Using Circ-ANAPC7 as a Novel Type of Biomarker in the Monitoring of Acute Myeloid Leukemia

Introduction: Circular RNAs (circRNAs) are a novel class of RNAs which occupy gene expression at the transcriptional or post-transcriptional level, involve in many physiological processes, and participate in many diseases, especially in cancer. Our previous study showed 1 altered circRNA named circ-anaphase promoting complex subunit 7 (ANAPC7) that was upregulated in acute myeloid leukemia (AML). To further clear the expression and clinical significance of circ-ANAPC7, we enlarged the sample size and illuminated the diagnostic and monitoring value of circ-ANAPC7 in AML. Methods: Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was supposed to confirm the expression of circ-ANAPC7 of AML patients. We assessed the correlation of circ-ANAPC7 and clinical variables using the Spearman correlation test. The receiver operating characteristic (ROC) curve was carried out to evaluate the diagnostic value. Results: Circ-ANAPC7 was first found to be upregulated in AML, and its expression was correlated to white blood cell counts in peripheral blood and blast percentage in bone marrow. ROC curve analysis revealed that circ-ANAPC7 has a significant value of auxiliary AML diagnosis (area under the curve = 0.915, p < 0.001). Furthermore, the expression level of circ-ANAPC7 was changed accompanied with disease condition transformation. Conclusion: Circ-ANAPC7 was upregulated in newly diagnosed and relapsed AML. It may serve as potential biomarkers for AML patient’s diagnosis and monitoring.


Introduction
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the clonal proliferation of immature myeloid progenitor cells in the bone marrow, compressing normal blood cell production and leading to bone marrow failure ultimately [1,2]. Although the overall survival (OS) rate of patients with AML has improved due to mature chemotherapy and novel multimodality therapy, such as FLT3-specific tyrosine kinase inhibitors [3], Bcl-2 family inhibitors [4], as well as hematopoietic stem cell transplantation [5], a number of patients with AML still show no response to the present therapy, or relapsed from transient complete remission (CR).
According to the National Comprehensive Cancer Network guideline, AML patients were divided into 3 risk statuses by monosomal karyotype, and poor-risk cytogenetic and molecular abnormalities. High-risk AML responds poorly to available induction therapy and is likely to relapse despite consolidation treatment [6,7]. Moreover, most AML patients with high risk die from progressive disease after relapse. Therefore, searching for ideal biomarkers is essential to help predict early relapse of AML. Hundreds of somatic mutations were validated using deep sequencing in relapsed AML several years ago [8]. Recently, researchers have focused on searching for new prognostic biomarkers on noncoding RNA, including circular RNA (circRNAs).
In the previous study, we have conducted a microarray screening of circRNA changes in bone marrow mononuclear cells from 5 AML patients and 5 iron deficiency anemia (IDA) controls. The result indicated that cir-cRNA named circ-anaphase promoting complex subunit 7 (ANAPC7) (ID: hsa_circ_0005785 in CircBase: http:// circbase.org/cgi-bin/simplesearch.cgi) was up-regulated in 87 AML patients [18]. Circ-ANAPC7 is located at chr12:110819556-110834257, and its associated-gene symbol is ANAPC7. Here, we analyze the relationship between the expression level of circ-ANAPC7 and clinical features of AML patients by enlarging the sample size, trying to demonstrate its value in the pathogenesis and disease monitoring of AML.

Patients and Clinical Specimens
Approval for this study was obtained from the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (Approval No. 2015186). After obtaining written informed consent from all patients, we collected bone marrow specimens from 144 newly diagnosed AML patients and 80 IDA control patients at the Department of Hematology, the Second Affiliated Hospital of Xi'an Jiaotong University, between 2015 and 2018. The diagnosis, stage, and risk status of AML were made in accordance with the National Comprehensive Cancer Network (2018 version 1).

Total RNA Extraction
Total RNA was collected from bone marrow samples of AML and IDA using TRIzol reagent (Invitrogen, Germany), according to manufacturer's instructions, and stored at −80°C until use. RNA purity and concentration were detected by NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA).

Reverse Transcription
RNA samples were reversely transcribed into cDNA using a PrimeScript RT Master mix with random primers in accordance with manufacturer's protocols (Takara, Kusatsu, Japan).

Statistical Analysis
Variable was compared between 2 groups using the Mann-Whitney Wilcoxon rank test. A receiver operating characteristic (ROC) curve was set up to evaluate the diagnostic value of circ-ANAPC7. The Spearman rank correlation coefficient was used to analyze the correlation between clinical variables and circANAPC7 expression of AML patients. The OS rate and disease-free survival (DFS) rate were estimated by the Kaplan-Meier analysis and compared using the log-rank test. Prognostic factors for survival were identified by Cox regression analysis. All tests were 2-sided, and p < 0.05 was defined as a significant difference. All statistical analyses were performed with Statistical Product and Service Solutions (SPSS) Statistics for Windows, version 18.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA).

Circ-ANAPC7 Was Upregulated in AML
The expression level of circ-ANAPC7 was detected in 144 newly diagnosed AML, 123 CR AML, and 80 IDA controls. Baseline characteristics of 144 newly diagnosed AML patients were detected and are presented in Table 1. As shown in Figure 1, its expression levels in newly diagnosed AML were significantly higher than those obtained DOI: 10.1159/000520446 in CR and IDA patients (p < 0.001). Furthermore, there was no significant difference in the expression of circ-ANAPC7 between CR and IDA patients.

Potential Diagnostic Value of Circ-ANAPC7 in AML
To evaluate the potential diagnostic value of circ-ANAPC7, we conducted an ROC curve analysis (shown in Fig. 2). The area under the curve was 0.915 (95% CI 0.865-0.966, p < 0.001), which means the expression level of circ-ANAPC7 in bone marrow of AML has the ability to separate the patients with AML from healthy controls.

Spearman Correlation Analysis of Clinical Variables and Circ-ANAPC7 Expression in AML Patients
We performed Spearman correlation analysis to evaluate the correlation between clinical characteristics of AML patients and the expression of circ-ANAPC7, in order to estimate whether the significance and differential expression of circ-ANAPC7 in AML was relevant biomarkers for the diagnosis and monitoring of AML.
As shown in Table 2, the expression level of circ-ANAPC7 was correlated to the white blood cell (WBC) count of AML patients (r = 0.545, p < 0.01) and the percentage of blasts in bone marrow (r = 0.470, p < 0.05). Furthermore, the WBC count was correlated with risk

Potential Ability of Monitoring the Disease Condition of AML
To clarify whether the expression level of circ-ANAPC7 changes with disease status, we dynamically monitored the expression of circ-ANAPC7 in bone marrow samples of 24 AML patients at initial diagnosis, CR, and recurrence. The results revealed that circ-ANAPC7 was highly expressed in newly diagnosed AML, decreased when patients acquired CR, and increased again when disease re-lapsed (p < 0.05) (shown in Fig. 3a). In the continuous CR patients, the expression level of circ-ANAPC7 remained at a minimal level always (shown in Fig. 3b).

Long-Term Effect of Circ-ANAPC7 in AML
By multivariate analysis, WBC count ≥10 × 10 9 /L, blast percentages in the bone marrow, and refractory AML were significantly associated with inferior survival (Table 3). To explore the long-term effect of circ-ANAPC7 expression in AML patients, we conducted a survival analysis. As shown in Figure 4, we found that the expression level of circ-ANAPC7 was not related to the OS and DFS of AML patients (p > 0.05).  FAB, French-American-British classification system; BM, bone marrow; AML, acute myeloid leukemia; WBC, white blood cell; circ-ANAPC7, circular-anaphase promoting complex subunit 7. * p < 0.05. ** p < 0.01. Fig. 3. a, b The expression level of circ-ANAPC7 was measured in matched-pair samples acquired from 24 available follow-up AML patients at the time when they were at ND, CR, and R stages. ND, newly diagnosed; CR, complete remission; R, relapsed; AML, acute myeloid leukemia; circ-ANAPC7, circular-anaphase promoting complex subunit 7. DOI: 10.1159/000520446

Discussion
Recently, circRNA, a large family of noncoding RNAs, received extensive attention of researchers along with the development of transcriptome sequencing technologies. CircRNAs are alternative transcripts from exonic backsplicing of coding genes (exonic circRNAs) in mammalian cells, with abundant, stable, evolutionarily conserved, and cell-type-specific characteristics [19]. Some cir-cRNAs called miRNA sponges have miRNA response elements and display important miRNA activities, enhancing the complexity of RNA regulatory networks and being involved in gene expression [20,21].
A growing number of studies highlight the diagnostic and therapeutic potential of circRNAs in many types of cancers. CircRNA_100269 was downregulated in gastric cancer and suppresses tumor cell growth by targeting miR-630, which comprised a novel pathway that regulates the proliferation of gastric cancer cells [22]. Cir-cRNA_100782 regulated the proliferation of pancreatic carcinoma by acting as miR-124 sponge through the IL6-STAT3 pathway [23]. In nonsmall-cell lung cancer, hsa_ circ_0007385 knockdown resulted in significant suppression of the proliferation, migration, and invasion of NSCLC cells [24]. Microarray profiling and bioinformatics analyses were also performed patients with in esophageal cancer [25] and cardiac fibroblasts [26].
Furthermore, the expression profile of circRNAs in AML patients has been done in 1 study, and a large number of circRNAs possibly expressed in a leukemia-specific manner are identified, especially hsa_circ_0004277, which was down-expressed in AML patients compared with healthy controls [17]. Another study showed that hsa_circ_0075001 expression correlates positively with total NPM1 expression in AML but is independent of the NPM1 mutational status [16].
In the present study, we conformed that circ-ANAPC7 was significantly upregulated in AML patients, which was in line with the result before [18]. Furthermore, we found that the expression level of circ-ANAPC7 was related to the count of WBC and the percentage of blasts in bone marrow, but not the FAB type or risk status, indicating that the altered expression level of circ-ANAPC7 may be related to the pathogenesis of AML, but not the types or risk status of AML. Survival analysis predicted the expression level of circ-ANAPC7 had no influence on the OS and DFS; that is, it could not be used as a prognostic factor in AML. The count of WBC, blast percentage in bone marrow, and refractory AML were the independent factors for the survival of AML patients, which were consistent with the previous study [27,28]. ROC analysis stated that circ-ANAPC7 has a great potential diagnostic value for AML. Ambulatory monitoring of the expression of circ-ANAPC7 in matched-pair AML samples showed its change is accompanied with the disease condition transformation. Therefore, we speculate that the expression level of circ-ANAPC7 might be used as a predictive index for supervising recurrence, advising inchoate treatment to prolong OS of AML patients.
According to bioinformatic analysis in our previous research, we have predicted that circ-ANAPC7 may function as a sponge to adsorb miR-181 family [18]. MiR-181 has been proven to be upregulated in AML, which was related to longer OS of AML patients [29,30]. Furthermore, elevated miR-181 expression was associated with increased survival in 395 American patients, and reduced survival in 325 Chinese one, which indicated that miR-181 can be used as a promising prognostic biomarker in AML patients, depending on the origin of patient population [31]. In a subsequent study, we will devote ourselves to dig out the mechanism between circ-ANAPC7 and miR-181.
However, there are still some limitations in our study. First, the sample size of our study is still relatively small, and these data should be confirmed in large-scale studies and populations with different races and regions. Second, we do not have data in the circ-ANAPC7 expression level of plasma or serum from AML patients, which is our next research plan.
In summary, we validated that circ-ANAPC7 was upregulated in AML patients. The clinical analysis revealed that circ-ANAPC7 may be a predictive index for diagnosing and supervising recurrence of AML. What is more, additional molecular mechanisms and biological functions of circ-ANAPC7 merit deeper investigation.