Leukemia & Lymphoma, 2014; Early Online: 1–5 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.977883
ORIGINAL ARTICLE: CLINICAL
Clinical significance of reduced SFRP1 expression in acute myeloid leukemia Cui An1*, Hong Guo2*, Xiang-mei Wen2, Chun-yan Tang1, Jing Yang1, Xiao-wen Zhu1, Jia-yu Yin1, Qing Liu1, Ji-chun Ma2, Zhao-qun Deng2, Jiang Lin2 & Jun Qian1 1Department of Hematology and 2Laboratory Center, Affiliated People’s Hospital of Jiangsu University, Zhenjiang,
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Jiangsu, People’s Republic of China
the understanding of leukemia pathogenesis will provide more molecules to join the cohort for diagnosis, risk stratification and therapeutic targets. The Wnt signaling system has crucial roles in the control of a number of developmental and cellular processes. Deregulation of Wnt signaling contributes to mammary tumorigenesis by means of its roles in promoting tumor cell proliferation, inhibiting differentiation and blocking apoptosis [8,9]. It is well known that Wnt modulates the canonical (β-catenin mediated) and non-canonical (β-catenin independent) signaling pathways [8,9]. Under physiologic conditions, Wnt signal transduction is regulated extracellularly by multiple secreted and transmembrane antagonists, such as Wnt inhibitory factor (WIF), Dickkopfs (DKKs) and secreted frizzled-related proteins (SFRPs) [10]. SFRPs, the largest family of secreted Wnt antagonists, inhibit Wnt signaling by interacting with and binding to Wnt ligands [10]. However, increasing studies indicate that SFRPs can also activate Wnt signaling and interfere with other signaling systems through Wnt-independent mechanisms [10,11]. A decade of studies has shown that SFRP deregulation contributes to the development of tumors [11,12]. Moreover, aberrant methylation of SFRP promoters has also been identified in clinical cancer samples [11,12]. SFRP1 is the most studied member of the SFRP family in cancers. A few studies in AML have revealed a distinct association between SFRP1 methylation and t(8;21) [13–15]. However, the pattern of SFRP1 expression and its clinical relevance in AML remain poorly defined. This study aimed to detect the status of SFRP1 expression and explore the clinical significance of SFRP1 expression in AML.
Abstract Deregulation of secreted frizzled-related protein 1 (SFRP1) has been found in many types of cancer. However, the pattern of SFRP1 expression in acute myeloid leukemia (AML) is still unclear. This study determined SFRP1 expression in patients with AML. SFRP1 expression was decreased markedly in patients with AML compared to controls (p ⬍ 0.001). White blood cell (WBC) counts increased as SFRP1 expression decreased in AML (p ⫽ 0.016). Patients with low SFRP1 expression showed a different distribution of French–American–British (FAB) subtypes M1/M2/M3 from those with high SFRP1 expression (p ⫽ 0.031). NPM1 mutation was mainly observed in patients with low SFRP1 expression (p ⫽ 0.011). There was a weak trend that patients with AML with low SFRP1 expression had shorter overall survival (OS) than those with high SFRP1 expression (p ⫽ 0.103). Our results indicate that reduced SFRP1 expression is found more frequently in the less well-differentiated subgroups of AML and is associated with NPM1 mutation in AML. Keywords: SFRP1, acute myeloid leukemia, NPM1 mutation
Introduction Acute myeloid leukemia (AML) represents a group of clonal disorders resulting from hematopoietic stem cells losing the capability of normal differentiation and proliferation [1]. The pathologic mechanism of AML can be mainly interpreted by non-random chromosomal translocations and somatic gene mutations, as well as epigenetic alterations occurring in hematopoietic stem/progenitor cells. Numerous studies have given evidence that these alterations provide important diagnostic and prognostic information for AML [2–5]. Chromosomal abnormalities and gene mutations have been successfully introduced into the risk stratification and treatment choice of AML [6,7]. The continuous advancement in
Materials and methods Patients and samples One hundred and eighteen patients with de novo AML were included in this study. The diagnosis and classifica-
*These authors contributed equally as first authors. Correspondence: Jun Qian, MD, Department of Hematology, Affiliated People’s Hospital of Jiangsu University, 8 Dianli Rd., 212002 Zhenjiang, People’s Republic of China. Fax: ⫹ 86-511-85234387. E-mail: [email protected]. Jiang Lin, PhD, Laboratory Center, Affiliated People’s Hospital of Jiangsu University, 8 Dianli Rd., 212002 Zhenjiang, People’s Republic of China. Fax: ⫹ 86-511-85234387. E-mail: [email protected] Received 19 June 2014; revised 8 October 2014; accepted 12 October 2014
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tion of these patients were based on French–American– British (FAB) and World Health Organization (WHO) criteria [6,16]. The treatment protocol was described as reported previously [17]. Written informed consent was obtained from all patients. The study was approved by the Institutional Review Board of the Affiliated People’s Hospital of Jiangsu University. The main clinical and laboratory characteristics of these patients are listed in Table I. Twenty-three healthy donors were collected as controls. Seven human leukemic cell lines (HL60, NB4, THP-1, SHI-1, U937, HEL and K562) were also studied. All cell lines were cultured in Iscoves’s modified Dulbecco’s medium (IMDM) containing 10% fetal calf serum and grown at 37°C in a 5% CO2 humidified atmosphere.
Cytogenetic analysis Chromosomes were prepared routinely by the direct method or 24 h short-term culture of bone marrow cells. Karyotypes were analyzed on R-banded metaphases. Chro-
mosome abnormalities were described according to the International System for Human Cytogenetic Nomenclature [18]. Karyotype risk was identified according to cytogenetic abnormalities [19].
RNA isolation and cDNA synthesis Mononuclear cells from bone marrows of patients with AML and healthy donors were separated by Ficoll–Hypaque gradient. Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer ’s instructions and resolved in diethylpyrocarbonate-treated water. Two micrograms of total RNA were reverse transcribed into cDNA in a total reaction volume of 40 μL containing 1 ⫻ buffer, dNTPs 10 mM each, random hexamers 10 μM, RNAsin 80 units and 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase (MBI Fermentas, Hanover, NH). The reverse transcription system was incubated for 10 min at 25°C, 60 min at 42°C, and then stored at ⫺ 20°C.
Table I. Clinical characteristics of patients with AML classified according to SFRP1 expression. Status of SFRP1 expression Patient parameters Sex, male/female Median age, years (range) Median WBC, ⫻ 109/L (range) Median hemoglobin, g/L (range) Median platelets, ⫻ 109/L (range) FAB M1 M2 M3 M4 M5 M6 WHO AML with t(8;21) APL with t(15;17) AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic and monocytic leukemia Acute erythroid leukemia Karyotype classification Favorable Intermediate Poor No data Karyotype Normal t(8;21) t(15;17) Complex Other No data Gene mutation* C/EBPA (⫹/⫺) NPM1 (⫹/⫺) FLT3-ITD (⫹/⫺) DNMT3A (⫹/⫺) IDH1/2 (⫹/⫺) C-KIT (⫹/⫺) N/K-RAS (⫹/⫺) CR (⫹/⫺) SFRP1 transcript
Low (n ⫽ 39)
Medium (n ⫽ 40)
24/15 58 (15–86) 23.5 (0.3–528.0) 68 (40–138) 32 (7–264)
26/14 52.5 (15–87) 12.2 (0.5–130.2) 87 (34–123) 40 (4–399)
High (n ⫽ 39) 16/23 54 (15–87) 5.4 (0.8–135.9) 75 (31–131) 44 (3–140)
Total (n ⫽ 118)
p-Value
66/52 54 (15–87) 10.7 (0.3–528.0) 76 (31–138) 39 (3–399)
0.070 0.564 0.016 0.054 0.764 0.059
5 (13%) 14 (36%) 4 (10%) 14 (36%) 2 (5%) 0 (0%)
2 (5%) 19 (48%) 10 (25%) 8 (20%) 1 (2%) 0 (0%)
2 (5%) 10 (26%) 13 (33%) 8 (20%) 5 (13%) 1 (3%)
9 (8%) 43 (36%) 27 (23%) 30 (25%) 8 (7%) 1 (1%)
2 (6%) 4 (8%) 5 (11%) 12 (30%) 15 (42%) 1 (3%) 0 (0%)
3 (8%) 10 (23%) 2 (5%) 16 (41%) 8 (20%) 1 (3%) 0 (0%)
2 (5%) 13 (34%) 2 (3%) 8 (21%) 8 (21%) 5 (13%) 1 (3%)
7 (6%) 27 (23%) 9 (8%) 36 (30%) 31 (26%) 7 (6%) 1 (1%)
6 (15%) 24 (62%) 3 (8%) 6 (15%)
13 (33%) 22 (55%) 4 (10%) 1 (2%)
15 (38%) 16 (41%) 7 (18%) 1 (3%)
34 (29%) 62 (52%) 14 (12%) 8 (7%)
19 (49%) 2 (5%) 4 (10%) 3 (8%) 5 (13%) 6 (15%)
15 (38%) 3 (7%) 10 (25%) 3 (8%) 8 (20%) 1 (2%)
15 (39%) 2 (5%) 13 (33%) 6 (15%) 2 (5%) 1 (3%)
49 (41%) 7 (6%) 27 (23%) 12 (10%) 15 (13%) 8 (7%)
0.072
0.129
0.229
0/34 (0%) 6/28 (18%) 6/28 (18%) 3/31 (9%) 2/32 (6%) 2/32 (6%) 1/33 (3%) 13/19 (41%) 0 (0–0.004)
2/33 (6%) 2/33 (6%) 5/30 (14%) 2/33 (6%) 3/32 (9%) 1/34 (3%) 4/31 (11%) 19/18 (51%) 0.137 (0.004–0.034)
1/32 (3%) 0/33 (0%) 2/31 (6%) 1/32 (3%) 1/32 (3%) 0/33 (0%) 4/29 (12%) 16/17 (48%) 0.212 (0.035–3.782)
3/99 (3%) 8/94 (8%) 13/89 (13%) 6/96 (6%) 6/96 (6%) 3/99 (3%) 9/93 (9%) 48/54 (47%) 0.014 (0–3.782)
0.653 0.011 0.396 0.779 0.869 0.653 0.350 0.680 ⬍ 0.001
AML, acute myeloid leukemia; WBC, white blood cells; FAB, French–American–British classification; WHO, World Health Organization; CR, complete remission. *Percentage equal to number of mutated patients divided by total cases in each group.
SFRP1 expression in AML 3
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Real-time quantitative polymerase chain reaction Real-time quantitative polymerase chain reaction (RQ-PCR) was carried out using a SYBR Premix Ex Taq™ II kit (Takara, Tokyo, Japan) on an ABI 7300 Thermo cycler (Applied Biosystems, Foster City, CA). Primers for the SFRP1 transcript were 5’-AGATGCTTAAG TGTGACAAGTTCC-3’ (forward) and 5’-TCAGATTTCAACTCGTTGTCACAG-3’ (reverse) with expected PCR products of 130 bp. The PCR reaction system was composed of 20 ng of cDNA, 0.8 μM of each primer, 10 μM of SYBRPremix Ex Taq II and 0.4 μM of ROX Reference Dye1 (Takara). The PCR procedure was as follows: initial predenaturation at 95°C for 30 s, then 45 cycles at 95°C for 5 s, 64°C for 30 s and 72°C for 30 s, and a fluorescence collection step at 85°C for 30 s, followed by a melting program at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s and 60°C for 15 s. Negative and positive controls were included in all experiments. The specificity of RQ-PCR products was verified by melting curves and DNA sequencing. The housekeeping gene ABL was used to calculate the abundance of SFRP1 mRNA. A standard curve was generated by amplifying plasmids cloned with SFRP1 or ABL in a series of dilutions. Relative SFRP1 expression was calculated using the following equation: NSFRP1 ⫽ (ESFRP1)ΔCT SFRP1 (control-sample) ⫼ (EABL)ΔCT ABL (control-sample) The parameter efficiency (E) is derived from the formula E ⫽ 10(⫺ 1/slope) (slope refers to CT versus plasmid concentration plot in the standard curve).
Statistical analysis Statistical analysis was applied using the SPSS 17.0 software package (SPSS, Chicago, IL). χ2 analysis or Fisher’s exact test was conducted to compare differences in categorical variables. The Kruskal–Wallis test or Mann–Whitney U-test was performed to compare differences in continuous variables. Survival was analyzed according to the Kaplan–Meier method.
Results SFRP1 expression in de novo AML and leukemic cell lines The level of SFRP1 expression was analyzed in patients with AML and healthy controls. It was found that SFRP1 was expressed in all controls (0.088–10.10, 2.595 ⫾ 2.580, median 1.750). The SFRP1 transcript was significantly decreased in patients with AML (0.000–3.782, median 0.0137) compared to controls (p ⬍ 0.001, Figure 1). Representative electrophoresis
Figure 1. Relative expression levels of SFRP1 in patients with AML and controls.
Figure 2. Electrophoresis results of RQ-PCR products in patients with AML. 1: Gene Ruler™ 100 bp DNA ladder; 2–3: normal controls; 4–5: AML samples in low expression group; 6–7: AML samples in medium expression group; 8–9: AML samples in high expression group; 10: cloned plasmid; 11: negative control. (A) SFRP1; (B) ABL.
results for RQ-PCR products are shown in Figure 2. Among the seven tested cell lines, only U937 and SHI-1 showed positive SFRP1 expression, and the remaining five cell lines presented loss of expression (Figure 3).
Clinical and laboratory characteristics of AML Patients with AML were divided into three subgroups according to tertile values of the SFRP1 level: low SFRP1 expression (n ⫽ 39, 33%), intermediate SFRP1 expression (n ⫽ 40, 34%) and high SFRP1 expression (n ⫽ 39, 33%). No differences were found in sex, age, or hemoglobin and platelet contents between these three groups (p ⬎ 0.05, Table I). However, white blood cell (WBC) count increased significantly as SFRP1 expression decreased (p ⫽ 0.016, Table I). Excluding FAB-M3 subtype, which is often accompanied by a low WBC level, there was also a significant difference in WBC count among the three subgroups (p ⫽ 0.022). No significant difference was observed in FAB subtype, WHO subtype or karyotype among the three subgroups. Among the FAB subtypes M1/M2/M3 (M1, AML without maturation; M2, AML with maturation; M3, acute promyelocytic leukemia), however, there was a trend of low SFRP1 expression toward the differentiation block of granulocytic lineage (p ⫽ 0.072). The subgroup with low SFRP1 expression had more obvious differentiation block of granulocytic lineage compared with the subgroup with high SFRP1 expression (p ⫽ 0.031). Six NPM1 mutations were observed in the low SFRP1 expression group while there were two in the intermediate SFRP1 expression group and none in the high SFRP1 expression group (p ⫽ 0.011) (Table I). No correlation was found between SFRP1 expression and other gene mutations (Table I).
Impact of SFRP1 expression on prognosis To investigate the prognostic impact of SFRP1 expression in AML, survival analysis was performed among the three subgroups with follow-up data. There was no significant difference in the rate of complete remission (CR) among the three subgroups (p ⫽ 0.680). Although no significant difference was observed in overall survival (OS) (p ⫽ 0.160), there was a weak trend that patients with AML with low SFRP1
Figure 3. Electrophoresis results of RQ-PCR products in leukemic cell lines. 1: Gene Ruler™ 100 bp DNA ladder; 2: U937; 3: K562; 4: SHI-1; 5: THP-1; 6: HL60; 7: HEL; 8: NB4; 9: cloned plasmid; 10: negative control. (A) SFRP1; (B) ABL.
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expression had shorter OS than those with high SFRP1 expression (p ⫽ 0.103) (Figure 4).
attributed to the observation that RUNX1–ETO fusion protein can result in endogenous SFRP1 transcriptional reduction through binding to the SFRP1 promoter [14]. It was interesting that most patients (6/8, 75%) with NPM1 mutations showed low SFRP1 expression in this study, which was contrary to the observation that the majority of NPM1 mutations were identified in patients with an unmethylated SFRP1 promoter [15]. However, two other studies did not find a close association of aberrant methylation of SFRP1 and NPM1 mutations [14,20]. Larger studies are needed to further determine this relationship in AML. In this study, we did not reveal the prognostic influence of SFRP1 down-regulation in AML, in accordance with three studies on SFRP1 methylation reported previously [13,15,21], although the independent prediction of SFRP1 methylation for prolonged event-free and relapse-free survivals was disclosed in patients with childhood non-M3 AML with nonadverse cytogenetics [14]. These discrepant results probably reflect the small number of patients and different patient selection. Large-scale studies with more patients with AML are needed to clarify this point. In summary, our findings indicate that reduced SFRP1 expression is found more frequently in the less welldifferentiated subgroups of AML and is associated with NPM1 mutation in AML.
Discussion
Acknowledgements
A few studies have focused on promoter methylation of SFRPs and other Wnt modulators in AML [13–15,20,21]. However, the expression of SFRPs, particularly its clinical relevance, remains seldom studied in AML to date. In this study, we confirmed that the SFRP mRNA level was significantly reduced in AML compared to normal controls [22]. In addition, we analyzed the clinical significance of deregulated SFRP1 expression in AML for the first time. Low SFRP1 expression was demonstrated to be associated with higher peripheral WBCs and with the differentiation blockage of granulocytic lineage. Cheng et al. also identified the predominant occurrence of SFRP1 methylation in FAB M0/M1/M2 subtypes and the trend of high WBC counts in patients with SFRP1 methylation [14]. Although the role of individual SFRP genes in normal and malignant hematopoiesis has remained poorly understood, the abovementioned observations may be supported by recent studies on SFRP1 function [14]. Cheng et al. indicated that SFRP1 inhibits leukemic cell proliferation by antagonizing canonical Wnt signaling [14]. Moreover, studies on solid tumors also showed that SFRP1 promotes apoptosis, inhibits proliferation in vitro and tumor growth in vivo, and suppresses invasion and metastasis [23–27]. SFRP1 loss may lead to the block of myeloblast differentiation and the expansion of leukemic cells. Findings obtained from previous studies on SFRP1 methylation in AML have been inconsistent. Although some groups did not find an association of SFRP1 methylation with specific chromosomal abnormalities [20,21], three studies identified that SFRP1 methylation occurs predominantly in patients with t(8;21) [13–15]. This association could be
This study was supported by the National Natural Science Foundation of China (81270630, 81172592), Science and Technology Special Project in Clinical Medicine of Jiangsu Province (BL2012056), 333 Project of Jiangsu Province (BRA2011085, BRA2013136), Science and Technology Infrastructure Program of Zhenjiang (SS2012003), Social Development Foundation of Zhenjiang (SH2013042, SH2013082), Research and Development Foundation of Clinical Medicine of Jiangsu University (JLY20120013, JLY20120011), Key Medical Talent Program of Zhenjiang City and Jiangsu Government Scholarship for Overseas Studies.
Figure 4. Overall survival of patients with AML.
Role of the funding source Research support played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
References [1] Lane SW, Scadden DT, Gilliland DG. The leukemic stem cell niche: current concepts and therapeutic opportunities. Blood 2009;114:1150–1157. [2] Marcucci G, Haferlach T, Döhner H. Molecular genetics of adult acute myeloid leukemia: prognostic and therapeutic implications. J Clin Oncol 2011;29:475–486.
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SFRP1 expression in AML 5 [3] Shih AH, Abdel-Wahab O, Patel JP, et al. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer 2012;12:599–612. [4] Chen J, Odenike O, Rowley JD. Leukaemogenesis: more than mutant genes. Nat Rev Cancer 2010;10:23–36. [5] Foran JM. New prognostic markers in acute myeloid leukemia: perspective from the clinic. Hematology Am Soc Hematol Educ Program 2010:47–55. [6] Swerdlow SH, Campo E, Harris NL, et al, editors. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press ; 2008. [7] Estey EH. Acute myeloid leukemia: 2013 update on risk-stratification and management. Am J Hematol 2013;88:318–327. [8] Barker N, Clevers H. Mining the Wnt pathway for cancer therapeutics. Nat Rev Drug Discov 2006;5:997–1014. [9] Klaus A , Birchmeier W. Wnt signalling and its impact on development and cancer. Nat Rev Cancer 2008;8:387–398. [10] Cruciat CM, Niehrs C. Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb Perspect Biol 2013;5:a015081. [11] Bovolenta P, Esteve P, Ruiz JM, et al. Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease. J Cell Sci 2008;121:737–746. [12] Surana R, Sikka S, Cai W, et al. Secreted frizzled related proteins: Implications in cancers. Biochim Biophys Acta 2014;1845:53–65. [13] Jost E, Schmid J, Wilop S, et al. Epigenetic inactivation of secreted Frizzled-related proteins in acute myeloid leukaemia. Br J Haematol 2008;142:745–753. [14] Cheng CK, Li L, Cheng SH, et al. Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia. Blood 2011;118:6638–6648. [15] Hou HA , Kuo YY, Liu CY, et al. Distinct association between aberrant methylation of Wnt inhibitors and genetic alterations in acute myeloid leukaemia. Br J Cancer 2011;105:1927–1933. [16] Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukaemia. A report of the French-American -British Cooperative Group. Ann Intern Med 1985;103:620–625.
[17] Li Y, Lin J, Yang J, et al. Overexpressed let-7a-3 is associated with poor outcome in acute myeloid leukemia. Leuk Res 2013;37: 1642–1647. [18] Mitelman F, editor. An international system for human cytogenetic nomenclature (1995). ISCN 1995. Basel: Karger ; 1995. [19] Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354–365. [20] Valencia A , Román-Gómez J, Cervera J, et al. Wnt signaling pathway is epigenetically regulated by methylation of Wnt antagonists in acute myeloid leukemia. Leukemia 2009;23:1658–1666. [21] Griffiths EA , Gore SD, Hooker C, et al. Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation. Leuk Lymphoma 2010;51:1711–1719. [22] Reins J, Mossner M, Neumann M, et al. Transcriptional down-regulation of the Wnt antagonist SFRP1 in haematopoietic cells of patients with different risk types of MDS. Leuk Res 2010;34: 1610–1616. [23] Suzuki H, Watkins DN, Jair KW, et al. Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat Genet 2004;36:417–422. [24] Suzuki H, Toyota M, Carraway H, et al. Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer. Br J Cancer 2008;98:1147–1156. [25] Chung MT, Lai HC, Sytwu HK , et al. SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway. Gynecol Oncol 2009;112:646–653. [26] Matsuda Y, Schlange T, Oakeley EJ, et al. WNT signaling enhances breast cancer cell motility and blockade of the WNT pathway by sFRP1 suppresses MDA-MB-231 xenograft growth. Breast Cancer Res 2009;11: R32. [27] Kongkham PN, Northcott PA , Croul SE, et al. The SFRP family of WNT inhibitors function as novel tumor suppressor genes epigenetically silenced in medulloblastoma. Oncogene 2010; 29:3017–3024.
ORIGINAL ARTICLE: CLINICAL
Clinical significance of reduced SFRP1 expression in acute myeloid leukemia Cui An1*, Hong Guo2*, Xiang-mei Wen2, Chun-yan Tang1, Jing Yang1, Xiao-wen Zhu1, Jia-yu Yin1, Qing Liu1, Ji-chun Ma2, Zhao-qun Deng2, Jiang Lin2 & Jun Qian1 1Department of Hematology and 2Laboratory Center, Affiliated People’s Hospital of Jiangsu University, Zhenjiang,
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Jiangsu, People’s Republic of China
the understanding of leukemia pathogenesis will provide more molecules to join the cohort for diagnosis, risk stratification and therapeutic targets. The Wnt signaling system has crucial roles in the control of a number of developmental and cellular processes. Deregulation of Wnt signaling contributes to mammary tumorigenesis by means of its roles in promoting tumor cell proliferation, inhibiting differentiation and blocking apoptosis [8,9]. It is well known that Wnt modulates the canonical (β-catenin mediated) and non-canonical (β-catenin independent) signaling pathways [8,9]. Under physiologic conditions, Wnt signal transduction is regulated extracellularly by multiple secreted and transmembrane antagonists, such as Wnt inhibitory factor (WIF), Dickkopfs (DKKs) and secreted frizzled-related proteins (SFRPs) [10]. SFRPs, the largest family of secreted Wnt antagonists, inhibit Wnt signaling by interacting with and binding to Wnt ligands [10]. However, increasing studies indicate that SFRPs can also activate Wnt signaling and interfere with other signaling systems through Wnt-independent mechanisms [10,11]. A decade of studies has shown that SFRP deregulation contributes to the development of tumors [11,12]. Moreover, aberrant methylation of SFRP promoters has also been identified in clinical cancer samples [11,12]. SFRP1 is the most studied member of the SFRP family in cancers. A few studies in AML have revealed a distinct association between SFRP1 methylation and t(8;21) [13–15]. However, the pattern of SFRP1 expression and its clinical relevance in AML remain poorly defined. This study aimed to detect the status of SFRP1 expression and explore the clinical significance of SFRP1 expression in AML.
Abstract Deregulation of secreted frizzled-related protein 1 (SFRP1) has been found in many types of cancer. However, the pattern of SFRP1 expression in acute myeloid leukemia (AML) is still unclear. This study determined SFRP1 expression in patients with AML. SFRP1 expression was decreased markedly in patients with AML compared to controls (p ⬍ 0.001). White blood cell (WBC) counts increased as SFRP1 expression decreased in AML (p ⫽ 0.016). Patients with low SFRP1 expression showed a different distribution of French–American–British (FAB) subtypes M1/M2/M3 from those with high SFRP1 expression (p ⫽ 0.031). NPM1 mutation was mainly observed in patients with low SFRP1 expression (p ⫽ 0.011). There was a weak trend that patients with AML with low SFRP1 expression had shorter overall survival (OS) than those with high SFRP1 expression (p ⫽ 0.103). Our results indicate that reduced SFRP1 expression is found more frequently in the less well-differentiated subgroups of AML and is associated with NPM1 mutation in AML. Keywords: SFRP1, acute myeloid leukemia, NPM1 mutation
Introduction Acute myeloid leukemia (AML) represents a group of clonal disorders resulting from hematopoietic stem cells losing the capability of normal differentiation and proliferation [1]. The pathologic mechanism of AML can be mainly interpreted by non-random chromosomal translocations and somatic gene mutations, as well as epigenetic alterations occurring in hematopoietic stem/progenitor cells. Numerous studies have given evidence that these alterations provide important diagnostic and prognostic information for AML [2–5]. Chromosomal abnormalities and gene mutations have been successfully introduced into the risk stratification and treatment choice of AML [6,7]. The continuous advancement in
Materials and methods Patients and samples One hundred and eighteen patients with de novo AML were included in this study. The diagnosis and classifica-
*These authors contributed equally as first authors. Correspondence: Jun Qian, MD, Department of Hematology, Affiliated People’s Hospital of Jiangsu University, 8 Dianli Rd., 212002 Zhenjiang, People’s Republic of China. Fax: ⫹ 86-511-85234387. E-mail: [email protected]. Jiang Lin, PhD, Laboratory Center, Affiliated People’s Hospital of Jiangsu University, 8 Dianli Rd., 212002 Zhenjiang, People’s Republic of China. Fax: ⫹ 86-511-85234387. E-mail: [email protected] Received 19 June 2014; revised 8 October 2014; accepted 12 October 2014
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C. An et al.
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tion of these patients were based on French–American– British (FAB) and World Health Organization (WHO) criteria [6,16]. The treatment protocol was described as reported previously [17]. Written informed consent was obtained from all patients. The study was approved by the Institutional Review Board of the Affiliated People’s Hospital of Jiangsu University. The main clinical and laboratory characteristics of these patients are listed in Table I. Twenty-three healthy donors were collected as controls. Seven human leukemic cell lines (HL60, NB4, THP-1, SHI-1, U937, HEL and K562) were also studied. All cell lines were cultured in Iscoves’s modified Dulbecco’s medium (IMDM) containing 10% fetal calf serum and grown at 37°C in a 5% CO2 humidified atmosphere.
Cytogenetic analysis Chromosomes were prepared routinely by the direct method or 24 h short-term culture of bone marrow cells. Karyotypes were analyzed on R-banded metaphases. Chro-
mosome abnormalities were described according to the International System for Human Cytogenetic Nomenclature [18]. Karyotype risk was identified according to cytogenetic abnormalities [19].
RNA isolation and cDNA synthesis Mononuclear cells from bone marrows of patients with AML and healthy donors were separated by Ficoll–Hypaque gradient. Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer ’s instructions and resolved in diethylpyrocarbonate-treated water. Two micrograms of total RNA were reverse transcribed into cDNA in a total reaction volume of 40 μL containing 1 ⫻ buffer, dNTPs 10 mM each, random hexamers 10 μM, RNAsin 80 units and 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase (MBI Fermentas, Hanover, NH). The reverse transcription system was incubated for 10 min at 25°C, 60 min at 42°C, and then stored at ⫺ 20°C.
Table I. Clinical characteristics of patients with AML classified according to SFRP1 expression. Status of SFRP1 expression Patient parameters Sex, male/female Median age, years (range) Median WBC, ⫻ 109/L (range) Median hemoglobin, g/L (range) Median platelets, ⫻ 109/L (range) FAB M1 M2 M3 M4 M5 M6 WHO AML with t(8;21) APL with t(15;17) AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic and monocytic leukemia Acute erythroid leukemia Karyotype classification Favorable Intermediate Poor No data Karyotype Normal t(8;21) t(15;17) Complex Other No data Gene mutation* C/EBPA (⫹/⫺) NPM1 (⫹/⫺) FLT3-ITD (⫹/⫺) DNMT3A (⫹/⫺) IDH1/2 (⫹/⫺) C-KIT (⫹/⫺) N/K-RAS (⫹/⫺) CR (⫹/⫺) SFRP1 transcript
Low (n ⫽ 39)
Medium (n ⫽ 40)
24/15 58 (15–86) 23.5 (0.3–528.0) 68 (40–138) 32 (7–264)
26/14 52.5 (15–87) 12.2 (0.5–130.2) 87 (34–123) 40 (4–399)
High (n ⫽ 39) 16/23 54 (15–87) 5.4 (0.8–135.9) 75 (31–131) 44 (3–140)
Total (n ⫽ 118)
p-Value
66/52 54 (15–87) 10.7 (0.3–528.0) 76 (31–138) 39 (3–399)
0.070 0.564 0.016 0.054 0.764 0.059
5 (13%) 14 (36%) 4 (10%) 14 (36%) 2 (5%) 0 (0%)
2 (5%) 19 (48%) 10 (25%) 8 (20%) 1 (2%) 0 (0%)
2 (5%) 10 (26%) 13 (33%) 8 (20%) 5 (13%) 1 (3%)
9 (8%) 43 (36%) 27 (23%) 30 (25%) 8 (7%) 1 (1%)
2 (6%) 4 (8%) 5 (11%) 12 (30%) 15 (42%) 1 (3%) 0 (0%)
3 (8%) 10 (23%) 2 (5%) 16 (41%) 8 (20%) 1 (3%) 0 (0%)
2 (5%) 13 (34%) 2 (3%) 8 (21%) 8 (21%) 5 (13%) 1 (3%)
7 (6%) 27 (23%) 9 (8%) 36 (30%) 31 (26%) 7 (6%) 1 (1%)
6 (15%) 24 (62%) 3 (8%) 6 (15%)
13 (33%) 22 (55%) 4 (10%) 1 (2%)
15 (38%) 16 (41%) 7 (18%) 1 (3%)
34 (29%) 62 (52%) 14 (12%) 8 (7%)
19 (49%) 2 (5%) 4 (10%) 3 (8%) 5 (13%) 6 (15%)
15 (38%) 3 (7%) 10 (25%) 3 (8%) 8 (20%) 1 (2%)
15 (39%) 2 (5%) 13 (33%) 6 (15%) 2 (5%) 1 (3%)
49 (41%) 7 (6%) 27 (23%) 12 (10%) 15 (13%) 8 (7%)
0.072
0.129
0.229
0/34 (0%) 6/28 (18%) 6/28 (18%) 3/31 (9%) 2/32 (6%) 2/32 (6%) 1/33 (3%) 13/19 (41%) 0 (0–0.004)
2/33 (6%) 2/33 (6%) 5/30 (14%) 2/33 (6%) 3/32 (9%) 1/34 (3%) 4/31 (11%) 19/18 (51%) 0.137 (0.004–0.034)
1/32 (3%) 0/33 (0%) 2/31 (6%) 1/32 (3%) 1/32 (3%) 0/33 (0%) 4/29 (12%) 16/17 (48%) 0.212 (0.035–3.782)
3/99 (3%) 8/94 (8%) 13/89 (13%) 6/96 (6%) 6/96 (6%) 3/99 (3%) 9/93 (9%) 48/54 (47%) 0.014 (0–3.782)
0.653 0.011 0.396 0.779 0.869 0.653 0.350 0.680 ⬍ 0.001
AML, acute myeloid leukemia; WBC, white blood cells; FAB, French–American–British classification; WHO, World Health Organization; CR, complete remission. *Percentage equal to number of mutated patients divided by total cases in each group.
SFRP1 expression in AML 3
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Real-time quantitative polymerase chain reaction Real-time quantitative polymerase chain reaction (RQ-PCR) was carried out using a SYBR Premix Ex Taq™ II kit (Takara, Tokyo, Japan) on an ABI 7300 Thermo cycler (Applied Biosystems, Foster City, CA). Primers for the SFRP1 transcript were 5’-AGATGCTTAAG TGTGACAAGTTCC-3’ (forward) and 5’-TCAGATTTCAACTCGTTGTCACAG-3’ (reverse) with expected PCR products of 130 bp. The PCR reaction system was composed of 20 ng of cDNA, 0.8 μM of each primer, 10 μM of SYBRPremix Ex Taq II and 0.4 μM of ROX Reference Dye1 (Takara). The PCR procedure was as follows: initial predenaturation at 95°C for 30 s, then 45 cycles at 95°C for 5 s, 64°C for 30 s and 72°C for 30 s, and a fluorescence collection step at 85°C for 30 s, followed by a melting program at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s and 60°C for 15 s. Negative and positive controls were included in all experiments. The specificity of RQ-PCR products was verified by melting curves and DNA sequencing. The housekeeping gene ABL was used to calculate the abundance of SFRP1 mRNA. A standard curve was generated by amplifying plasmids cloned with SFRP1 or ABL in a series of dilutions. Relative SFRP1 expression was calculated using the following equation: NSFRP1 ⫽ (ESFRP1)ΔCT SFRP1 (control-sample) ⫼ (EABL)ΔCT ABL (control-sample) The parameter efficiency (E) is derived from the formula E ⫽ 10(⫺ 1/slope) (slope refers to CT versus plasmid concentration plot in the standard curve).
Statistical analysis Statistical analysis was applied using the SPSS 17.0 software package (SPSS, Chicago, IL). χ2 analysis or Fisher’s exact test was conducted to compare differences in categorical variables. The Kruskal–Wallis test or Mann–Whitney U-test was performed to compare differences in continuous variables. Survival was analyzed according to the Kaplan–Meier method.
Results SFRP1 expression in de novo AML and leukemic cell lines The level of SFRP1 expression was analyzed in patients with AML and healthy controls. It was found that SFRP1 was expressed in all controls (0.088–10.10, 2.595 ⫾ 2.580, median 1.750). The SFRP1 transcript was significantly decreased in patients with AML (0.000–3.782, median 0.0137) compared to controls (p ⬍ 0.001, Figure 1). Representative electrophoresis
Figure 1. Relative expression levels of SFRP1 in patients with AML and controls.
Figure 2. Electrophoresis results of RQ-PCR products in patients with AML. 1: Gene Ruler™ 100 bp DNA ladder; 2–3: normal controls; 4–5: AML samples in low expression group; 6–7: AML samples in medium expression group; 8–9: AML samples in high expression group; 10: cloned plasmid; 11: negative control. (A) SFRP1; (B) ABL.
results for RQ-PCR products are shown in Figure 2. Among the seven tested cell lines, only U937 and SHI-1 showed positive SFRP1 expression, and the remaining five cell lines presented loss of expression (Figure 3).
Clinical and laboratory characteristics of AML Patients with AML were divided into three subgroups according to tertile values of the SFRP1 level: low SFRP1 expression (n ⫽ 39, 33%), intermediate SFRP1 expression (n ⫽ 40, 34%) and high SFRP1 expression (n ⫽ 39, 33%). No differences were found in sex, age, or hemoglobin and platelet contents between these three groups (p ⬎ 0.05, Table I). However, white blood cell (WBC) count increased significantly as SFRP1 expression decreased (p ⫽ 0.016, Table I). Excluding FAB-M3 subtype, which is often accompanied by a low WBC level, there was also a significant difference in WBC count among the three subgroups (p ⫽ 0.022). No significant difference was observed in FAB subtype, WHO subtype or karyotype among the three subgroups. Among the FAB subtypes M1/M2/M3 (M1, AML without maturation; M2, AML with maturation; M3, acute promyelocytic leukemia), however, there was a trend of low SFRP1 expression toward the differentiation block of granulocytic lineage (p ⫽ 0.072). The subgroup with low SFRP1 expression had more obvious differentiation block of granulocytic lineage compared with the subgroup with high SFRP1 expression (p ⫽ 0.031). Six NPM1 mutations were observed in the low SFRP1 expression group while there were two in the intermediate SFRP1 expression group and none in the high SFRP1 expression group (p ⫽ 0.011) (Table I). No correlation was found between SFRP1 expression and other gene mutations (Table I).
Impact of SFRP1 expression on prognosis To investigate the prognostic impact of SFRP1 expression in AML, survival analysis was performed among the three subgroups with follow-up data. There was no significant difference in the rate of complete remission (CR) among the three subgroups (p ⫽ 0.680). Although no significant difference was observed in overall survival (OS) (p ⫽ 0.160), there was a weak trend that patients with AML with low SFRP1
Figure 3. Electrophoresis results of RQ-PCR products in leukemic cell lines. 1: Gene Ruler™ 100 bp DNA ladder; 2: U937; 3: K562; 4: SHI-1; 5: THP-1; 6: HL60; 7: HEL; 8: NB4; 9: cloned plasmid; 10: negative control. (A) SFRP1; (B) ABL.
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4
C. An et al.
expression had shorter OS than those with high SFRP1 expression (p ⫽ 0.103) (Figure 4).
attributed to the observation that RUNX1–ETO fusion protein can result in endogenous SFRP1 transcriptional reduction through binding to the SFRP1 promoter [14]. It was interesting that most patients (6/8, 75%) with NPM1 mutations showed low SFRP1 expression in this study, which was contrary to the observation that the majority of NPM1 mutations were identified in patients with an unmethylated SFRP1 promoter [15]. However, two other studies did not find a close association of aberrant methylation of SFRP1 and NPM1 mutations [14,20]. Larger studies are needed to further determine this relationship in AML. In this study, we did not reveal the prognostic influence of SFRP1 down-regulation in AML, in accordance with three studies on SFRP1 methylation reported previously [13,15,21], although the independent prediction of SFRP1 methylation for prolonged event-free and relapse-free survivals was disclosed in patients with childhood non-M3 AML with nonadverse cytogenetics [14]. These discrepant results probably reflect the small number of patients and different patient selection. Large-scale studies with more patients with AML are needed to clarify this point. In summary, our findings indicate that reduced SFRP1 expression is found more frequently in the less welldifferentiated subgroups of AML and is associated with NPM1 mutation in AML.
Discussion
Acknowledgements
A few studies have focused on promoter methylation of SFRPs and other Wnt modulators in AML [13–15,20,21]. However, the expression of SFRPs, particularly its clinical relevance, remains seldom studied in AML to date. In this study, we confirmed that the SFRP mRNA level was significantly reduced in AML compared to normal controls [22]. In addition, we analyzed the clinical significance of deregulated SFRP1 expression in AML for the first time. Low SFRP1 expression was demonstrated to be associated with higher peripheral WBCs and with the differentiation blockage of granulocytic lineage. Cheng et al. also identified the predominant occurrence of SFRP1 methylation in FAB M0/M1/M2 subtypes and the trend of high WBC counts in patients with SFRP1 methylation [14]. Although the role of individual SFRP genes in normal and malignant hematopoiesis has remained poorly understood, the abovementioned observations may be supported by recent studies on SFRP1 function [14]. Cheng et al. indicated that SFRP1 inhibits leukemic cell proliferation by antagonizing canonical Wnt signaling [14]. Moreover, studies on solid tumors also showed that SFRP1 promotes apoptosis, inhibits proliferation in vitro and tumor growth in vivo, and suppresses invasion and metastasis [23–27]. SFRP1 loss may lead to the block of myeloblast differentiation and the expansion of leukemic cells. Findings obtained from previous studies on SFRP1 methylation in AML have been inconsistent. Although some groups did not find an association of SFRP1 methylation with specific chromosomal abnormalities [20,21], three studies identified that SFRP1 methylation occurs predominantly in patients with t(8;21) [13–15]. This association could be
This study was supported by the National Natural Science Foundation of China (81270630, 81172592), Science and Technology Special Project in Clinical Medicine of Jiangsu Province (BL2012056), 333 Project of Jiangsu Province (BRA2011085, BRA2013136), Science and Technology Infrastructure Program of Zhenjiang (SS2012003), Social Development Foundation of Zhenjiang (SH2013042, SH2013082), Research and Development Foundation of Clinical Medicine of Jiangsu University (JLY20120013, JLY20120011), Key Medical Talent Program of Zhenjiang City and Jiangsu Government Scholarship for Overseas Studies.
Figure 4. Overall survival of patients with AML.
Role of the funding source Research support played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
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