloid leukemia cell lines [9]. These studies suggest that miRNAs located at the regions of genomic alterations can be deregulated in MDS patients. For instance, miR-145 and miR-146a located at 5q have been reported to be downregulated in patients with 5q-syndrome and to be involved in the mediation of the 5q-syndrome phenotype [10]. Similarly, miRNAs located at chromosomes 8 and 1q, which most frequently carry abnormalities detected in Korean MDS patients, could also be deregulated. Currently, MDS diagnostics is based on the dysplasia of one or more myeloid cell lines, the presence of ring sideroblasts, and blast counts. Conventional cytogenetics and fluorescence in situ hybridization (FISH) can assist the diagnosis of MDS. However,
http://dx.doi.org/10.1016/j.leukres.2015.04.013 0145-2126/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Choi J-S, et al. MicroRNA-194-5p could serve as a diagnostic and prognostic biomarker in myelodysplastic syndromes. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.04.013
G Model LR-5384; No. of Pages 6
ARTICLE IN PRESS J.-S. Choi et al. / Leukemia Research xxx (2015) xxx–xxx
2
these methods have limitations: cytogenetics requires long cell culturing (>2 days) and FISH needs multiple expensive probes to detect frequent aneuploidy. In contrast, the detection of miRNAs using real-time PCR is fast and simple, and may be successfully used for MDS diagnostics. Thus, miRNAs located at chromosomes 8 and 1q could be the biomarkers specific to Korean MDS patients, as +8 and +1q are the most frequent cytogenetic abnormalities in this population. The aim of the present study was to investigate whether the expression of miRNAs located at chromosomes 8 and 1q could be used as diagnostic as well as outcome prediction biomarkers for MDS in Korean patients. 2. Material and methods 2.1. Patients The present study included 65 MDS patients and 11 controls. MDS diagnosis was based on the WHO classification of MDS [11]. Controls exhibited no evidence of cancer, and had normocellular bone marrow with no signs of hematologic disease. Clinical and laboratory data (karyotyping, complete blood cell counts, and analysis of bone marrow blasts) were obtained from patients’ medical records. This research was approved by the Korea University Anam Hospital Institutional Review Board and Korea University Ansan Hospital Institutional Review Board. 2.2. MiRNA extraction and quantification MiRNA was extracted from archived unstained bone marrow aspirate slides of the participants using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction, and used to evaluate the relative expression of nine miRNAs encoded by chromosome 8 (miR-30b-5p, miR-30d-5p, miR-1013p, miR-124-3p, miR-151a-5p, miR-320a, miR-486-5p, miR-596, and miR-875-5p) and three miRNAs encoded by chromosome 1q (miR-29c-3p, miR-194-5p, and miR214-3p). Multiplex reverse transcription was performed using the primer pool of the TaqMan MicroRNA Assay (Applied Biosystems, Carlsbad, CA, USA) and the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instruction. Because the amount of total RNA extracted from each sample was less than 350 ng, the synthesized cDNA was pre-amplified prior to performing real-time PCR using the primer pool and TaqMan probe of the TaqMan MicroRNA Assay and TaqMan PreAmp Master Mix (Applied Biosystems) according to the manufacturer’s instruction. MiRNA expression was evaluated by real-time PCR using the TaqMan MicroRNA Assay and a StepOne Real-Time PCR system (Applied Biosystems) according to the manufacturer’s instruction. The relative expression of each miRNA was quantified using the 2−Ct method [12]. To compare miRNA expression between MDS patients and controls, 2–Ct of each miRNA was calculated, where Ct = CtmiRNA – Ctendogenous control and Ct is the threshold cycle defined as the number of cycles at which the fluorescent signal exceeds the threshold. RNU48 expression was used as an endogenous control. 2.3. Statistics The relative expression of each miRNA was compared between MDS patients and controls using the non-parametric Mann Whitney test. The Revised International Prognostic Scoring System (IPSS-R) for myelodysplasia score was used as previously described [13] and the IPSS-R score was calculated using an IPSS-R calculator tool: (http://www.ipss-com, or http://mds-foundation.org/calcaulator/index.php). Overall survival was determined as the time from initial diagnosis to death and analyzed by the Kaplan–Meier method. SPSS version 21.0 (SPSS, Chicago, IL, USA) was used for the statistical analysis. The data are expressed as the mean ± statistical significance (SD).
3. Results 3.1. Patients’ characteristics Patients’ clinical and hematological parameters are presented in Table 1. The study included 35 male and 30 female MDS patients with a median age of 68 years (range, 21–88 years). MDS diagnosis based on the WHO criteria included 26 cases (34.2%) of refractory cytopenia with multilineage dysplasia (RCMD), 12 cases (15.8%) of myelodysplastic syndrome-unclassified (MDS-U), 10 cases (13.2%) of refractory anemia with excess blasts-2 (RAEB-2), nine cases (11.8%) of RAEB-1, seven cases (9.2%) of refractory anemia with unilineage dysplasia (RCUD), and one case (1.3%) of refractory anemia with ring sideroblasts (RARS). Of the 65 MDS patients, 28 patients
Table 1 Patients’ characteristics. Descriptive statistics Total patients Median age, years (minimum–maximum)
65 68 (21–88)
Age 61
6 (9.2%) 14 (21.5%) 45 (69.2%)
Gender Male Female
35 (53.8%) 30 (46.2%)
Marrow blasts (%) ≤2 >2–10
35 (53.8%) 13 (20.0%) 10 (15.4%) 7 (10.8%)
Hemoglobin (g/dL)
