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The prognostic significance of the long non-coding RNAs “CCAT1, PVT1” in t(8;21) associated Acute Myeloid Leukemia
Gene 707 (2019) 172–177
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
The prognostic significance of the long non-coding RNAs “CCAT1, PVT1” in t(8;21) associated Acute Myeloid Leukemia
T
Nashwa El-Khazragya, , Wael Elayatb, Safa Matboulyc, Sarah Selimand, Ashraqat Samid, Gehan Safwatd, Ayman Diabd ⁎
a
Clinical Pathology and Hematology Department, Faculty of Medicine, Ain Shams University Biomedical Research Department, Cairo, P.O. Box 11381, Egypt Department of Medical Biochemistry, Faculty of Medicine, Ain Shams University, Egypt c Department of Pediatrics, Faculty of Medicine, Ain Shams University, Egypt d Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA), Cairo, Egypt b
ARTICLE INFO
ABSTRACT
Keywords: Lnc-CCAT1 Lnc-PVT1 Prognostic factor t(8;21) Acute Myeloid Leukemia (AML)
Long non-coding RNA (LncRNA) is recently linked to various types of cancers, CCAT and PVT1 are two LncRNAs linked to t(8;21) associated Acute Myeloid Leukemia, the interplay between CCAT, PVT1 and the MYC protooncogene implicated in t(8;21) could present an opportunity for using LncRNA as prognostic biomarker or a target for therapy, We investigated the expression levels of LncRNAs in 70 patients; 30 with t(8;21) positive AML and 40 with t(8;21) negative AML, We found that CCAT1 and PVT1 are expressed in higher levels in t(8;21) positive –AML by 5.3 folds compared to t(8;21) negative group; the expression values were significantly associated with high-risk clinical criteria; moreover, they are associated with lower overall survival (OS) rate and leukemia-free survival (LFS), however we didn't find a statistically significant cut-off value of LncRNAs using the Cox regression analysis for Lnc_PVT1 except with LFS, we conclude that high expression levels of CCAT1 and PVT1 are associated with poor prognosis while being poor prognostic biomarkers in t(8;21) associated AML.
1. Introduction Acute Myeloid Leukemia (AML) accounts for 80% of acute leukemia in adults and 15% in children (Siegel et al., 2017; Gamis et al., 2013). The balanced translocation t(8;21) is one of the cytogenic abnormalities associated with AML; seen in 7% of adult AML patients and is the most common cause of AML in children (Grimwade et al., 2010; Nucifora and Rowley, 1995). t(8;21) in AML has a favorable prognosis in adults, while children show poor response to treatment (Klein et al., 2015). Chemotherapy alone for AML achieves complete remission in up to 60% of patients (Wheatley et al., 1999). Resistance to chemotherapy is associated with unfavorable cytogenic features; The RT-PCR quantification of RUNX1-RUNX1T1 transcripts - the product of the t(8;21) - were linked to high risk of relapse (Tobal et al., 2000). The MYC-MAX transcriptional complex regulates transcription of various genes across different cell types. Overexpression of this complex leads to many types of cancer. AML has been linked to abnormal expression of the MYC proto-oncogene, various mechanisms for this association includes the up-regulation of MYC gene by the AML-
associated fusion protein RUNX1-RUNX1T1 (Müller-Tidow et al., 2004), the mutation of the CCAAT/enhancer binding protein alpha (C/ EBPα) transcription factor which normally down-regulate the MYC gene (Johansen et al., 2001), and the amplification of the 8q24 region where the MYC gene is located (Slovak et al., 1994). Most of the human genome is non-coding sequences which were considered to be of no use, but recently this view is changing as some non-coding sequences were found to have a regulatory role on gene transcription and post-transcriptional process. Long noncoding RNA (LncRNA) is a 200 nucleotide molecule which doesn't code for a specific protein product (Hamilton et al., 2015), it is the focus of many recent studies investigating the role of t (8;21) in AML (Pan et al., 2017; Quek et al., 2015). Colon cancer associated transcript (CCAT) is a LncRNA present together with the MYC in the 8q24 region. CCAT1 acts as a super-enhancer to the enhancer region present 335 KB upstream from the MYC gene and have been implicated in gastric, Hepatocellular carcinoma (Yang et al., 2015) and cancer colon (Fan et al., 2018). Although CCAT1 has been involved in different solid cancers, limited knowledge is
Abbreviations: AML, Acute Myeloid Leukemia; LncRNA, Long noncoding RNA; RUNX1, Runt-Related transcription factor 1; CCAT1, colon cancer associated transcript 1; PVT1, plasmacytoma variant translocation 1; SWI/SNF, Switch/Sucrose non-fermentable ⁎ Corresponding author. E-mail address: [email protected] (N. El-Khazragy). https://doi.org/10.1016/j.gene.2019.03.055 Received 18 November 2018; Received in revised form 13 March 2019; Accepted 25 March 2019 Available online 31 March 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Shows the interplay between CCAT1, PVT1, and MYC in AML could produce an abnormally overexpressed MYC protein involved in the pathogenesis of AML.
competing endogenous RNA (ceRNA) for miR-155 which is a direct target for c-myc (Hamilton et al., 2015). PVT-1 is another LncRNA present in the 8q24 region required for MYC gene expression (Hamilton et al., 2015). It was evident that aberrant overexpression of PVT1 has been discovered in many cancers such as cancer ovary and hepatocellular carcinoma (Yang et al., 2015; Saus et al., 2016), moreover, it has been demonstrated that PVT1 has a major role in c-Myc protein expression (Hamilton et al., 2015). The interplay between CCAT1, PVT1, and MYC in AML could produce an abnormally overexpressed MYC protein involved in the pathogenesis of AML as shown in (Fig. 1), this presents an opportunity to use the CCAT1 or the PVT1 as a potential target for therapy or as a prognostic marker of AML.
Table 1 Descriptive analysis of the studied subjects. Demographic data
Age (years) Gender Age subgroups TLC (×106/L) Hemoglobin (grm/ dl) Platelet count (×1012/L) BM blasts (%) MRD at day 15 Clinical response Status Overall survival (OS) Leukemia free survival (LFS)
Mean ± SD Range Male Female Favorable (≤40) Unfavorable (> 40) ≤50 > 50 >6 ≤6 ≥30 < 30 ≤25% > 25% ≤0.01 > 0.01 Remission Relapse Died alive Median (IQR) Range Median (IQR) Range
AML-t(8;21) positive N = 30
AML-negative for t(8;21) N = 40
50.0 ± 14.0 28–73 19 (37%) 11 (58%) 12 (37%) 18 (51%)
49.4 ± 14.5 22–77 32 (63%) 8 (42%) 32 (63%) 27 (49%)
19 (35%) 11 (73%) 18 (37%) 12 (86%) 11 (73%) 19 (35%) 15 (29%) 15 (83%) 13 (27%) 17 (81%) 10 (22%) 5 (15%) 15 (65%) 15 (32%) 15 (11) 2–24 8 (10) 0.8–22
38 (66%) 4 (27%) 38 (68%) 2 (14%) 4 (27%) 36 (65%) 37 (71%) 3 (17%) 36 (73%) 4 (19%) 11 (24%) 36 (78%) 8 (35%) 32 (68%) 20 (4) 5–24 16 (1.8) 0.4–7.7
2. Results 2.1. Demographic and clinical features of studied subjects The Mean age of Subjects participating in this study was 50.0 ± 14.0 for the t(8;21) positive group and 49.4 ± 14.5 for the t (8;21) negative group, 37% of the male participants were t(8;21) positive, 58% of female participants were t(8;21) positive. Considering the favorable age group to be (≤40), only 37% of the favorable age group were t(8;21) positive, while in the unfavorable age group (> 40) t (8;21) positive and t(8;21) negative expression was nearly equal. High TLC (> 50 × 106/L), Low Hemoglobin level (≤6 g/dl) and High Blasts count (> 25%) were observed in the t(8;21) positive group, while low platelet counts (< 30 × 1012/L) were observed in the t(8;21) negative group (65%) (Table 1).
BM: bone marrow; MRD: minimal residual disease.
known about its role in AML pathogenesis as well as its prognostic value. Moreover, up to date, the real mechanism by which CCAT1 exerts its oncogenic effect remains unknown. Recently, in a study conducted by Chen et al. (2016), he demonstrated higher expression levels of CCAT1 in M4 and M5 AML subtypes compared to normal control. Furtherly, in vitro studies demonstrated that myeloid proliferation differentiation and is inhibited by high expression of CCAT1 through a
2.2. High expression of CCAT1 and PVT1 in t(8;21) positive AML Expression levels of the Lnc-CCAT1, Inc-PVT1 and MYC were significantly higher among the AML group compared to the control group (P value ≤ 0.01) and significantly higher among the t(8;21) positive 173
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Table 2 Comparative analysis for CCAT1, PVT1 and c-Myc between different studied subgroups. Variable
Median (IQR) Range
Lnc-CCAT1(log10) Lnc-PVT1(log10) c-Myc (log10)
Statistics: Ⱬ Mann Whitney test value((p value)
AML n = 70
t(8;21) positive-AML n = 30
t(8;21) negative-AML n = 40
Control n = 20
AML/control
t(8;21) positive-AML/ control
t(8;21) negative AML/ control
t(8;21) positive/ negative AML
3.1 (4.1) 0.4–12.0 20.6 (31) 2.2–99 13.4(7.5) 6.8–22.0
5.5 (2.3) 0.9–12.0 40.2 (31) 5.1–99.0 50.0 (38) 21.7–88
1.6 (1.8) 0.4–7.7 11.4 (14.6) 2.2–67 30.4 (13) 8.6–61.0
0.8 (2.8) 0.1–5.7 2.8 (3.2) 1.0–11.0 13.4 (7.5) 6.8–22.0
4.2 (≤0.01)⁎
4.8 (≤0.01)⁎
3.0 (≤0.01)⁎
5.3 (≤0.01)⁎
6.0 (≤0.01)⁎
5.7 (≤0.01)⁎
5.2 (≤0.01)⁎
4.6 (≤0.01)⁎
6.4 (≤0.01)⁎
5.9 (≤0.01)⁎
5.6 (≤0.01)⁎
4.3 (≤0.01)⁎
⁎ Test is significant at level ≤ 0.01, IQR: Interquartile ratio; AML: Acute Myeloid Leukemia; t(8:21): translocation between chromosome 8 and 21; n:number of subjects; Lnc: long non coding RNA.
statistical significance. On investigating the difference in the individual expression levels between the t(8;21) positive and negative groups, the Lnc-CCAT1 and myc expression levels were correlated to low OS and LFS in both groups with statistical significance, the Lnc-PVT1 expression levels were correlated to low OS and LFS in both groups without statistical significance (Supplementary Table 2 & Fig. 3).
60 50 40 30
2.5. CCAT1, PVT1 and c-Myc as independent prognostic factors in t(8;21)AML
20 10
Univariate analysis of Cox proportional hazards modeling of potential prognostic factors for OS and LFS in the study population revealed that gene expression levels exceeding the assigned cut-off value for CCAT1, PVT1 and c-Myc were associated with an adverse prognosis and shorter overall survival (p < 0.05). Unless PVT1; the same findings were obtained for LFS; higher expression values are significantly associated with shorter duration of LFS (p < 0.05) (Table 4).
0 Lnc-CCAT1 (log10)
t(8;21)-positive AML
Lnc-PVT1 (log10)
c-Myc (log10)
t(8;21)-negative AML
Healthy control
Fig. 2. Expression level of CCAT1, PVT1, and c-Myc in t(8;21)-AML positive, negative patients and healthy control.
group compared to t(8;21) negative group (P value ≤ 0.01) (Table 2) (Fig. 2).
3. Subjects & methods 3.1. Study cohort
2.3. Higher expression levels of-of CCAT1 and PVT1 is positively correlated with up regulation of c-Myc and associated with high-risk factors in t(8;21) positive AML
The present study enrolled seventy patients with de-novo diagnosed AML, attended at Clinical Hematology Department, Ain Shams Internal Medicine Hospital, Cairo, Egypt between March 2015 and August 2017, twenty patients not suffering from any hematologic or other type of malignancy consisting our control cohort. Patients' clinic pathological characteristics are presented in (Table 1). All patients provided their written informed consent in accordance with the Declaration of Helsinki. Peripheral blood (PB) samples were obtained, with a minimum blast infiltration of 25%. The sample was collected before the receiving of chemotherapy induction protocol; All sample handling, procedure and storage was the same for leukemic and control subjects. Patients follow up and risk groups stratification of AML was done according to the Berlin-Frankfurt-Munster (BFM) guidelines; it includes; assessment of BM blasts on day 8, BM blasts % on day 15 and minimal residual disease (MRD) measurement at day 28 of induction (Zeng et al., 2015). The diagnosis of t(8;21) associated AML was made according to a morphologic assessment of the Leishman stained smears of the bone marrow aspirates along with special immune-cytochemical stains, immune-phenotyping, and Cytogenetic analysis. Assessment of minimal residual disease (MRD) was performed using a lineage-specific monoclonal panel; MRD was considered positive when leukemic cells exceeded 0.01% of all marrow nucleated cells on days 15 and 28 (Table 3).
We found that high expression levels of the Lnc-CCAT1, Inc-PVT1, and MYC among the t(8;21) positive group were correlated to low hemoglobin level (≤6 g/dl) and low platelet count (≤30 × 1012/L). Also, the expression levels of the Lnc-CCAT1 and MYC were higher in patients with BM blast cells (> 70%) and minimal residual disease (MRD) (> 0.01) with statistical significance (Supplementary Table 1). 2.4. Association of CCAT1 and PVT1 with t(8;21)-AML patients overall survival (OS) and progression-free survival (PFS) In order to assess the influence of higher gene expression levels on t (8;21) associated AML disease's outcome; the median expression value for each tested biomarker (CCAT1, PVT1 and c-myc) was selected as a cut-off value of (5.5, 40 and 50); respectively. Furtherly, the Kaplan Meir survival analysis was performed to find out the association between expression's level of biomarker and patient's overall (OS) and leukemia free survival (LFS). Generally, gene expression with values higher than the cut off value was significantly associated with shorter OS for CCAT1 and c-Myc (p < 0.01) compared to those with expression values beyond the cut off value. However, PVT1 expression was insignificantly associated with OS (p = 0.7). On the other hand; patients showed higher expression levels of Lnc-CCAT1, Inc-PVT1, and c-Myc have shorter time to relapse than those with mild increase, therefore, correlate with leukemia-free survival (LFS) in t(8;21)AML patients with
3.2. Bioinformatics analysis In the present study, the association of lncRNA–CCAT1, PVT1 and 174
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Fig. 3. Kaplan-Meier survival curves of patients with high and low Lnc-CCAT1, Lnc_PVT1 and c-myc expression in t(8; 21) associated AML. Patients were dichotomized into high and low genes expression groups based on median expression value. Patients with expression levels for “Lnc_CCAT1 and c-myc” less than the optimum cut-off values had significantly better survival (a, e) and leukemia free survival (b. f): respectively than those with higher expression; Meanwhile, the LncPVT1 expression was significantly correlated with LFS (d), no significant association was reached with OS (c) (p > 0.05).
mRNA c-Myc with t(8;21) associated AML pathogenesis was investigated using the lncRNA disease database (http://www.cuilab.cn/ lncrnadisease) and [37] (http://www.cuilab.cn/files/images/ldd/ rnaseq/HOTAIR.txt). The interaction between lncRNAs “CCAT1 and PVT1” was predicted from the crosslinking immunoprecipitation RNA sequencing (CLIP-Seq) data using the star Base platform (http:// starbase.sysu.edu.cn/) (Quek et al., 2015). Finally, the interlink pathway in t(8;21)-AML related lncRNAs “CCAT1, PVT1” and c-Myc are further confirmed using PubMed (http://www.ncbi.nlm.nih.gov/ pubmed/), (http://oncodb.hcc.ibms.sinica.edu.tw/index.htm. On the basis of the genomic information that was collected, the regulatory network of the lncRNA-CCAT1, PVT1 and mRNA-cMyc in AML was constructed by Cytoscape software version 3.5.0 (http://www. cytoscape.org/index.html).
samples in EDTA bone marrow cells from newly diagnosed patients. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, Total RNA concentration and purity were evaluated spectrophotometric ally at 260 and 280 nm. RNA integrity was visually confirmed by agarose gel electrophoresis. cDNA was synthesized by reverse transcription reaction using QuantiTect RT Kit (Qiagen, Hilden, Germany). 3.4. Long non-coding RNAs “CCAT1, PVT1” and c-Myc expression analysis For detection of genes expression; cDNA was amplified using a Hs_CCAT1 RT2 Primer Assay cat no: 330701, assay ID: LPH15969A, Hs_PVT1 RT2 Primer Assay cat no: 330701, assay ID: LPH17013A, Hs_cMyc_SG QuantiTect primer assay cat no: 249900, assay ID: QT01663361 and Hs_ACTB_1_SG QuantiTect Primer assay (NM_001101) used as reference gene. Each sample was amplified by both primers using QuantiTect Syber green Master Mix (Qiagen, Hilden, Germany). The 25 μl reaction mixture/reaction consist of 2× QuantiTect Syber green PCR mastermix, 0.3 μM primer assay and
3.3. Total RNA extraction and purification Total mRNA was extracted from mononuclear cells (MNCs) that it is isolated by Ficoll Hypaque density gradient centrifugation from 2 ml PB
Table 3 Impact of high/low expression levels of CCAT1.PVT1, c-Myc on overall and progression free survival in t(8;21) positive AML. Variable
Overall survival (months) Cut-off
10
Lnc-CCAT1 (log ) Lnc-PVT1 (log10) c-Myc (log10)
≤5.5 > 5.5 ≤40 > 40 ≤50 > 50
Estimate OS
22 9.8 16 14 21 10
Leukemia free survival (months)
95% Confidence Interval
Log Rank (Mantel-Cox) 2
Lower
Upper
Χ
19 6.4 12 10 18 7
24 13 21 17 24 12
14
0.001
0.2
0.7
11.9
0.001
Estimate OS
P value
175
13 5 17 7 16 5
95% Confidence Interval
Log Rank (Mantel-Cox)
Lower
Upper
Χ2
P value
12 3 11 5 13 3
20 6.8 22 9 21 7.2
20
0.001
9.5
0.002
12
0.001
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Table 4 Univariate Cox proportional hazards analysis of survival analysis for t(8;21) positive AML. Variable
Lnc-CCAT1 ≤5.5 vs > 5.5 Lnc-PVT1 ≤40 vs > 40 Lnc-c-Myc ≤50 vs > 50 Overall model coefficients
Cox Regression analysis Overall survival (months)
Cox Regression analysis Leukemia free survival (months)
HR
95% CI
P value
HR
95% CI
P value
0.2
0.4–0.6
0.009
0.1
0.03–0.5
0.002
4.7
1.3–16.5
0.02
0.8
0.2–3.2
0.8
0.09
0.02–0.5
0.004
0.2
0.05–0.8
0.02
0.001
2
2
Χ :25.4; df:3
Χ :27.2; df:3
0.001
HR: Hazards ratio, CI: confidence interval, Χ2: Chi-square.
500 ng cDNA. Both targets were amplified in duplicates for each sample. The thermal protocol consists 15 min for HotStar-Taq DNA Polymerase activation at 95 °C followed by 45 cycles of denaturation at 94 °C for 15 min, primer annealing for 30 s at 52 °C and extension at 72 °C for 30 s). The fluorescence data were collected at extension step. Following amplification, gene normalization was calculated using ΔCt equation; ΔCt = mean value Ct (reference) − mean value Ct (interesting gene). Finally, the gene expression levels were calculated by 2−∆∆Ct calculation using Rotor-Gene software version 1.2.3 (Qiagen).
especially with the t(8;21)- and MYC proto-oncogene is a very interesting link to study as a therapeutic target or even prognostic marker. Many studies have studied the link between these LncRNAs and different types of leukemia; Zeng et al. found significantly upregulated PVT1 in Acute Promyelocytic leukemia patients and showed that knocking either PVT1 or MYC reduced the expression of the other (Zeng et al., 2015), while Hu et al. found that PVT1 was highly expressed in Acute Lymphoblastic leukemia patients (Hu et al., 2018). In contrary, the expression levels of CCAT1 and PVT1 didn't differ between AML of different phenotypes and healthy controls. Meanwhile; when high risk AML-M3 patients, higher PVT1 expression level was detected and it was significantly associated with poor prognosis (Zeng et al., 2015; Izadifard et al., 2018). On the other hand, higher expression levels of CCAT1 were detected in AML-M4 and M5. However, to our knowledge, CCAT1 and PVT1 deregulation have not be investigated in t(8;21)-AML on clinical samples. These findings support the phenomena that the dysregulation of CCAT1 and PVT1 transcripts may be useful to understand the leukemogenic process and furtherly could serve as targeted therapy (Izadifard et al., 2018). CCAT1 and PVT 1 binds to MYC preventing its phosphorylation and degradation (Hamilton et al., 2015), this creates an up-regulated MYC that promotes leukemic cell proliferation and block myeloid differentiation. Saus et al. found that CCAT-1 and PVT-1 behave as oncogenes in colorectal cancer. CCAT-1 induces tumor progression and metastasis which is mediated by c-Myc binding to the CCAT1 promoter region. While PVT-1 induces tumor proliferation, invasion, and an antiapoptotic effect via modulation of chromatin remodeling complex SWI/ SNF in CRC (Saus et al., 2016). Over-expression of PVT1 has been suggested as a powerful predictor of tumor progression and patient survival in a diverse range of cancer types, such as pancreatic cancer, gastric cancer, hepatocellular cancer. Also, the CCAT-1 has been shown to be upregulated in these cancers and also in lung and breast cancer (Huang et al., 2015). It was demonstrated by Saus et al. that CCAT1 was significantly correlated with histopathological grade, lymph nodes metastasis, and survival time after surgery. In vivo studies highlighted the expected pathways; they suggested that CCAT1 transcription could be promoted by c-Myc; this fact explained how CCAT1 overexpression promotes cell proliferation and invasion in colon cancer cells (Fan et al., 2018). Furthermore; in gastric carcinoma tissues, CCAT1 has a pathogenic role in gastric carcinoma; therefore, it has been implicated in the treatment of gastric carcinoma (Saus et al., 2016). Unfortunately, up to date, the role of lncRNA CCAT1 expression progression of breast cancer is not fully understood (Fatima et al., 2015). Many recent studies have investigated the potential of LncRNA to be used as a therapeutic target (Fatima et al., 2015; Boon et al., 2016), knocking down the up-regulated CCAT1 and PVT1 in AML could theoretically decrease MYC over-expression and improve the survival outcome.
3.5. Statistical analysis Statistical analysis was performed using SPSS v.22 (Chicago, IL, USA).The non-parametric Mann–Whitney U test and Wilcoxon Signed Rank test were performed to evaluate the differences of CCAT1, PVT1 and c-Myc expression between leukemic vs healthy specimen, and between t(8;21) positive AML vs t(8;21) negative, respectively. Moreover, to assess the correlation of CCAT1, PVT1 and c-Myc expression with patients' clinic-pathological features. Spearman's correlation was used to assess the expression levels of lnc_CCAT1, lnc_PVT1 and c_Myc with patient outcome. Overall survival (OS) was calculated from diagnosis to death or last follow-up and Leukemic-free survival (LFS) from complete remission (CR) to relapse or death. Both OS and LFS were estimated with the Kaplan–Meier method and comparisons among subgroups of patients with high and low values of genes expressions at diagnosis were performed using the log-rank test. Significance was set at ≤0.05. 4. Discussion We found the Long noncoding RNAs CCAT1 and PVT1 to be overexpressed and associated with lower overall survival rate and leukemiafree survival in Acute Myeloid Leukemia patients with the translocation (8; 21); a high expression of CCAT1 and PVT1 to be linked to high-risk t (8;21) leukemia, high blasts count, low platelets and low hemoglobin were significantly higher in t(8;21) positive patients. This suggests a role of CCAT1 and PVT1 in the progression of the disease, moreover; high expression levels were linked to high minimal residual disease in the t(8;21) positive patients, suggesting that chemotherapy resistant AML may be induced by the high expression of CCAT1 and PVT1, our results were in agreement with some studies that reported PVT1 association with multidrug resistance in certain cancer cases (Pan et al., 2017; Yang et al., 2015), most anticancer drugs exert their effect by inducing apoptosis, hence inhibiting apoptosis by CCAT1 or The PVT1 is one of the main mechanisms of multidrug resistance (Fan et al., 2018). LncRNAs were thought previously to be translational jargon. This view is changing recently with them emerging as a contributor to the regulation of many types of genes. The interplay between the CCAT1 and PVT1 – which we found to be highly expressed in AML patients 176
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5. Conclusion
expression and its roles in acute lymphoblastic leukemia. Epigenomics 10 (6), 723–732. (Internet). (cited 2018 Jul 31). Available from: http://www.ncbi.nlm.nih. gov/pubmed/29693417. Huang, C., Yu, W., Wang, Q., Cui, H., Wang, Y., Zhang, L., et al., 2015 Jun. Increased expression of the lncRNA PVT1 is associated with poor prognosis in pancreatic cancer patients. Minerva Med. 106 (3), 143–149. (Internet). (cited 2018 Jul 21). Available from: http://www.ncbi.nlm.nih.gov/pubmed/25668599. Izadifard, M., Pashaiefar, H., Yaghmaie, M., Montazeri, M., Sadraie, M., Momeny, M., Jalili, M., Ahmadvand, M., Ghaffari, S.H., Mohammadi, S., Alimoghaddam, K., Ghavamzadeh, A., 2018. Expression analysis of PVT1, CCDC26, and CCAT1 long noncoding RNAs in acute myeloid leukemia patients. Genet. Test. Mol. Biomarkers 22 (10), 593–598. https://doi.org/10.1089/gtmb.2018.0143. 2018 Oct. Epub 2018 Sep 14. https://www.ncbi.nlm.nih.gov/pubmed/30222365. Johansen, L.M., Iwama, A., Lodie, T.A., Sasaki, K., Felsher, D.W., Golub, T.R., et al., 2001 Jun 1. c-Myc is a critical target for C/EBP in granulopoiesis. Mol. Cell. Biol. 21 (11), 3789–3806. Internet. (cited 2018 Jun 27). Available from. http://www.ncbi.nlm. nih.gov/pubmed/11340171. Klein, K., Kaspers, G., Harrison, C.J., Beverloo, H.B., Reedijk, A., Bongers, M., et al., 2015 Dec 20. Clinical impact of additional cytogenetic aberrations, cKIT and RAS mutations, and treatment elements in pediatric t(8;21)-AML: results from an international retrospective study by the international Berlin-Frankfurt-Münster study group. J. Clin. Oncol. 33 (36), 4247–4258. (Internet). (cited 2018 Jun 27). Available from: http://ascopubs.org/doi/10.1200/JCO.2015.61.1947. Müller-Tidow, C., Steffen, B., Cauvet, T., Tickenbrock, L., Ji, P., Diederichs, S., et al., 2004 Apr. Translocation products in acute myeloid leukemia activate the Wnt signaling pathway in hematopoietic cells. Mol. Cell. Biol. 24 (7), 2890–2904. Internet. (cited 2018 Jun 27). Available from. http://www.ncbi.nlm.nih.gov/pubmed/15024077. Nucifora, G., Rowley, J.D., 1995 Jul 1. AML1 and the 8;21 and 3;21 translocations in acute and chronic myeloid leukemia. Blood 86 (1), 1–14. (Internet). (cited 2018 Jun 27). Available from: http://www.ncbi.nlm.nih.gov/pubmed/7795214. Pan, J.-Q., Zhang, Y.-Q., Wang, J.-H., Xu, P., Wang, W., 2017. lncRNA co-expression network model for the prognostic analysis of acute myeloid leukemia. Int. J. Mol. Med (Internet). Available from. http://www.spandidos-publications.com/10.3892/ ijmm.2017.2888. Quek, X.C., et al., 2015. lncRNAdb v2.0: expanding the reference database for functional long noncoding RNAs. Nucleic Acids Res. 43 (D1), D168–D173. Saus, E., Brunet-Vega, A., Iraola-Guzmán, S., Pegueroles, C., Gabaldón, T., Pericay, C., 2016. Long non-coding RNAs as potential novel prognostic biomarkers in colorectal cancer. Front. Genet. 7, 54. (Internet). (cited 2018 Jul 21). Available from. http:// www.ncbi.nlm.nih.gov/pubmed/27148353. Siegel, R.L., Miller, K.D., Jemal, A., 2017. Cancer statistics. CA Cancer J. Clin. 67 (1), 7–30. (Internet). 2017 Jan (cited 2018 Jun 27). Available from: https://doi.org/10. 3322/caac.21387. Slovak, M.L., Ho, J.P., Pettenati, M.J., Khan, A., Douer, D., Lal, S., et al., 1994 Jan. Localization of amplified MYC gene sequences to double minute chromosomes in acute myelogenous leukemia. Genes Chromosomes Cancer 9 (1), 62–67. (Internet). (cited 2018 Jun 27). Available from: http://www.ncbi.nlm.nih.gov/pubmed/ 7507702. Tobal, K., Newton, J., Machida, M., Chang, J., Morgenstern, G., Evans, P.A., et al., 2000 Feb 1. Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood 95 (3), 815–819. Internet. (cited 2018 Jun 27). Available from. http://www. ncbi.nlm.nih.gov/pubmed/10648391. Wheatley K, Burnett AK, Goldstone AH, Gray RG, Hann IM, Harrison CJ, et al. A simple, robust, validated and highly predictive index for the determination of risk-directed therapy in acute myeloid leukemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council's Adult and Childhood Leukaemia Working Parties. Br. J. Haematol. (Internet). 1999 Oct (cited 2018 Jun 27);107(1):69–79. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10520026 Yang, X., Xie, X., Xiao, Y.-F., Xie, R., Hu, C.-J., Tang, B., et al., 2015 May 1. The emergence of long non-coding RNAs in the tumorigenesis of hepatocellular carcinoma. Cancer Lett. 360 (2), 119–124. (Internet). (cited 2018 Jul 31). Available from: http://www.ncbi.nlm.nih.gov/pubmed/25721084. Zeng, C., Yu, X., Lai, J., Yang, L., Chen, S., Li, Y., 2015 Nov 6. Overexpression of the long non-coding RNA PVT1 is correlated with leukemic cell proliferation in acute promyelocytic leukemia. J. Hematol. Oncol. 8, 126. (Internet). (cited 2018 Jul 31). Available from: http://www.ncbi.nlm.nih.gov/pubmed/26545364.
Over-expression of CCAT1 and PVT1 is correlated with poor prognosis in t(8;21)-AML; But, Both markers are poor prognostic biomarkers. New trials are needed to assess the potential of both as therapeutic targets. Acknowledgements We thank our colleagues from [Clinical Haematology Department, Ain Shams University Hospital] who provided insight and expertise that greatly assisted the research, although they may not involve with all of the interpretations/conclusions of this paper. We would also like to show our gratitude to the patients who are the backbone of this study for their cooperation and sharing their pearls of wisdom with us during the course of this research, and we thank 3 “anonymous” reviewers for their so-called insights. We are also immensely grateful for their comments on an earlier version of the manuscript, although any errors are our own and should not tarnish the reputations of these esteemed persons. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.03.055. References Boon, R.A., Jaé, N., Holdt, L., Dimmeler, S., 2016 Mar 15. Long noncoding RNAs: from clinical genetics to therapeutic targets? J. Am. Coll. Cardiol. 67 (10), 1214–1226. (Internet). (cited 2018 Jul 21). Available from: https://www.sciencedirect.com/ science/article/pii/S0735109716002242. Chen, Lianxiang, Wang, Wei, Cao, Lixia, Li, Zhijun, Wang, Xing, 2016. Long non-coding CCAT1 acts as a competing endogenous RNA to regulate cell growth and differentiation in acute myeloid leukemia. Mol. Cell 39 (4), 330–336. http://dx.doi.org/10. 14348/molcells.2016.2308. Fan, H., Zhu, J.-H., Yao, X.-Q., 2018 Jun. Knockdown of long non-coding RNA PVT1 reverses multidrug resistance in colorectal cancer cells. Mol. Med. Rep. 17 (6), 8309–8315. (Internet). (cited 2018 Jul 31). Available from: http://www.ncbi.nlm. nih.gov/pubmed/29693171. Fatima, R., Akhade, V.S., Pal, D., Rao, S.M., 2015 Dec 12. Long noncoding RNAs in development and cancer: potential biomarkers and therapeutic targets. Mol. Cell. Ther. 3 (1), 5. (Internet). (cited 2018 Jul 21). Available from: http://molcelltherapies. com/article/view/30. Gamis, A.S., Alonzo, T.A., Perentesis, J.P., Meshinchi, S., 2013 Jun. Children's Oncology Group's 2013 blueprint for research: acute myeloid leukemia. Pediatr. Blood Cancer 60 (6), 964–971. (Internet). (cited 2018 Jun 27). Available from: http://doi.wiley. com/10.1002/pbc.24432. Grimwade, D., Hills, R.K., Moorman, A.V., Walker, H., Chatters, S., Goldstone, A.H., et al., 2010 Jul 22. 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 116 (3), 354–365. (Internet). (cited 2018 Jun 27). Available from: http://www.bloodjournal.org/cgi/doi/10.1182/blood-2009-11254441. Hamilton, M.J., Young, M.D., Sauer, S., Martinez, E., 2015. The interplay of long noncoding RNAs and MYC in cancer. AIMS Biophys. 2 (4), 794–809. (Internet). Available from: http://www.aimspress.com/article/10.3934/biophy.2015.4.794. Hu, J., Han, Q., Gu, Y., Ma, J., McGrath, M., Qiao, F., et al., 2018 Jun. Circular RNA PVT1
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Research paper
The prognostic significance of the long non-coding RNAs “CCAT1, PVT1” in t(8;21) associated Acute Myeloid Leukemia
T
Nashwa El-Khazragya, , Wael Elayatb, Safa Matboulyc, Sarah Selimand, Ashraqat Samid, Gehan Safwatd, Ayman Diabd ⁎
a
Clinical Pathology and Hematology Department, Faculty of Medicine, Ain Shams University Biomedical Research Department, Cairo, P.O. Box 11381, Egypt Department of Medical Biochemistry, Faculty of Medicine, Ain Shams University, Egypt c Department of Pediatrics, Faculty of Medicine, Ain Shams University, Egypt d Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA), Cairo, Egypt b
ARTICLE INFO
ABSTRACT
Keywords: Lnc-CCAT1 Lnc-PVT1 Prognostic factor t(8;21) Acute Myeloid Leukemia (AML)
Long non-coding RNA (LncRNA) is recently linked to various types of cancers, CCAT and PVT1 are two LncRNAs linked to t(8;21) associated Acute Myeloid Leukemia, the interplay between CCAT, PVT1 and the MYC protooncogene implicated in t(8;21) could present an opportunity for using LncRNA as prognostic biomarker or a target for therapy, We investigated the expression levels of LncRNAs in 70 patients; 30 with t(8;21) positive AML and 40 with t(8;21) negative AML, We found that CCAT1 and PVT1 are expressed in higher levels in t(8;21) positive –AML by 5.3 folds compared to t(8;21) negative group; the expression values were significantly associated with high-risk clinical criteria; moreover, they are associated with lower overall survival (OS) rate and leukemia-free survival (LFS), however we didn't find a statistically significant cut-off value of LncRNAs using the Cox regression analysis for Lnc_PVT1 except with LFS, we conclude that high expression levels of CCAT1 and PVT1 are associated with poor prognosis while being poor prognostic biomarkers in t(8;21) associated AML.
1. Introduction Acute Myeloid Leukemia (AML) accounts for 80% of acute leukemia in adults and 15% in children (Siegel et al., 2017; Gamis et al., 2013). The balanced translocation t(8;21) is one of the cytogenic abnormalities associated with AML; seen in 7% of adult AML patients and is the most common cause of AML in children (Grimwade et al., 2010; Nucifora and Rowley, 1995). t(8;21) in AML has a favorable prognosis in adults, while children show poor response to treatment (Klein et al., 2015). Chemotherapy alone for AML achieves complete remission in up to 60% of patients (Wheatley et al., 1999). Resistance to chemotherapy is associated with unfavorable cytogenic features; The RT-PCR quantification of RUNX1-RUNX1T1 transcripts - the product of the t(8;21) - were linked to high risk of relapse (Tobal et al., 2000). The MYC-MAX transcriptional complex regulates transcription of various genes across different cell types. Overexpression of this complex leads to many types of cancer. AML has been linked to abnormal expression of the MYC proto-oncogene, various mechanisms for this association includes the up-regulation of MYC gene by the AML-
associated fusion protein RUNX1-RUNX1T1 (Müller-Tidow et al., 2004), the mutation of the CCAAT/enhancer binding protein alpha (C/ EBPα) transcription factor which normally down-regulate the MYC gene (Johansen et al., 2001), and the amplification of the 8q24 region where the MYC gene is located (Slovak et al., 1994). Most of the human genome is non-coding sequences which were considered to be of no use, but recently this view is changing as some non-coding sequences were found to have a regulatory role on gene transcription and post-transcriptional process. Long noncoding RNA (LncRNA) is a 200 nucleotide molecule which doesn't code for a specific protein product (Hamilton et al., 2015), it is the focus of many recent studies investigating the role of t (8;21) in AML (Pan et al., 2017; Quek et al., 2015). Colon cancer associated transcript (CCAT) is a LncRNA present together with the MYC in the 8q24 region. CCAT1 acts as a super-enhancer to the enhancer region present 335 KB upstream from the MYC gene and have been implicated in gastric, Hepatocellular carcinoma (Yang et al., 2015) and cancer colon (Fan et al., 2018). Although CCAT1 has been involved in different solid cancers, limited knowledge is
Abbreviations: AML, Acute Myeloid Leukemia; LncRNA, Long noncoding RNA; RUNX1, Runt-Related transcription factor 1; CCAT1, colon cancer associated transcript 1; PVT1, plasmacytoma variant translocation 1; SWI/SNF, Switch/Sucrose non-fermentable ⁎ Corresponding author. E-mail address: [email protected] (N. El-Khazragy). https://doi.org/10.1016/j.gene.2019.03.055 Received 18 November 2018; Received in revised form 13 March 2019; Accepted 25 March 2019 Available online 31 March 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Shows the interplay between CCAT1, PVT1, and MYC in AML could produce an abnormally overexpressed MYC protein involved in the pathogenesis of AML.
competing endogenous RNA (ceRNA) for miR-155 which is a direct target for c-myc (Hamilton et al., 2015). PVT-1 is another LncRNA present in the 8q24 region required for MYC gene expression (Hamilton et al., 2015). It was evident that aberrant overexpression of PVT1 has been discovered in many cancers such as cancer ovary and hepatocellular carcinoma (Yang et al., 2015; Saus et al., 2016), moreover, it has been demonstrated that PVT1 has a major role in c-Myc protein expression (Hamilton et al., 2015). The interplay between CCAT1, PVT1, and MYC in AML could produce an abnormally overexpressed MYC protein involved in the pathogenesis of AML as shown in (Fig. 1), this presents an opportunity to use the CCAT1 or the PVT1 as a potential target for therapy or as a prognostic marker of AML.
Table 1 Descriptive analysis of the studied subjects. Demographic data
Age (years) Gender Age subgroups TLC (×106/L) Hemoglobin (grm/ dl) Platelet count (×1012/L) BM blasts (%) MRD at day 15 Clinical response Status Overall survival (OS) Leukemia free survival (LFS)
Mean ± SD Range Male Female Favorable (≤40) Unfavorable (> 40) ≤50 > 50 >6 ≤6 ≥30 < 30 ≤25% > 25% ≤0.01 > 0.01 Remission Relapse Died alive Median (IQR) Range Median (IQR) Range
AML-t(8;21) positive N = 30
AML-negative for t(8;21) N = 40
50.0 ± 14.0 28–73 19 (37%) 11 (58%) 12 (37%) 18 (51%)
49.4 ± 14.5 22–77 32 (63%) 8 (42%) 32 (63%) 27 (49%)
19 (35%) 11 (73%) 18 (37%) 12 (86%) 11 (73%) 19 (35%) 15 (29%) 15 (83%) 13 (27%) 17 (81%) 10 (22%) 5 (15%) 15 (65%) 15 (32%) 15 (11) 2–24 8 (10) 0.8–22
38 (66%) 4 (27%) 38 (68%) 2 (14%) 4 (27%) 36 (65%) 37 (71%) 3 (17%) 36 (73%) 4 (19%) 11 (24%) 36 (78%) 8 (35%) 32 (68%) 20 (4) 5–24 16 (1.8) 0.4–7.7
2. Results 2.1. Demographic and clinical features of studied subjects The Mean age of Subjects participating in this study was 50.0 ± 14.0 for the t(8;21) positive group and 49.4 ± 14.5 for the t (8;21) negative group, 37% of the male participants were t(8;21) positive, 58% of female participants were t(8;21) positive. Considering the favorable age group to be (≤40), only 37% of the favorable age group were t(8;21) positive, while in the unfavorable age group (> 40) t (8;21) positive and t(8;21) negative expression was nearly equal. High TLC (> 50 × 106/L), Low Hemoglobin level (≤6 g/dl) and High Blasts count (> 25%) were observed in the t(8;21) positive group, while low platelet counts (< 30 × 1012/L) were observed in the t(8;21) negative group (65%) (Table 1).
BM: bone marrow; MRD: minimal residual disease.
known about its role in AML pathogenesis as well as its prognostic value. Moreover, up to date, the real mechanism by which CCAT1 exerts its oncogenic effect remains unknown. Recently, in a study conducted by Chen et al. (2016), he demonstrated higher expression levels of CCAT1 in M4 and M5 AML subtypes compared to normal control. Furtherly, in vitro studies demonstrated that myeloid proliferation differentiation and is inhibited by high expression of CCAT1 through a
2.2. High expression of CCAT1 and PVT1 in t(8;21) positive AML Expression levels of the Lnc-CCAT1, Inc-PVT1 and MYC were significantly higher among the AML group compared to the control group (P value ≤ 0.01) and significantly higher among the t(8;21) positive 173
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Table 2 Comparative analysis for CCAT1, PVT1 and c-Myc between different studied subgroups. Variable
Median (IQR) Range
Lnc-CCAT1(log10) Lnc-PVT1(log10) c-Myc (log10)
Statistics: Ⱬ Mann Whitney test value((p value)
AML n = 70
t(8;21) positive-AML n = 30
t(8;21) negative-AML n = 40
Control n = 20
AML/control
t(8;21) positive-AML/ control
t(8;21) negative AML/ control
t(8;21) positive/ negative AML
3.1 (4.1) 0.4–12.0 20.6 (31) 2.2–99 13.4(7.5) 6.8–22.0
5.5 (2.3) 0.9–12.0 40.2 (31) 5.1–99.0 50.0 (38) 21.7–88
1.6 (1.8) 0.4–7.7 11.4 (14.6) 2.2–67 30.4 (13) 8.6–61.0
0.8 (2.8) 0.1–5.7 2.8 (3.2) 1.0–11.0 13.4 (7.5) 6.8–22.0
4.2 (≤0.01)⁎
4.8 (≤0.01)⁎
3.0 (≤0.01)⁎
5.3 (≤0.01)⁎
6.0 (≤0.01)⁎
5.7 (≤0.01)⁎
5.2 (≤0.01)⁎
4.6 (≤0.01)⁎
6.4 (≤0.01)⁎
5.9 (≤0.01)⁎
5.6 (≤0.01)⁎
4.3 (≤0.01)⁎
⁎ Test is significant at level ≤ 0.01, IQR: Interquartile ratio; AML: Acute Myeloid Leukemia; t(8:21): translocation between chromosome 8 and 21; n:number of subjects; Lnc: long non coding RNA.
statistical significance. On investigating the difference in the individual expression levels between the t(8;21) positive and negative groups, the Lnc-CCAT1 and myc expression levels were correlated to low OS and LFS in both groups with statistical significance, the Lnc-PVT1 expression levels were correlated to low OS and LFS in both groups without statistical significance (Supplementary Table 2 & Fig. 3).
60 50 40 30
2.5. CCAT1, PVT1 and c-Myc as independent prognostic factors in t(8;21)AML
20 10
Univariate analysis of Cox proportional hazards modeling of potential prognostic factors for OS and LFS in the study population revealed that gene expression levels exceeding the assigned cut-off value for CCAT1, PVT1 and c-Myc were associated with an adverse prognosis and shorter overall survival (p < 0.05). Unless PVT1; the same findings were obtained for LFS; higher expression values are significantly associated with shorter duration of LFS (p < 0.05) (Table 4).
0 Lnc-CCAT1 (log10)
t(8;21)-positive AML
Lnc-PVT1 (log10)
c-Myc (log10)
t(8;21)-negative AML
Healthy control
Fig. 2. Expression level of CCAT1, PVT1, and c-Myc in t(8;21)-AML positive, negative patients and healthy control.
group compared to t(8;21) negative group (P value ≤ 0.01) (Table 2) (Fig. 2).
3. Subjects & methods 3.1. Study cohort
2.3. Higher expression levels of-of CCAT1 and PVT1 is positively correlated with up regulation of c-Myc and associated with high-risk factors in t(8;21) positive AML
The present study enrolled seventy patients with de-novo diagnosed AML, attended at Clinical Hematology Department, Ain Shams Internal Medicine Hospital, Cairo, Egypt between March 2015 and August 2017, twenty patients not suffering from any hematologic or other type of malignancy consisting our control cohort. Patients' clinic pathological characteristics are presented in (Table 1). All patients provided their written informed consent in accordance with the Declaration of Helsinki. Peripheral blood (PB) samples were obtained, with a minimum blast infiltration of 25%. The sample was collected before the receiving of chemotherapy induction protocol; All sample handling, procedure and storage was the same for leukemic and control subjects. Patients follow up and risk groups stratification of AML was done according to the Berlin-Frankfurt-Munster (BFM) guidelines; it includes; assessment of BM blasts on day 8, BM blasts % on day 15 and minimal residual disease (MRD) measurement at day 28 of induction (Zeng et al., 2015). The diagnosis of t(8;21) associated AML was made according to a morphologic assessment of the Leishman stained smears of the bone marrow aspirates along with special immune-cytochemical stains, immune-phenotyping, and Cytogenetic analysis. Assessment of minimal residual disease (MRD) was performed using a lineage-specific monoclonal panel; MRD was considered positive when leukemic cells exceeded 0.01% of all marrow nucleated cells on days 15 and 28 (Table 3).
We found that high expression levels of the Lnc-CCAT1, Inc-PVT1, and MYC among the t(8;21) positive group were correlated to low hemoglobin level (≤6 g/dl) and low platelet count (≤30 × 1012/L). Also, the expression levels of the Lnc-CCAT1 and MYC were higher in patients with BM blast cells (> 70%) and minimal residual disease (MRD) (> 0.01) with statistical significance (Supplementary Table 1). 2.4. Association of CCAT1 and PVT1 with t(8;21)-AML patients overall survival (OS) and progression-free survival (PFS) In order to assess the influence of higher gene expression levels on t (8;21) associated AML disease's outcome; the median expression value for each tested biomarker (CCAT1, PVT1 and c-myc) was selected as a cut-off value of (5.5, 40 and 50); respectively. Furtherly, the Kaplan Meir survival analysis was performed to find out the association between expression's level of biomarker and patient's overall (OS) and leukemia free survival (LFS). Generally, gene expression with values higher than the cut off value was significantly associated with shorter OS for CCAT1 and c-Myc (p < 0.01) compared to those with expression values beyond the cut off value. However, PVT1 expression was insignificantly associated with OS (p = 0.7). On the other hand; patients showed higher expression levels of Lnc-CCAT1, Inc-PVT1, and c-Myc have shorter time to relapse than those with mild increase, therefore, correlate with leukemia-free survival (LFS) in t(8;21)AML patients with
3.2. Bioinformatics analysis In the present study, the association of lncRNA–CCAT1, PVT1 and 174
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Fig. 3. Kaplan-Meier survival curves of patients with high and low Lnc-CCAT1, Lnc_PVT1 and c-myc expression in t(8; 21) associated AML. Patients were dichotomized into high and low genes expression groups based on median expression value. Patients with expression levels for “Lnc_CCAT1 and c-myc” less than the optimum cut-off values had significantly better survival (a, e) and leukemia free survival (b. f): respectively than those with higher expression; Meanwhile, the LncPVT1 expression was significantly correlated with LFS (d), no significant association was reached with OS (c) (p > 0.05).
mRNA c-Myc with t(8;21) associated AML pathogenesis was investigated using the lncRNA disease database (http://www.cuilab.cn/ lncrnadisease) and [37] (http://www.cuilab.cn/files/images/ldd/ rnaseq/HOTAIR.txt). The interaction between lncRNAs “CCAT1 and PVT1” was predicted from the crosslinking immunoprecipitation RNA sequencing (CLIP-Seq) data using the star Base platform (http:// starbase.sysu.edu.cn/) (Quek et al., 2015). Finally, the interlink pathway in t(8;21)-AML related lncRNAs “CCAT1, PVT1” and c-Myc are further confirmed using PubMed (http://www.ncbi.nlm.nih.gov/ pubmed/), (http://oncodb.hcc.ibms.sinica.edu.tw/index.htm. On the basis of the genomic information that was collected, the regulatory network of the lncRNA-CCAT1, PVT1 and mRNA-cMyc in AML was constructed by Cytoscape software version 3.5.0 (http://www. cytoscape.org/index.html).
samples in EDTA bone marrow cells from newly diagnosed patients. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, Total RNA concentration and purity were evaluated spectrophotometric ally at 260 and 280 nm. RNA integrity was visually confirmed by agarose gel electrophoresis. cDNA was synthesized by reverse transcription reaction using QuantiTect RT Kit (Qiagen, Hilden, Germany). 3.4. Long non-coding RNAs “CCAT1, PVT1” and c-Myc expression analysis For detection of genes expression; cDNA was amplified using a Hs_CCAT1 RT2 Primer Assay cat no: 330701, assay ID: LPH15969A, Hs_PVT1 RT2 Primer Assay cat no: 330701, assay ID: LPH17013A, Hs_cMyc_SG QuantiTect primer assay cat no: 249900, assay ID: QT01663361 and Hs_ACTB_1_SG QuantiTect Primer assay (NM_001101) used as reference gene. Each sample was amplified by both primers using QuantiTect Syber green Master Mix (Qiagen, Hilden, Germany). The 25 μl reaction mixture/reaction consist of 2× QuantiTect Syber green PCR mastermix, 0.3 μM primer assay and
3.3. Total RNA extraction and purification Total mRNA was extracted from mononuclear cells (MNCs) that it is isolated by Ficoll Hypaque density gradient centrifugation from 2 ml PB
Table 3 Impact of high/low expression levels of CCAT1.PVT1, c-Myc on overall and progression free survival in t(8;21) positive AML. Variable
Overall survival (months) Cut-off
10
Lnc-CCAT1 (log ) Lnc-PVT1 (log10) c-Myc (log10)
≤5.5 > 5.5 ≤40 > 40 ≤50 > 50
Estimate OS
22 9.8 16 14 21 10
Leukemia free survival (months)
95% Confidence Interval
Log Rank (Mantel-Cox) 2
Lower
Upper
Χ
19 6.4 12 10 18 7
24 13 21 17 24 12
14
0.001
0.2
0.7
11.9
0.001
Estimate OS
P value
175
13 5 17 7 16 5
95% Confidence Interval
Log Rank (Mantel-Cox)
Lower
Upper
Χ2
P value
12 3 11 5 13 3
20 6.8 22 9 21 7.2
20
0.001
9.5
0.002
12
0.001
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Table 4 Univariate Cox proportional hazards analysis of survival analysis for t(8;21) positive AML. Variable
Lnc-CCAT1 ≤5.5 vs > 5.5 Lnc-PVT1 ≤40 vs > 40 Lnc-c-Myc ≤50 vs > 50 Overall model coefficients
Cox Regression analysis Overall survival (months)
Cox Regression analysis Leukemia free survival (months)
HR
95% CI
P value
HR
95% CI
P value
0.2
0.4–0.6
0.009
0.1
0.03–0.5
0.002
4.7
1.3–16.5
0.02
0.8
0.2–3.2
0.8
0.09
0.02–0.5
0.004
0.2
0.05–0.8
0.02
0.001
2
2
Χ :25.4; df:3
Χ :27.2; df:3
0.001
HR: Hazards ratio, CI: confidence interval, Χ2: Chi-square.
500 ng cDNA. Both targets were amplified in duplicates for each sample. The thermal protocol consists 15 min for HotStar-Taq DNA Polymerase activation at 95 °C followed by 45 cycles of denaturation at 94 °C for 15 min, primer annealing for 30 s at 52 °C and extension at 72 °C for 30 s). The fluorescence data were collected at extension step. Following amplification, gene normalization was calculated using ΔCt equation; ΔCt = mean value Ct (reference) − mean value Ct (interesting gene). Finally, the gene expression levels were calculated by 2−∆∆Ct calculation using Rotor-Gene software version 1.2.3 (Qiagen).
especially with the t(8;21)- and MYC proto-oncogene is a very interesting link to study as a therapeutic target or even prognostic marker. Many studies have studied the link between these LncRNAs and different types of leukemia; Zeng et al. found significantly upregulated PVT1 in Acute Promyelocytic leukemia patients and showed that knocking either PVT1 or MYC reduced the expression of the other (Zeng et al., 2015), while Hu et al. found that PVT1 was highly expressed in Acute Lymphoblastic leukemia patients (Hu et al., 2018). In contrary, the expression levels of CCAT1 and PVT1 didn't differ between AML of different phenotypes and healthy controls. Meanwhile; when high risk AML-M3 patients, higher PVT1 expression level was detected and it was significantly associated with poor prognosis (Zeng et al., 2015; Izadifard et al., 2018). On the other hand, higher expression levels of CCAT1 were detected in AML-M4 and M5. However, to our knowledge, CCAT1 and PVT1 deregulation have not be investigated in t(8;21)-AML on clinical samples. These findings support the phenomena that the dysregulation of CCAT1 and PVT1 transcripts may be useful to understand the leukemogenic process and furtherly could serve as targeted therapy (Izadifard et al., 2018). CCAT1 and PVT 1 binds to MYC preventing its phosphorylation and degradation (Hamilton et al., 2015), this creates an up-regulated MYC that promotes leukemic cell proliferation and block myeloid differentiation. Saus et al. found that CCAT-1 and PVT-1 behave as oncogenes in colorectal cancer. CCAT-1 induces tumor progression and metastasis which is mediated by c-Myc binding to the CCAT1 promoter region. While PVT-1 induces tumor proliferation, invasion, and an antiapoptotic effect via modulation of chromatin remodeling complex SWI/ SNF in CRC (Saus et al., 2016). Over-expression of PVT1 has been suggested as a powerful predictor of tumor progression and patient survival in a diverse range of cancer types, such as pancreatic cancer, gastric cancer, hepatocellular cancer. Also, the CCAT-1 has been shown to be upregulated in these cancers and also in lung and breast cancer (Huang et al., 2015). It was demonstrated by Saus et al. that CCAT1 was significantly correlated with histopathological grade, lymph nodes metastasis, and survival time after surgery. In vivo studies highlighted the expected pathways; they suggested that CCAT1 transcription could be promoted by c-Myc; this fact explained how CCAT1 overexpression promotes cell proliferation and invasion in colon cancer cells (Fan et al., 2018). Furthermore; in gastric carcinoma tissues, CCAT1 has a pathogenic role in gastric carcinoma; therefore, it has been implicated in the treatment of gastric carcinoma (Saus et al., 2016). Unfortunately, up to date, the role of lncRNA CCAT1 expression progression of breast cancer is not fully understood (Fatima et al., 2015). Many recent studies have investigated the potential of LncRNA to be used as a therapeutic target (Fatima et al., 2015; Boon et al., 2016), knocking down the up-regulated CCAT1 and PVT1 in AML could theoretically decrease MYC over-expression and improve the survival outcome.
3.5. Statistical analysis Statistical analysis was performed using SPSS v.22 (Chicago, IL, USA).The non-parametric Mann–Whitney U test and Wilcoxon Signed Rank test were performed to evaluate the differences of CCAT1, PVT1 and c-Myc expression between leukemic vs healthy specimen, and between t(8;21) positive AML vs t(8;21) negative, respectively. Moreover, to assess the correlation of CCAT1, PVT1 and c-Myc expression with patients' clinic-pathological features. Spearman's correlation was used to assess the expression levels of lnc_CCAT1, lnc_PVT1 and c_Myc with patient outcome. Overall survival (OS) was calculated from diagnosis to death or last follow-up and Leukemic-free survival (LFS) from complete remission (CR) to relapse or death. Both OS and LFS were estimated with the Kaplan–Meier method and comparisons among subgroups of patients with high and low values of genes expressions at diagnosis were performed using the log-rank test. Significance was set at ≤0.05. 4. Discussion We found the Long noncoding RNAs CCAT1 and PVT1 to be overexpressed and associated with lower overall survival rate and leukemiafree survival in Acute Myeloid Leukemia patients with the translocation (8; 21); a high expression of CCAT1 and PVT1 to be linked to high-risk t (8;21) leukemia, high blasts count, low platelets and low hemoglobin were significantly higher in t(8;21) positive patients. This suggests a role of CCAT1 and PVT1 in the progression of the disease, moreover; high expression levels were linked to high minimal residual disease in the t(8;21) positive patients, suggesting that chemotherapy resistant AML may be induced by the high expression of CCAT1 and PVT1, our results were in agreement with some studies that reported PVT1 association with multidrug resistance in certain cancer cases (Pan et al., 2017; Yang et al., 2015), most anticancer drugs exert their effect by inducing apoptosis, hence inhibiting apoptosis by CCAT1 or The PVT1 is one of the main mechanisms of multidrug resistance (Fan et al., 2018). LncRNAs were thought previously to be translational jargon. This view is changing recently with them emerging as a contributor to the regulation of many types of genes. The interplay between the CCAT1 and PVT1 – which we found to be highly expressed in AML patients 176
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Over-expression of CCAT1 and PVT1 is correlated with poor prognosis in t(8;21)-AML; But, Both markers are poor prognostic biomarkers. New trials are needed to assess the potential of both as therapeutic targets. Acknowledgements We thank our colleagues from [Clinical Haematology Department, Ain Shams University Hospital] who provided insight and expertise that greatly assisted the research, although they may not involve with all of the interpretations/conclusions of this paper. We would also like to show our gratitude to the patients who are the backbone of this study for their cooperation and sharing their pearls of wisdom with us during the course of this research, and we thank 3 “anonymous” reviewers for their so-called insights. We are also immensely grateful for their comments on an earlier version of the manuscript, although any errors are our own and should not tarnish the reputations of these esteemed persons. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.03.055. References Boon, R.A., Jaé, N., Holdt, L., Dimmeler, S., 2016 Mar 15. Long noncoding RNAs: from clinical genetics to therapeutic targets? J. Am. Coll. Cardiol. 67 (10), 1214–1226. (Internet). (cited 2018 Jul 21). Available from: https://www.sciencedirect.com/ science/article/pii/S0735109716002242. Chen, Lianxiang, Wang, Wei, Cao, Lixia, Li, Zhijun, Wang, Xing, 2016. Long non-coding CCAT1 acts as a competing endogenous RNA to regulate cell growth and differentiation in acute myeloid leukemia. Mol. Cell 39 (4), 330–336. http://dx.doi.org/10. 14348/molcells.2016.2308. Fan, H., Zhu, J.-H., Yao, X.-Q., 2018 Jun. Knockdown of long non-coding RNA PVT1 reverses multidrug resistance in colorectal cancer cells. Mol. Med. Rep. 17 (6), 8309–8315. (Internet). (cited 2018 Jul 31). Available from: http://www.ncbi.nlm. nih.gov/pubmed/29693171. Fatima, R., Akhade, V.S., Pal, D., Rao, S.M., 2015 Dec 12. Long noncoding RNAs in development and cancer: potential biomarkers and therapeutic targets. Mol. Cell. Ther. 3 (1), 5. (Internet). (cited 2018 Jul 21). Available from: http://molcelltherapies. com/article/view/30. Gamis, A.S., Alonzo, T.A., Perentesis, J.P., Meshinchi, S., 2013 Jun. Children's Oncology Group's 2013 blueprint for research: acute myeloid leukemia. Pediatr. Blood Cancer 60 (6), 964–971. (Internet). (cited 2018 Jun 27). Available from: http://doi.wiley. com/10.1002/pbc.24432. Grimwade, D., Hills, R.K., Moorman, A.V., Walker, H., Chatters, S., Goldstone, A.H., et al., 2010 Jul 22. 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 116 (3), 354–365. (Internet). (cited 2018 Jun 27). Available from: http://www.bloodjournal.org/cgi/doi/10.1182/blood-2009-11254441. Hamilton, M.J., Young, M.D., Sauer, S., Martinez, E., 2015. The interplay of long noncoding RNAs and MYC in cancer. AIMS Biophys. 2 (4), 794–809. (Internet). Available from: http://www.aimspress.com/article/10.3934/biophy.2015.4.794. Hu, J., Han, Q., Gu, Y., Ma, J., McGrath, M., Qiao, F., et al., 2018 Jun. Circular RNA PVT1
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