G Model
ARTICLE IN PRESS
LR-5355; No. of Pages 7
Leukemia Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia Ying-zhi He a,1 , Zhao Liang a,1 , Mei-rong Wu b,1 , Qi Wen b , Lan Deng a , Chao-yang Song a , Bing-yi Wu a , San-Fang Tu a , Rui Huang a , Yu-hua Li a,∗ a b
Department of Hematology, Zhujiang Hospital, Southern Medical University, 510282 Guangzhou, China Second School of Clinical Medicine, Southern Medical University, 510515 Guangzhou, China
a r t i c l e
i n f o
Article history: Received 29 October 2014 Received in revised form 29 January 2015 Accepted 10 March 2015 Available online xxx Keywords: EPS8 MDR1 WT1 Acute lymphoblastic leukemia mRNA expression Minimal residual disease
a b s t r a c t Molecular markers have become an invaluable tool in monitoring disease status particularly of leukemias, as bone marrow samples can be easily collected for analysis during all stages of disease development including diagnosis, treatment, and follow-up. Two genes that have been used as prognostic markers in acute leukemia are Wilms’ tumor (WT1) and multidrug resistance-1 (MDR1). A novel gene, epidermal growth factor receptor pathway substrate 8 (EPS8), is often over-expressed and associated with poor outcome in some solid tumor types. However, whether EPS8 is also associated with the development of acute lymphoblastic leukemia (ALL) is unclear. Here, quantitative real-time PCR was used to evaluate the expression of EPS8, MDR1, and WT1 in bone marrow samples of adult ALL patients (n = 107) and non-leukemia controls (n = 22). EPS8, MDR1, and WT1 were detected in ALL patients, and significant correlations were found between expression profiles for EPS8 and MDR1, EPS8 and WT1, and MDR1 and WT1. In general, high expression of EPS8, MDR1, or WT1 in patients was associated with a higher risk of relapse. Furthermore, when patients were stratified based on high or low expression of the genes, Kaplan–Meier survival analysis indicated that disease-free survival of patients with the high-EPS8/high-WT1/highMDR1 profile was significantly shorter than in patients with the low-EPS8/low-WT1/low-MDR1 profile or those excluded from either of these groups (P < 0.0001). Thus, EPS8, as MDR1 and WT1, may be a clinically valuable biomarker for assessing the outcome of ALL patients. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Acute lymphoblastic leukemia (ALL) is a malignant disease most commonly diagnosed in adolescents and young adults, especially in patients younger than 15 years. The five-year survival rate for childhood ALL has increased to about 90% recently [1,2]. However, despite advances in therapy, leukemia relapse or no response to therapy remains the major cause of treatment failure for ALL patients [3]. Minimal residual disease (MRD) has been regarded as the most powerful prognostic indicator in adult and childhood ALL [2,4–8]. MRD+ patients have been shown to have a poorer outcome
∗ Corresponding author at: Department of Hematology, Zhujiang Hospital, Southern Medical University, No. 253 Gongye Avenue, Haizhu, 510282 Guangzhou, China. Tel.: +86 2061643188; fax: +86 2061643188. E-mail address: [email protected]. 1 These authors (Ying-zhi He, Zhao Liang and Mei-rong Wu) contributed equally to this work and should be considered as co-first authors.
compared to MRD− patients [4]. Several studies have suggested that the remission status as assessed by MRD might be helpful to identify high risk patients [5,6]. Thus, MRD monitoring has become routine in risk assessment and selection of therapeutic regimens for leukemia patients. Molecular techniques based on the analysis of nucleic acids, such as quantitative real-time polymerase chain reaction (qRT-PCR), appear to be highly sensitive and specific for the detection of MRD [9]. Therefore, intensive efforts are being undertaken in order to identify a combination of molecular markers that is highly sensitive for the detection of MRD. The Wilms’ tumor gene (WT1), which was originally identified as a tumor suppressor gene, is highly expressed in the cells of the majority of leukemia patients, especially in acute myeloid leukemia (AML) [10]. Previous studies have indicated that WT1 appears to be highly promising as a molecular MRD marker also in ALL [11,12]. Overexpression of a second gene, multidrug resistance-1 (MDR1), is thought to confer resistance to chemotherapy, and studies have indicated that the overexpression of MDR1 is associated with a worse prognosis in childhood ALL [13–15]. Moreover, results from a small scale study indicated that simultaneous detection of WT1
http://dx.doi.org/10.1016/j.leukres.2015.03.007 0145-2126/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: He Y-z, et al. Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.03.007
G Model
ARTICLE IN PRESS
LR-5355; No. of Pages 7
Y.-z. He et al. / Leukemia Research xxx (2015) xxx–xxx
2 Table 1 Clinical and biological characteristics of ALL patients. Patients (n) Sex (male/female) Splenomegaly/hepatomegaly (+/−)
107 72/35 30/77
Median (range) Age (years) WBC count (×103 /L) Platelet count (×103 /L) Hemoglobin (g/L)
30 (10–74) 55.21 (1.89–373.13) 50.00 (5–948) 79.50 (48–140)
n (%) Stage NDPs CRPs RPs
40 (37%) 41 (38%) 26 (25%)
+, positive; −, negative; NDPs, newly diagnosed patients; CRPs, complete remission patients; RPs, relapsed patients.
and MDR1 in leukemia may provide useful information for clinical management of ALL patients [16]. Although WT1 expression has been extensively studied in AML and MDR1 expression has been widely investigated in childhood ALL, reports in adult ALL remain limited. Epidermal growth factor receptor pathway substrate 8 (EPS8) is a tyrosine kinase substrate, involved in many signaling pathways that promote proliferation, tumorigenesis, and the development of metastases [17–19]. Growing evidence indicates that the overexpression of EPS8 is associated with a worse outcome in solid tumors and hematological malignances, such as cervical cancer, oral squamous cell carcinoma (OSCC), and ALL in infants [20–22]. However, whether EPS8 might be a molecular marker for monitoring MRD in adult ALL patients is currently unknown. In the current study, the mRNA levels of the EPS8 gene, in addition to MDR1 and WT1 genes, were evaluated in a cohort of Chinese ALL patients and non-leukemia controls, with qRT-PCR. Potential prognostic and therapeutic implications of the molecular markers in ALL were examined based on survival data of the patients.
cohort consisted of newly diagnosed patients (NDPs; n = 40), complete remission patients (CRPs; n = 41), and relapsed patients (RPs; n = 26). Some of the NDPs were monitored from primary diagnosis to complete remission (CRPs; n = 27) or from primary diagnosis to relapse (RPs; n = 15). The human acute myeloid leukemia cell line KG1a was cultured in RPMI 1640 medium (Hyclone; Beijing, China) supplemented with 10% fetal bovine serum (Sijiqing Bioengineering Company; Hangzhou, China). Cells were seeded at a density of 4 × 105 cells per mL and incubated for 48 h at 37 ◦ C in a humidified chamber containing 5% CO2 , before being collected for further use. 2.3. Isolation of RNA and cDNA synthesis Total RNA was extracted from all BM samples and KG1a cells with TRIzol® Reagent (Invitrogen; Carlsbad, CA, USA), following the manufacturer’s instructions. The concentration and purity of isolated RNA was evaluated with a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies Inc.; Wilimington, DE, USA); purity was estimated from the absorbance ratio 260 nm/280 nm (mean ratio = 1.94; range, 1.88–2.02). cDNA was synthesized from RNA (1.0 g) in a 20 L reaction volume with the PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa Bio Inc.; Tokyo, Japan) according to the manufacturer’s instructions and stored at −80 ◦ C until further use. 2.4. qRT-PCR The NCBI Sequence database and Primer Express 3.0 (Applied Biosystems; Foster City, CA, USA) were used to design four pairs of gene-specific primers. The sequences of all qRT-PCR primers and the lengths of the PCR products are listed in Table 2. The primers were synthesized by Invitrogen (Shanghai, China). qRT-PCR was performed in a 20 L reaction for EPS8, MDR1, WT1, and glyceraldehyde-3phosphatedehydrogenase (GAPDH) mRNAs, with the SYBR® Green Dye detection system (Applied Biosystems), in 96-well plates on an ABI Prism 7500 Thermal Cycler (Applied Biosystems). The reaction mixture contained cDNA (2 L) diluted in DEPCtreated water (6.8 L), 0.6 L of gene-specific primers (final concentration, 0.3 M each), and 10 L of 2× SYBR® Select Master Mix (Applied Biosystems) containing AmpliTaq Gold® hot-start DNA polymerase. The thermal cycle profile included a UDG incubation at 50 ◦ C for 2 min with activation of hot-start DNA polymerase activation at 95 ◦ C for 2 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, primer annealing at 59 ◦ C for 15 s, and extension at 72 ◦ C for 15 s. The increase in fluorescence emission (Rn) was measured during the course of PCR amplification, and the difference (Rn) between the fluorescence emission of the PCR product and the baseline was calculated and plotted versus the cycle number. All samples were performed in duplicate to evaluate data reproducibility, and the average threshold cycle (CT) values were calculated for expression analysis. 2.5. Dissociation curve analysis
2. Materials and methods
The study was approved by the ethical review board of Zhujiang Hospital (Guangzhou, China). Written informed consent was obtained from all participants.
Following amplification, a dissociation curve was generated to distinguish PCR products of interest from primer-dimers or other non-specific products, according to the melting temperature (Tm ). The Tm of the EPS8, MDR1, WT1, and GAPDH products are shown in Table 2. Primer-dimers and other nonspecific products were not detected after melting of the PCR products.
2.2. Patients and cell line
2.6. Calculations and validation of the comparative CT (2−CT ) method
Bone marrow (BM) samples from ALL patients (n = 107) were collected from the Hematology Department in Zhujiang Hospital, Guangzhou, China between January 2012 and July 2014. The clinical characteristics of patients are shown in Table 1. In addition to the ALL patients (male = 72; female = 35), non-leukemia patients (NLPs, n = 22; male = 14; female = 9) were included in the study as the control group. Overall survival (OS) was calculated as the time from the primary diagnosis to death or the last contact. Follow-up information was available for eight patients. All patients received two cycles of a standard induction with VDLP [23], followed by consolidation chemotherapy [24] alternating with high-dose MTX or Ara-C therapy. The ALL
The relative quantification of expression of the target gene mRNA was calculated with the 2−CT method. GAPDH served as the internal control to normalize amplification reactions for the amount of cDNA added. Amplification of the target genes and GAPDH in the human acute myeloid leukemia cell line KG1a comprised the calibrator for our system in order to compare results from distinct runs [25]. The CT is the difference between the CT of the target gene and the CT of the endogenous reference gene (GAPDH) of the same sample. The CT represents the difference between mean values of the target gene in a BM sample and in the calibrator, as calculated from a single PCR run.
2.1. Ethics statement
Table 2 Primers used for real-time PCR amplification of EPS8, MDR1, WT1 and GAPDH. Gene
Sequence (5 -3 )
GenBank
Length
Tm of the product
EPS8
F-CTGCTCCATCACCTCCTCCAA R-CGATACTGCCACCACTGTCACT F-GGGAGCTTAACACCCGACTTA R-GCCAAAATCACAAGGGTTAGCTT F-CACAGCACAGGGTACGAGAG R-CAAGAGTCGGGGCTACTCCA F-GGAGCGAGATCCCTCCAAAAT R-GGCTGTTGTCATACTTCTCATGG
XM 006719057.1
141 bp
83.8
NM 000927.4
154 bp
78.4
NM 001198552.1
133 bp
85.3
NM 001289746.1
197 bp
86.8
MDR1 WT1 GAPDH
F, forward; R, reverse; EPS8, epidermal growth factor receptor pathway substrate No.8; MDR1, multidrug-resistance 1; WT1, Wilm’s tumor gene 1; GAPDH, reduced glyceraldehyde-phosphate dehydrogenase.
Please cite this article in press as: He Y-z, et al. Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.03.007
G Model
ARTICLE IN PRESS
LR-5355; No. of Pages 7
Y.-z. He et al. / Leukemia Research xxx (2015) xxx–xxx
3
3. Results 3.1. EPS8, MDR1, and WT1 expression analysis in ALL
Fig. 1. Relative expressions levels of EPS8, MDR1, and WT1 in newly diagnosed patients (NDPs). Gene expression levels of EPS8 (circles), MDR1 (squares), and WT1 (triangles), as determined by qRT-PCR, in NDPs relative to the leukemia cell line KG1a.
The application of the 2−CT method requires that the PCR amplification efficiencies of the target and the reference genes are approximately equal and close to 1 [25]. For determination of the efficiencies of the target gene (EPS8, MDR1, and WT1) and the endogenous reference gene (GAPDH), a validation experiment was conducted using serial dilutions of the KG1a cDNA as template over five orders of magnitude. The efficiency of each gene was calculated with the following formula: E = −1 + 10(−1/ ␣ ) , where ˛ is the slope of the corresponding amplification plot. The slopes of EPS8, MDR1, WT1, and GAPDH amplification plots were similar (−3.087, −3.089, −3.145, and −3.210, respectively), yielding high efficiencies for the corresponding amplicons (110.82%, 110.75%, 107.93%, and 104.90%, respectively).
2.7. Statistical analysis Statistical analysis was performed with SPSS version 17.0 (released 2008; SPSS Inc., Chicago, IL, USA). Analyses of the differences in EPS8, MDR1, and WT1 expression profiles between the ALL patients (including 40 NDPs, 41 CRPs, and 26 RPs) and NLPs (n = 22) were performed with a nonparametric Mann–Whitney U test. Patients were separated into high and low gene expression groups based on the median values [26,27] for EPS8, MDR1, and WT1 expression levels (106.6, 68.1, and 51.1, respectively) in the NDPs. Spearman correlation analysis was used to identify associations between EPS8, MDR1, or WT1 mRNA expression and clinical characteristics of NDPs. Survival analyses were performed by constructing Kaplan–Meier diseasefree survival (DFS) curves. The differences between the curves were evaluated by the log-rank test. Statistical significance was defined as a P value < 0.05.
Expression of the target genes was first determined for patients based on disease status: newly diagnosed, complete remission, or relapse. The levels of EPS8, WT1, and MDR1 mRNA expression at diagnosis were shown in Fig. 1. When the data were compared between the 107 ALL patients and the 22 NLPs, expression of EPS8, MDR1, and WT1 was more frequently detected and higher in NDPs and RPs compared to CRPs or NLPs (Table 3). Differences in the expression levels of EPS8, MDR1, and WT1 were statistically significant between NDPs, CRPs, RPs, and NLPs (Table 3). Furthermore, correlations in gene expression between EPS8 and MDR1 (rs = 0.759, P < 0.0001), EPS8 and WT1 (rs = 0.764, P < 0.0001), and between MDR1 and WT1 (rs = 0.617, P < 0.0001; Fig. 2) were significant. MRD detection by eight-color flow cytometry was performed on 29 CRPs. The method of multiparameter flow cytometry used was that as standardized by the EU-supported EuroFlow Consortium [28,29]. MRD assessment was dependent on leukemiaassociated immunophenotypes (LAIPs) at diagnosis. The 29 CRPs were divided into two groups: MRD < 0.1% and MRD ≥ 0.1%. The levels of EPS8 transcript in the MRD < 0.1% group (n = 21, median = 7.6, range: 3.0–17.5) was lower than in the MRD ≥ 0.1% group (n = 8, median = 26.9, range: 20.5–32.9; P < 0.0001).
3.2. High expression of EPS8, MDR1, and WT1 is associated with relapse in ALL patients One of the goals of the experiments was to identify relationships between gene expression and clinical characteristics, which might reveal mechanisms underlying pathogenesis and/or guide the clinical management of ALL patients. Therefore, data were evaluated for any possible associations between expression levels of EPS8, MDR1, and WT1, and clinical characteristics in the NDPs from the cohort. No significant differences were observed for expression of EPS8, MDR1, or WT1 based on age, gender, WBC count, presence of the BCR/ABL fusion gene, T/B immunophenotype, risk degree, or whether CR was achieved after one course of induction chemotherapy. However, higher
Table 3 EPS8, MDR1, and WT1 expression analysis in 107 ALL patients and 22 non-leukemia patients. Variable median (range)
NDPs (n = 40)
CRPs (n = 41)
RPs (n = 26)
NLPs (n = 22)
P value
EPS8 MDR1 WT1
106.6 (10.5–48867.6) 68.1 (3.9–3361.0) 51.1 (1.2–2514.6)
9.5 (3.0–32.9) 11.7 (2.2–44.6) 13.0 (4.6–35.7)
70.2 (6.1–2421.8) 89.0 (10.2–516.1) 72.0 (6.3–340.6)
4.7 (0.5–21.5) 4.2 (0.3–13.6) 4.31 (0–11.4)
ARTICLE IN PRESS
LR-5355; No. of Pages 7
Leukemia Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia Ying-zhi He a,1 , Zhao Liang a,1 , Mei-rong Wu b,1 , Qi Wen b , Lan Deng a , Chao-yang Song a , Bing-yi Wu a , San-Fang Tu a , Rui Huang a , Yu-hua Li a,∗ a b
Department of Hematology, Zhujiang Hospital, Southern Medical University, 510282 Guangzhou, China Second School of Clinical Medicine, Southern Medical University, 510515 Guangzhou, China
a r t i c l e
i n f o
Article history: Received 29 October 2014 Received in revised form 29 January 2015 Accepted 10 March 2015 Available online xxx Keywords: EPS8 MDR1 WT1 Acute lymphoblastic leukemia mRNA expression Minimal residual disease
a b s t r a c t Molecular markers have become an invaluable tool in monitoring disease status particularly of leukemias, as bone marrow samples can be easily collected for analysis during all stages of disease development including diagnosis, treatment, and follow-up. Two genes that have been used as prognostic markers in acute leukemia are Wilms’ tumor (WT1) and multidrug resistance-1 (MDR1). A novel gene, epidermal growth factor receptor pathway substrate 8 (EPS8), is often over-expressed and associated with poor outcome in some solid tumor types. However, whether EPS8 is also associated with the development of acute lymphoblastic leukemia (ALL) is unclear. Here, quantitative real-time PCR was used to evaluate the expression of EPS8, MDR1, and WT1 in bone marrow samples of adult ALL patients (n = 107) and non-leukemia controls (n = 22). EPS8, MDR1, and WT1 were detected in ALL patients, and significant correlations were found between expression profiles for EPS8 and MDR1, EPS8 and WT1, and MDR1 and WT1. In general, high expression of EPS8, MDR1, or WT1 in patients was associated with a higher risk of relapse. Furthermore, when patients were stratified based on high or low expression of the genes, Kaplan–Meier survival analysis indicated that disease-free survival of patients with the high-EPS8/high-WT1/highMDR1 profile was significantly shorter than in patients with the low-EPS8/low-WT1/low-MDR1 profile or those excluded from either of these groups (P < 0.0001). Thus, EPS8, as MDR1 and WT1, may be a clinically valuable biomarker for assessing the outcome of ALL patients. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Acute lymphoblastic leukemia (ALL) is a malignant disease most commonly diagnosed in adolescents and young adults, especially in patients younger than 15 years. The five-year survival rate for childhood ALL has increased to about 90% recently [1,2]. However, despite advances in therapy, leukemia relapse or no response to therapy remains the major cause of treatment failure for ALL patients [3]. Minimal residual disease (MRD) has been regarded as the most powerful prognostic indicator in adult and childhood ALL [2,4–8]. MRD+ patients have been shown to have a poorer outcome
∗ Corresponding author at: Department of Hematology, Zhujiang Hospital, Southern Medical University, No. 253 Gongye Avenue, Haizhu, 510282 Guangzhou, China. Tel.: +86 2061643188; fax: +86 2061643188. E-mail address: [email protected]. 1 These authors (Ying-zhi He, Zhao Liang and Mei-rong Wu) contributed equally to this work and should be considered as co-first authors.
compared to MRD− patients [4]. Several studies have suggested that the remission status as assessed by MRD might be helpful to identify high risk patients [5,6]. Thus, MRD monitoring has become routine in risk assessment and selection of therapeutic regimens for leukemia patients. Molecular techniques based on the analysis of nucleic acids, such as quantitative real-time polymerase chain reaction (qRT-PCR), appear to be highly sensitive and specific for the detection of MRD [9]. Therefore, intensive efforts are being undertaken in order to identify a combination of molecular markers that is highly sensitive for the detection of MRD. The Wilms’ tumor gene (WT1), which was originally identified as a tumor suppressor gene, is highly expressed in the cells of the majority of leukemia patients, especially in acute myeloid leukemia (AML) [10]. Previous studies have indicated that WT1 appears to be highly promising as a molecular MRD marker also in ALL [11,12]. Overexpression of a second gene, multidrug resistance-1 (MDR1), is thought to confer resistance to chemotherapy, and studies have indicated that the overexpression of MDR1 is associated with a worse prognosis in childhood ALL [13–15]. Moreover, results from a small scale study indicated that simultaneous detection of WT1
http://dx.doi.org/10.1016/j.leukres.2015.03.007 0145-2126/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: He Y-z, et al. Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.03.007
G Model
ARTICLE IN PRESS
LR-5355; No. of Pages 7
Y.-z. He et al. / Leukemia Research xxx (2015) xxx–xxx
2 Table 1 Clinical and biological characteristics of ALL patients. Patients (n) Sex (male/female) Splenomegaly/hepatomegaly (+/−)
107 72/35 30/77
Median (range) Age (years) WBC count (×103 /L) Platelet count (×103 /L) Hemoglobin (g/L)
30 (10–74) 55.21 (1.89–373.13) 50.00 (5–948) 79.50 (48–140)
n (%) Stage NDPs CRPs RPs
40 (37%) 41 (38%) 26 (25%)
+, positive; −, negative; NDPs, newly diagnosed patients; CRPs, complete remission patients; RPs, relapsed patients.
and MDR1 in leukemia may provide useful information for clinical management of ALL patients [16]. Although WT1 expression has been extensively studied in AML and MDR1 expression has been widely investigated in childhood ALL, reports in adult ALL remain limited. Epidermal growth factor receptor pathway substrate 8 (EPS8) is a tyrosine kinase substrate, involved in many signaling pathways that promote proliferation, tumorigenesis, and the development of metastases [17–19]. Growing evidence indicates that the overexpression of EPS8 is associated with a worse outcome in solid tumors and hematological malignances, such as cervical cancer, oral squamous cell carcinoma (OSCC), and ALL in infants [20–22]. However, whether EPS8 might be a molecular marker for monitoring MRD in adult ALL patients is currently unknown. In the current study, the mRNA levels of the EPS8 gene, in addition to MDR1 and WT1 genes, were evaluated in a cohort of Chinese ALL patients and non-leukemia controls, with qRT-PCR. Potential prognostic and therapeutic implications of the molecular markers in ALL were examined based on survival data of the patients.
cohort consisted of newly diagnosed patients (NDPs; n = 40), complete remission patients (CRPs; n = 41), and relapsed patients (RPs; n = 26). Some of the NDPs were monitored from primary diagnosis to complete remission (CRPs; n = 27) or from primary diagnosis to relapse (RPs; n = 15). The human acute myeloid leukemia cell line KG1a was cultured in RPMI 1640 medium (Hyclone; Beijing, China) supplemented with 10% fetal bovine serum (Sijiqing Bioengineering Company; Hangzhou, China). Cells were seeded at a density of 4 × 105 cells per mL and incubated for 48 h at 37 ◦ C in a humidified chamber containing 5% CO2 , before being collected for further use. 2.3. Isolation of RNA and cDNA synthesis Total RNA was extracted from all BM samples and KG1a cells with TRIzol® Reagent (Invitrogen; Carlsbad, CA, USA), following the manufacturer’s instructions. The concentration and purity of isolated RNA was evaluated with a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies Inc.; Wilimington, DE, USA); purity was estimated from the absorbance ratio 260 nm/280 nm (mean ratio = 1.94; range, 1.88–2.02). cDNA was synthesized from RNA (1.0 g) in a 20 L reaction volume with the PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa Bio Inc.; Tokyo, Japan) according to the manufacturer’s instructions and stored at −80 ◦ C until further use. 2.4. qRT-PCR The NCBI Sequence database and Primer Express 3.0 (Applied Biosystems; Foster City, CA, USA) were used to design four pairs of gene-specific primers. The sequences of all qRT-PCR primers and the lengths of the PCR products are listed in Table 2. The primers were synthesized by Invitrogen (Shanghai, China). qRT-PCR was performed in a 20 L reaction for EPS8, MDR1, WT1, and glyceraldehyde-3phosphatedehydrogenase (GAPDH) mRNAs, with the SYBR® Green Dye detection system (Applied Biosystems), in 96-well plates on an ABI Prism 7500 Thermal Cycler (Applied Biosystems). The reaction mixture contained cDNA (2 L) diluted in DEPCtreated water (6.8 L), 0.6 L of gene-specific primers (final concentration, 0.3 M each), and 10 L of 2× SYBR® Select Master Mix (Applied Biosystems) containing AmpliTaq Gold® hot-start DNA polymerase. The thermal cycle profile included a UDG incubation at 50 ◦ C for 2 min with activation of hot-start DNA polymerase activation at 95 ◦ C for 2 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, primer annealing at 59 ◦ C for 15 s, and extension at 72 ◦ C for 15 s. The increase in fluorescence emission (Rn) was measured during the course of PCR amplification, and the difference (Rn) between the fluorescence emission of the PCR product and the baseline was calculated and plotted versus the cycle number. All samples were performed in duplicate to evaluate data reproducibility, and the average threshold cycle (CT) values were calculated for expression analysis. 2.5. Dissociation curve analysis
2. Materials and methods
The study was approved by the ethical review board of Zhujiang Hospital (Guangzhou, China). Written informed consent was obtained from all participants.
Following amplification, a dissociation curve was generated to distinguish PCR products of interest from primer-dimers or other non-specific products, according to the melting temperature (Tm ). The Tm of the EPS8, MDR1, WT1, and GAPDH products are shown in Table 2. Primer-dimers and other nonspecific products were not detected after melting of the PCR products.
2.2. Patients and cell line
2.6. Calculations and validation of the comparative CT (2−CT ) method
Bone marrow (BM) samples from ALL patients (n = 107) were collected from the Hematology Department in Zhujiang Hospital, Guangzhou, China between January 2012 and July 2014. The clinical characteristics of patients are shown in Table 1. In addition to the ALL patients (male = 72; female = 35), non-leukemia patients (NLPs, n = 22; male = 14; female = 9) were included in the study as the control group. Overall survival (OS) was calculated as the time from the primary diagnosis to death or the last contact. Follow-up information was available for eight patients. All patients received two cycles of a standard induction with VDLP [23], followed by consolidation chemotherapy [24] alternating with high-dose MTX or Ara-C therapy. The ALL
The relative quantification of expression of the target gene mRNA was calculated with the 2−CT method. GAPDH served as the internal control to normalize amplification reactions for the amount of cDNA added. Amplification of the target genes and GAPDH in the human acute myeloid leukemia cell line KG1a comprised the calibrator for our system in order to compare results from distinct runs [25]. The CT is the difference between the CT of the target gene and the CT of the endogenous reference gene (GAPDH) of the same sample. The CT represents the difference between mean values of the target gene in a BM sample and in the calibrator, as calculated from a single PCR run.
2.1. Ethics statement
Table 2 Primers used for real-time PCR amplification of EPS8, MDR1, WT1 and GAPDH. Gene
Sequence (5 -3 )
GenBank
Length
Tm of the product
EPS8
F-CTGCTCCATCACCTCCTCCAA R-CGATACTGCCACCACTGTCACT F-GGGAGCTTAACACCCGACTTA R-GCCAAAATCACAAGGGTTAGCTT F-CACAGCACAGGGTACGAGAG R-CAAGAGTCGGGGCTACTCCA F-GGAGCGAGATCCCTCCAAAAT R-GGCTGTTGTCATACTTCTCATGG
XM 006719057.1
141 bp
83.8
NM 000927.4
154 bp
78.4
NM 001198552.1
133 bp
85.3
NM 001289746.1
197 bp
86.8
MDR1 WT1 GAPDH
F, forward; R, reverse; EPS8, epidermal growth factor receptor pathway substrate No.8; MDR1, multidrug-resistance 1; WT1, Wilm’s tumor gene 1; GAPDH, reduced glyceraldehyde-phosphate dehydrogenase.
Please cite this article in press as: He Y-z, et al. Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.03.007
G Model
ARTICLE IN PRESS
LR-5355; No. of Pages 7
Y.-z. He et al. / Leukemia Research xxx (2015) xxx–xxx
3
3. Results 3.1. EPS8, MDR1, and WT1 expression analysis in ALL
Fig. 1. Relative expressions levels of EPS8, MDR1, and WT1 in newly diagnosed patients (NDPs). Gene expression levels of EPS8 (circles), MDR1 (squares), and WT1 (triangles), as determined by qRT-PCR, in NDPs relative to the leukemia cell line KG1a.
The application of the 2−CT method requires that the PCR amplification efficiencies of the target and the reference genes are approximately equal and close to 1 [25]. For determination of the efficiencies of the target gene (EPS8, MDR1, and WT1) and the endogenous reference gene (GAPDH), a validation experiment was conducted using serial dilutions of the KG1a cDNA as template over five orders of magnitude. The efficiency of each gene was calculated with the following formula: E = −1 + 10(−1/ ␣ ) , where ˛ is the slope of the corresponding amplification plot. The slopes of EPS8, MDR1, WT1, and GAPDH amplification plots were similar (−3.087, −3.089, −3.145, and −3.210, respectively), yielding high efficiencies for the corresponding amplicons (110.82%, 110.75%, 107.93%, and 104.90%, respectively).
2.7. Statistical analysis Statistical analysis was performed with SPSS version 17.0 (released 2008; SPSS Inc., Chicago, IL, USA). Analyses of the differences in EPS8, MDR1, and WT1 expression profiles between the ALL patients (including 40 NDPs, 41 CRPs, and 26 RPs) and NLPs (n = 22) were performed with a nonparametric Mann–Whitney U test. Patients were separated into high and low gene expression groups based on the median values [26,27] for EPS8, MDR1, and WT1 expression levels (106.6, 68.1, and 51.1, respectively) in the NDPs. Spearman correlation analysis was used to identify associations between EPS8, MDR1, or WT1 mRNA expression and clinical characteristics of NDPs. Survival analyses were performed by constructing Kaplan–Meier diseasefree survival (DFS) curves. The differences between the curves were evaluated by the log-rank test. Statistical significance was defined as a P value < 0.05.
Expression of the target genes was first determined for patients based on disease status: newly diagnosed, complete remission, or relapse. The levels of EPS8, WT1, and MDR1 mRNA expression at diagnosis were shown in Fig. 1. When the data were compared between the 107 ALL patients and the 22 NLPs, expression of EPS8, MDR1, and WT1 was more frequently detected and higher in NDPs and RPs compared to CRPs or NLPs (Table 3). Differences in the expression levels of EPS8, MDR1, and WT1 were statistically significant between NDPs, CRPs, RPs, and NLPs (Table 3). Furthermore, correlations in gene expression between EPS8 and MDR1 (rs = 0.759, P < 0.0001), EPS8 and WT1 (rs = 0.764, P < 0.0001), and between MDR1 and WT1 (rs = 0.617, P < 0.0001; Fig. 2) were significant. MRD detection by eight-color flow cytometry was performed on 29 CRPs. The method of multiparameter flow cytometry used was that as standardized by the EU-supported EuroFlow Consortium [28,29]. MRD assessment was dependent on leukemiaassociated immunophenotypes (LAIPs) at diagnosis. The 29 CRPs were divided into two groups: MRD < 0.1% and MRD ≥ 0.1%. The levels of EPS8 transcript in the MRD < 0.1% group (n = 21, median = 7.6, range: 3.0–17.5) was lower than in the MRD ≥ 0.1% group (n = 8, median = 26.9, range: 20.5–32.9; P < 0.0001).
3.2. High expression of EPS8, MDR1, and WT1 is associated with relapse in ALL patients One of the goals of the experiments was to identify relationships between gene expression and clinical characteristics, which might reveal mechanisms underlying pathogenesis and/or guide the clinical management of ALL patients. Therefore, data were evaluated for any possible associations between expression levels of EPS8, MDR1, and WT1, and clinical characteristics in the NDPs from the cohort. No significant differences were observed for expression of EPS8, MDR1, or WT1 based on age, gender, WBC count, presence of the BCR/ABL fusion gene, T/B immunophenotype, risk degree, or whether CR was achieved after one course of induction chemotherapy. However, higher
Table 3 EPS8, MDR1, and WT1 expression analysis in 107 ALL patients and 22 non-leukemia patients. Variable median (range)
NDPs (n = 40)
CRPs (n = 41)
RPs (n = 26)
NLPs (n = 22)
P value
EPS8 MDR1 WT1
106.6 (10.5–48867.6) 68.1 (3.9–3361.0) 51.1 (1.2–2514.6)
9.5 (3.0–32.9) 11.7 (2.2–44.6) 13.0 (4.6–35.7)
70.2 (6.1–2421.8) 89.0 (10.2–516.1) 72.0 (6.3–340.6)
4.7 (0.5–21.5) 4.2 (0.3–13.6) 4.31 (0–11.4)
