Background: Acute promyelocytic leukaemia (APL) is a unique subtype of acute myeloid leukaemia (AML) characterized by haematopoietic failure caused by the accumulation of abnormal promyelocytic cells in bone marrow (BM). However, indispensable BM biopsy frequently afflicts patients in leukaemia surveillance, which increases the burden on patients and reduces compliance. This study aimed to explore whether the novel circulating long noncoding RNA LOC100506453 (lnc-LOC) could be a target in diagnosis, assess the treatment response and supervise the minimal residual disease (MRD) of APL, thereby blazing a trail in noninvasive lncRNA biomarkers of APL. Methods: Our study comprised 100 patients (40 with APL and 60 with non-APL AML) and 60 healthy donors. BM and peripheral blood (PB) sample collection was accomplished from APL patients at diagnosis and postinduction. Quantitative real-time PCR (qRT–PCR) was conducted to evaluate lnc-LOC expression. A receiver operating characteristic (ROC) analysis was implemented to analyse the value of lnc-LOC in the diagnosis of APL and treatment monitoring. For statistical analysis, the Mann–Whitney U test, a t test, and Spearman's rank correlation test were utilized. Results: Our results showed that BM lnc-LOC expression was significantly different between APL and healthy donors and non-APL AML. lnc-LOC was drastically downregulated in APL patients' BM after undergoing induction therapy. Lnc-LOC was upregulated in APL cell lines and downregulated after all-trans retinoic acid (ATRA)-induced myeloid differentiation, preliminarily verifying that lnc-LOC has the potential to be considered a treatment monitoring biomarker. PB lnc-LOC was positively correlated with BM lnc-LOC in APL patients, non-APL AML patients and healthy donors and decreased sharply after complete remission (CR). However, upregulated lnc-LOC was manifested in relapsed-refractory patients. A positive correlation was revealed between PB lnc-LOC and PML-RARα transcript levels in BM samples. Furthermore, we observed a positive correlation between PB lnc-LOC and BM lnc-LOC expression in APL patients, suggesting that lnc-LOC can be utilized as a noninvasive biomarker for MRD surveillance. Conclusions: Our study demonstrated that PB lnc-LOC might serve as a novel noninvasive biomarker in the treatment surveillance of APL, and it innovated the investigation and application of newly found lncRNAs in APL noninvasive biomarkers used in diagnosis and detection.
Keywords: Lnc-LOC; Acute promyelocytic leukaemia; Minimal residual disease; Noninvasive biomarker; Surveillance
Guiran Wang, Guiling Yan and Kanru Sang contributed equally to this work.
Acute promyelocytic leukaemia (APL) is a unique subtype of acute leukaemia characterized by balanced chromosomal ectopic t (15;17) (q22; q12), leading to promyelocytic (PML) genes and retinol receptor alpha (RARα) gene fusion [[
Previous works have shown that circulating tumour nucleic acids, circulating tumour cells and exosomes can be applied to liquid biopsy and are highly likely to become part of future clinical practice [[
The identification of dysregulated lncRNAs may provide new targets for the diagnosis and treatment of APL. Based on previous studies, we systematically integrated the gene expression profiles of APL and significantly coexpressed gene pairs by using GeneChip analysis. Then, we first selected one candidate differentially expressed long noncoding RNA, LOC100506453 (lnc-LOC), whose gene symbol is LOC100506453 and gene accession number is ENST00000424415, in BM samples from APL patients with ATRA-based targeted therapy [[
In this study, we investigated lnc-LOC expression in APL by establishing a sensitive and specific TaqMan probe-based quantitative real-time PCR (qRT–PCR) and then assessed its potential value. Our data showed that after the induction of ATRA, the expression of lnc-LOC was downregulated in both APL cell lines (NB
In this study, we enrolled 100 leukaemia patients, including 40 APL patients and 60 non-APL AML patients, from The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University between April 2018 and October 2019 (Table 1). All leukaemia cases met the WHO 2016 acute leukaemia classification criteria (Table 2) [[
Table 1 Characteristics of 100 patients with AML
Variable at diagnosis APL patients ( Non-APL patients ( N (%) N (%) Female 25 (62.5) 30 (50) Male 15 (37.5) 30 (50) 30 (75.0) 35 (58.3) ≥ 50 10 (25.0) 25 (41.7) 35 (87.5) 45 (75.0) ≥ 10 5 (12.5) 15 (25.0) Low/intermediate 35 (87.5) High 5 (12.5) ATO 38 (95.0) Cytarabine + ATRA 2 (5.0)
Abbreviations: AML acute myeloid leukaemia, APL acute promyelocytic leukaemia, ATO Arsenic Trioxide, ATRA all-trans retinoic acid
Table 2 WHO classification of 100 patients with AML
WHO classification N (%) APL with PML-RARα 40 (40.0) AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 10 (10.0) AML with inv.(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 7 (7.0) AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A 12 (12.0) AML with minimal differentiation 6 (6.0) Acute myelomonocytic leukaemia 25 (25.0)
Abbreviations: AML acute myeloid leukaemia, APL acute promyelocytic leukaemia
The human leukaemia cell lines NB4, HL-60, THP-1, U937, K562, Kasumi-6 and HEL were purchased from the Shanghai Institution of Haematology and cultured in RPMI 1640 (Gibco BRL, USA) containing 10% foetal bovine serum (Gibco BRL, USA) at 37 °C in a humidified 5% CO
PB samples and BM samples were collected from patients and healthy donors using EDTA-K
qRT–PCR was performed with TaqMan Universal PCR Master Mix II, no UNG (Applied Biosystems, USA), and GAPDH was used as a reference for lncRNAs. After the extensive test, the final 20 μL optimized amplification system of lnc-LOC or GAPDH was as follows: 10 μL TaqMan Universal PCR Master Mix II, NO UNG (2×), 0.6 μL upstream primer (10 μM), 0.6 μL downstream primer (10 μM), 0.4 μL MGB probe (10 μM), 6.4 μL enzyme-free water, and 2 μL cDNA. Forward (F) and reverse (R) primers were synthesized by Shanghai Yiyue Biological Technology Company (Shanghai, China) as follows: lnc-LOC, lnc-LOC100506453-F GAGACCCTGGAAATAAAC, and lnc-LOC100506453-R CGATGGAATCAGTTAGAC; and GAPDH, hGAPDH-F TGCACCACCAACTGCTTAGC, and hGAPDH-R TCTTCTGGGTGGCAGTGATG. The probe sequences were as follows: lnc-LOC100506453-MGB FAM-TGGCTTCAGCGTCACCTAGT-MGB and GAPDH-MGB FAM- ACTGTGGTCATGAGTC-MGB. The PCR program was run as follows: denaturation at 95 °C for 10 min, 45 cycles of extension at 95 °C for 5 s, and 59 °C for 1 min. qRT–PCR was repeated three times. The expression level relative to the control samples was calculated by the 2
MRD analysis was based on 40 BM samples collected from APL patients before and after induction therapy. qRT–PCR was used to detect PML-RARα fusion gene transcripts from all BM samples on an ABI7500 System with a TaqMan probe as previously described [[
SPSS 21.0 was used for statistical analysis, and graph plotting was performed using GraphPad Prism 8.01 (GraphPad Software, USA). The nonparametric Mann–Whitney U test and a t test were performed to assess the differences in lnc-LOC expression between experimental samples and control samples. The receiver operating characteristic (ROC) curve was used to evaluate the value of lnc-LOC for APL diagnosis. Spearman's rank correlation test and rank-sum test were performed for the correlation analysis. P < 0.05 was considered statistically significant.
Based on previous studies, we explored some important lncRNAs in APL patients with ATRA-based targeted therapy through a combined computational and experimental approach [[
To confirm whether BM lnc-LOC is a potential biomarker for APL diagnosis, we used qRT–PCR to detect BM lnc-LOC expression in APL, non-APL AML and healthy donors. Representative amplification plots of APL, non-APL AML, and healthy donors by qRT–PCR are shown in Supplemental Fig. 1. The results suggested that lnc-LOC expression was higher in BM samples from APL patients than in BM samples from non-APL AML patients or healthy donors (P < 0.001; Fig. 1A). To investigate whether BM lnc-LOC expression was qualified for APL diagnosis, we used ROC curve analysis to reveal the diagnostic accuracy of lnc-LOC. When the optimal cut-off values were 2.070, 3.010 and 1.790, the areas under the ROC curves (AUCs) were 0.937 (95% CI = 0.888–0.985, P < 0.001; sensitivity = 0.975; specificity = 0.767) for distinguishing APL from healthy donors, 0.901 (95% CI = 0.839–0.963, P < 0.001; sensitivity = 0.825; specificity = 0.850) for distinguishing APL from non-APL AML, and 0.588 (95% CI = 0.486–0.691, P = 0.095; sensitivity = 0.533; specificity = 0.683) for distinguishing non-APL AML from healthy donors (Fig. 1B-D). Taken together, BM lnc-LOC expression was significantly different between APL and healthy donors and non-APL AML, which indicated that BM lnc-LOC might be a potential biomarker for APL diagnosis.
Graph: Fig. 1 BM lnc-LOC expression and diagnostic value in APL patients. a Comparison of BM lnc-LOC expression in different groups, including APL, non-APL AML and healthy donors. b ROC curve analysis of BM lnc-LOC expression for the discrimination of APL from healthy donors. c ROC curve analysis of lnc-LOC expression for the discrimination of APL from non-APL AML. d ROC curve analysis of lnc-LOC expression for the discrimination of non-APL AML from healthy donors. * Statistically significant
To clarify the specific expression of lnc-LOC in the development of haematopoietic cells, we assessed lnc-LOC expression in different leukaemia cell lineages by qRT–PCR. The results showed that lnc-LOC expression was significantly higher in NB4 cells than in non-APL cells (HL-60, U937, THP-1, K562, Kasumi-6 and HEL) (P < 0.001; Fig. 2A). Next, we selected NB4 and HL-60 cells as models of BM differentiation. Lnc-LOC was downregulated in ATRA-treated HL-60 (P < 0.001; Fig. 2B) and NB4 cells (P < 0.001; Fig. 2C). Besides, the expression of PML-RARα in NB4 after ATRA treatment was downregulated (P < 0.001; Fig. 2D), and the expression of lnc-LOC was positively correlated with PML-RARα in NB4 after ATRA treatment (Fig. 2E). However, it was not observed in other non-APL cell lines (P > 0.05; Supplemental Fig. 2). In summary, lnc-LOC was specifically upregulated in APL cell lines and downregulated in ATRA-induced myeloid differentiation, suggesting that lnc-LOC could be used to measure the response to ATRA-based APL therapy.
Graph: Fig. 2 Differential expression of lnc-LOC in myeloid lineage and ATRA-treated APL cells. a lnc-LOC expression in myeloid lineage cells, including NB4, HL-60, U937, THP-1, K562, Kasumi-6, and HEL cells. b lnc-LOC expression in HL-60 cells treated with ATRA as indicated. c lnc-LOC expression in NB4 cells treated with ATRA as indicated. d PML-RARα transcript expression in NB4 cells treated with ATRA as indicated. e lnc-LOC expression in NB4 cells was positively correlated with PML-RARα transcript expression after ATRA treatment in NB4 cells. * Statistically significant
To confirm whether PB samples can replace BM samples for lnc-LOC detection, we first evaluated PB lnc-LOC expression in different groups by qRT–PCR. As shown in Fig. 3A, PB lnc-LOC expression in the APL patient group was higher than that in either the non-APL AML patient group (P < 0.001) or the healthy donor group (P < 0.001). Then, we analysed the correlation between PB lnc-LOC and BM lnc-LOC expression in APL at diagnosis. The results showed a positive correlation between PB lnc-LOC and BM lnc-LOC expression in APL at diagnosis (R
Graph: Fig. 3 Correlation between lnc-LOC expression in PB samples and that in BM samples from APL patients. a Comparison of PB lnc-LOC expression in APL, non-APL AML and healthy donors. b PB lnc-LOC expression was positively correlated with BM lnc-LOC expression in APL at diagnosis (R2 = 0.936, P < 0.001*). c PB lnc-LOC expression was positively correlated with BM lnc-LOC expression in non-APL AML (R2 = 0.918, P < 0.001*). d PB lnc-LOC expression was positively correlated with BM lnc-LOC expression in healthy donors (R2 = 0.910, P < 0.001*). e A significant positive correlation was observed between all PB samples and BM samples in lnc-LOC expression (R2 = 0.963, P < 0.001*). * Statistically significant
To explore whether PB lnc-LOC can be applied for monitoring APL treatment response, we collected 40 pairs of PB samples from APL patients at initial diagnosis, after induction therapy and at the three-year clinical follow-up. The statistical results showed that after induction therapy, lnc-LOC expression decreased compared with newly diagnosed APL patients (P < 0.001; Fig. 4A). Moreover, lnc-LOC expression was continually reduced even after achieving remission (P < 0.001; Fig. 4A). Furthermore, lnc-LOC expression remained low every 3 months after remission (data not shown). However, when patients relapsed, lnc-LOC was upregulated (P = 0.001; Fig. 4B) and similar to the initial diagnosis (P = 0.103; Fig. 4B). Taken together, these results provide important insights into PB lnc-LOC, which could be a potential biomarker for treatment surveillance. Furthermore, these findings indicated that PB lnc-LOC downregulation after targeted therapy could reflect the response to treatment in APL.
Graph: Fig. 4 Utilization of PB lnc-LOC in monitoring the treatment response of APL patients. a PB lnc-LOC expression in 40 APL patients at new diagnosis, postinduction and CR. b PB lnc-LOC expression in 5 representative relapsed APL patients at different periods (at diagnosis, CR and relapse). * Statistically significant
To evaluate whether PB lnc-LOC can be used for MRD surveillance, we analysed the correlation between BM PML-RARα/ABL NCN and PB lnc-LOC before and after APL-induced differentiation. The results showed that lnc-LOC expression at diagnosis was positively correlated with PML-RARα/ABL NCN (R
Graph: Fig. 5 PB lnc-LOC as a potential MRD monitoring marker for APL patients. a PB lnc-LOC levels were positively correlated with PML-RARα transcript expression in APL patients at diagnosis (R2 = 0.949, P < 0.001*). b PB lnc-LOC levels were positively correlated with PML-RARα transcript expression in APL patients postinduction (R2 = 0.934, P < 0.001*). c PB lnc-LOC levels were consistently correlated with MRD values in APL patients postinduction (R2 = 0.904, P < 0.001*). * Statistically significant
In this study, we innovatively proposed lnc-LOC in PB samples, a newly found lncRNA, as a probable noninvasive biomarker for APL diagnosis and treatment surveillance. Our previous study found that lnc-LOC (primary ID ENST00000424415) from BM samples was a dysregulated lncRNA associated with ATRA-induced APL differentiation [[
Currently, technological advances and analysis of biomarkers provide new methods for haematological diseases, including APL. The common identification of APL-specific genetic lesions can be made by conventional karyotyping, fluorescence in situ hybridization (FISH), or comparable nucleic acid-based techniques [[
lncRNAs have been demonstrated to have diverse functions in multiple biological processes and play important roles in cell differentiation and development, and it may implicate several cancers [[
In a previous study, early detection of APL molecular relapse using BM was the preferred approach [[
In our previous study, we proved that miR-638 may be an ideal novel target for APL diagnosis and long-term surveillance [[
PB represents an attractive specimen source for MRD surveillance, allowing for frequent sampling attributed to easier sample collection. There are still scarce studies showing that PB lncRNAs may be detected as potential noninvasive biomarkers for leukaemia. In addition, PB lnc-LOC expression showed a significantly positive correlation with BM lnc-LOC expression, indicating lnc-LOC as a valid biomarker in noninvasive tracking for APL, which still requires multicentre validation. Therefore, our results showed the possibility of utilizing lnc-LOC as a noninvasive target for monitoring APL recurrence in patients with clinical suspicion of extramedullary relapse that may not be amenable to biopsy. In addition, many researchers have proposed that various regulatory correlations exist between lncRNAs and miRNAs. According to our previous study, lnc-LOC may change the function of miR-638 to initiate, maintain, and develop APL. Regrettably, the specific mechanism remains to be further studied. However, there are some limitations to the present study, including a relatively small sample size (especially high-risk cases), individual heterogeneity, and shorter observation duration. Therefore, our results still require multicentre validation. Meanwhile, whether PB lnc-LOC can be used to predict clinical outcomes, such as the relapse rates and survival of APL patients, needs to be further studied by expanding the sample size.
In conclusion, this study provides the first evidence that lnc-LOC may be a circulating biomarker for APL, including for diagnosis, treatment response, and MRD surveillance. Our study provided evidence that PB lnc-LOC could serve as a potential biomarker for APL, which set a precedent in the field of lncRNA noninvasive biomarkers of APL.
The authors thank all of the participants for donating BM/PB samples and all of the researchers for their contributions to this work.
ZGC and XQZ conceived and designed the study. GRW, GLY and KRS analysed the data and drafted the manuscript. ZGC, KRS and FX critically revised the manuscript. HJY and YYB performed the experiments. GLY and HJY collected samples. FX and XQZ provided technical support. NS provided samples. All authors approved the final manuscript submitted for publication.
This work was supported by the Basic Public Welfare Technology Research Project of Zhejiang Province (LGF20H200005), the Medical and Health Research Science and Technology Plan Project of Zhejiang Province (2021KY216), the Basic Scientific Research Project of Wenzhou City (Y20190090), and the Lin He's New Medicine and Clinical Translation Academician Workstation Research Fund (18331203).
The datasets generated during and analyzed during the current study are not publicly available due to patient privacy reasons but are available from the corresponding author on reasonable request.
The study was approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. All participants had given written informed consent. Research involving human participants and human cell lines have been performed in accordance with the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations.
Not applicable.
The authors declare no competing interests.
Graph: Additional file 1: Supplemental Figure 1. The representative lnc-LOC and GAPDH amplification plots by qRT-PCR based on TaqMan probe. a The amplification plot of lnc-LOC from APL patients. b The amplification plot of GAPDH from APL patients. c The amplification plot of lnc-LOC from non-APL AML patients. d The amplification plot of GAPDH from non-APL AML patients. e The amplification plot of lnc-LOC from healthy donors. f The amplification plot of GAPDH from healthy donors. The x-axis represents the cycle number, and the y-axis represents the relative change in the fluorescence values. The threshold of amplification plot is set at 0.100.
Graph: Additional file 2: Supplemental Figure 2. Expression of lnc-LOC in non-APL cells treated with ATRA. lnc-LOC expression in all cell lines were measured by qRT–PCR. qRT–PCR results are expressed as mean ± standard deviation.
Graph: Additional file 3: Supplemental Table 1. Genetic characteristic of lnc-LOC.
• APL
- Acute promyelocytic leukaemia
• PML
- Promyelocytic
- RARα
- Retinol receptor alpha
• ATRA
- All-trans retinoic acid
• MRD
- Minimal residual disease
• BM
- Bone marrow
• PB
- Peripheral blood
• AML
- Acute myeloid leukaemia
• lncRNAs
- Long noncoding RNAs
• lnc-LOC
- Long noncoding RNA LOC100506453
- qRT–PCR
- Quantitative real-time PCR
• CR
- Complete remission
• DMSO
- Dimethyl sulfoxide
• ROC
- Receiver operating characteristic
• CG
- Control gene
• NCN
- Normalized copy number
• bp
- Base pair
• AUCs
- Areas under the curves
• FISH
- Fluorescence in situ hybridization
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By Guiran Wang; Guiling Yan; Kanru Sang; Huijie Yang; Ni Sun; Yuanyuan Bai; Feng Xu; Xiaoqun Zheng and Zhanguo Chen
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