The diagnostic and prognostic values of circulating miRNA-1246 in multiple myeloma

ABSTRACT Background/objective Bone marrow biopsy, the gold standard for the diagnosis of multiple myeloma (MM), has main limitation of the invasiveness. Here, we explored the diagnostic and prognostic values of circulating miR-1246 in patients with MM. Material and methods Ninety MM patients and 30 healthy donors (control group) were recruited in this study. The expression of miR-1246 in the peripheral blood samples was detected using qPCR. The receiver operating characteristic (ROC) curve was used to assess the diagnostic value of miR-1246 in MM. The Kaplan–Meier survival analyze was performed to evaluate the prognostic value of miR-1246. Results The expression level of serum miR-1246 from newly diagnosed MM patients was significantly higher than that of the control group. Circulating miR-1246 level was decreased after treatment in remission patients, but remained high levels in relapsed patients (P < 0.05). ROC analysis demonstrated that miR-1246 showed a high diagnostic value in MM with an area under the curve (AUC) of 0.952, the sensitivity of 87%, and the specificity of 95% [95% confidence interval (CI) 0.902-1.007; P < 0.001]. Kaplan–Meier analysis showed that the progression-free survival (PFS) (14.0 months vs. 26.5 months, P = 0.045) and overall survival (OS) (20.5 months vs. 55.5 months, P = 0.014) were significantly shorter in patients with high miR-1246 expression as compared with those in patients with miR-1246 low expression. Multiple Cox regression model analysis showed that circulating miR-1246 was an independent prognostic factor for PFS (HR 2.786, 95% CI: 1.420–5.467, P = 0.003) and OS (HR 2.995, 95% CI: 1.166–7.689, P = 0.023) in MM patients. Conclusion This study demonstrates that circulating miR-1246 level is elevated in MM patients, which shows high values in the diagnosis and prognosis prediction in patients with MM.


Introduction
Multiple myeloma (MM) is the second most prevalent hematologic malignancy, accounting for 1.3% of all malignancies and 15% of hematologic neoplasms, with an incidence of 4.5-6 cases per 100,000 people annually [1]. It is characterized by the monoclonal proliferation of plasma cells in bone marrow (BM) microenvironment, triggering the overproduction of nonfunctional intact immunoglobulins or immunoglobulin chains [2,3]. This disease is more common in adults over 40 years old, with a median age at diagnosis of 70 years and the 5-year survival of about 46.6% [4][5][6]. Bone marrow biopsy is the traditional gold standard for the diagnosis of MM, but it may not be accepted by a number of patients mainly because it is invasive method which can cause pain and thereafter bring psychological stress to the patients. Limitations in accessing fresh bone marrow material from MM prevent the diagnosis and delay the optimal treatment period. Thus, it is an urgent need to find a more sensitive, convenient, and noninvasive biomarker for the early clinical diagnosis of MM.
Liquid biopsy is a comprehensive approach as it is noninvasive, simple, more accessible, less painful and contains all the biologically relevant information about the disease. MicroRNA (miRNA) is a type of small and non-coding RNA molecules with about 19-25 bases in length and high stability in blood [7,8]. miRNAs are implicated in regulating of multiple biological processes, such as cell proliferation, survival, apoptosis, stemness, and cell cycle through inducing the degradation and translational repression of target mRNAs via complementary sequences [9][10][11]. Masri et al. [12] was the first group who revealed the involvement of miRNAs in MM pathogenesis, that the expression of miR-125b, miR-133a, miR-1, miR-124a, miR-15, and miR-16 were decreased in MM patients and cells. Soon afterwards, many miRNAs were verified to be closely involved in the pathogenesis of MM [13,14]. In addition, increasing evidence has demonstrated the potential diagnosis value of circulatory miRNAs in MM [15,16]. For instance, circulating miR-125b-5p level was elevated in MM patients, and the receiver operating characteristic (ROC) analysis demonstrated that miR-125b-5p displayed a high diagnostic accuracy for MM with an area under the curve (AUC) of 0.954 (P < 0.001) [17]. Also, Gupta et al. [17] reported miR-203 level was decreased in the blood samples of MM patients, which showed a high diagnostic value in MM. miR-1246 was first identified in human embryonic stem cells through a high throughput sequencing technique [18]. Subsequent studies have mapped the human miR-1246-coding gene, i.e. MIR1246 gene on chromosome 2q31.1 and demonstrated p53 effect on the modulation of its expression [19]. Recently, Ren et al. [20] demonstrated that miR-1246 expression was significantly increased in MM patients, which was independent of age, gender, stage, serum levels of β2-microglobulin (β2-MG), albumin, calcium, creatinine, myeloma protein, hemoglobin, as well as the population of bone marrow plasma cells and chromosome 13 deletion, indicating that miR-1246 may be a useful marker in the diagnosis of MM. In the present study, we aimed to determine the diagnostic and prognostic values of circulating miR-1246 in patients with MM.

Clinical information
A total of 90 serum samples were collected from 90 newly diagnosed MM patients without any treatment in the Department of Hematology, the Fourth Hospital of Hebei Medical University, between July 2013 and October 2020. Inclusion criteria were: [1] patients with a precise diagnosis of primary MM according to the 2015 International Myeloma Working Group guidelines; [2] the age of patients ≥ 18 years; [3] patients with no treatment before blood sample collection; [4] patients signed the informed contents. Exclusion criteria: [1] patients with MM developing into plasma cell leukemia; [2] patients accompanied by other malignancies or organ failures; [3] patients suffered from immune system defects, or other hematologic diseases; [4] patients treated prior to the blood sample collection. Disease remission and relapse were defined according to the International Myeloma Working Group criteria [21]. Another 30 healthy donors with no family history of hematological diseases were included in this study as control. This study got the approval of the Medical Ethics Committee of the Fourth Hospital of Hebei Medical University to use the clinical material for research purposes.

Serum collection and miRNA extraction
Five ml peripheral venous blood samples from 90 MM and 30 healthy controls were placed in EDTA (Ethylene Diamine Tetraacetic Acid) anticoagulation test tubes. RNA was extracted using the miRNeasy serum/ plasma separation kit provided by Qiagen (Hilden, Germany) according to the manufacturer's instructions. Then, the NanoDrop spectrophotometer (ND-2000, Thermo Fisher Scientific, Waltham, MA) was used to assess the concentration and purity of the isolated RNAs. RNA samples were stored at −80°C for further analysis.

microRNA qPCR analysis
The miRcute Plus miRNA first-strand cDNA synthesis kit (Tiangen Co., Beijing, China) was applied to synthesize the cDNA of miR-1246. Then, the PCR was carried out using the using TaqMan miRNA Assay Kits (Applied Biosystems, Foster, CA, USA) to specially quantify mature miR-1246 according to manufacturer's instructions. The PCR conditions were as follows: pre-denaturation at 95°C for 15 min, and then 45 cycles of 94°C (20 s) and 60°C (34 s) using ABI 9700 real-time PCR system (Applied Biosystems). U6 was used as an internal control. Relative miR-1246 expression levels were determined by the 2 −ΔΔ(CT) method [22]. The primers for miR-1246 forward was 5 ′ -AAUG-GAUUUUUGGAGCAGG-3 ′ .

Statistical analysis
All statistical analyses were performed using the SPSS 20 software package. The student's t and one-way ANOVA tests were used to analyze the relationships between clinical characteristics and miR-1246 expression levels. ROC analysis was used to evaluate the diagnostic value of miR-1246 in MM. Patient survival curves were plotted using the Kaplan-Meier method, and comparisons between curves were made using the log-rank test. Multifactorial survival analysis was performed using the Cox proportional risk model. Progression-free survival (PFS) was calculated from the date of diagnosis to disease progression or death, and overall survival (OS) was calculated from the date of diagnosis to death. P < 0.05 or P < 0.01 showed that the differences were statistically significant.  Table 1. The patients were followed up for 3.0-79.2 months, with a median follow-up time of 45.5 months. Eight patients (8.9%) died during the study, among which 2 patients died of primary disease progression, 4 patients died of severe infection, and 2 patients died of multiple organ dysfunction syndrome (MODS). In addition, 2 patients (2.2%) were lost to follow-up.

Characteristics of the patients
miR-1246 expression levels were increased in the peripheric blood samples of MM patients To reveal the diagnostic and prognostic value of miR-1246 in MM, we first compared the expression levels of miR-1246 in the peripheral blood from MM patients and healthy donors. As shown in Figure 1, miR-1246 levels were significantly higher in patients with newly diagnosed MM compared with those of the healthy donors (P < 0.05). In addition, we found that miR-1246 expression levels in MM patients in remission after chemotherapy were decreased as compared with the primary MM samples, which were close to those in normal controls, while miR-1246 levels in relapse samples were elevated which were similar to the levels at the time of new diagnosis (P < 0.05). These results indicated that miR-1246 level was increased in MM patients, which was also associated with the curative effect.

Correlation between miR-1246 expression and the clinical characteristics of MM patients
We then evaluated the correlation between the expression levels of miR-1246 and the clinical characteristics of MM patients. As shown in Table 2, miR-1246 level was significantly increased in patients with extramedullary infiltration (P < 0.05), while showed no obvious difference in patients with different ages, gender, R-ISS stages, light chain types, heavy chain types, renal injury, LDH (lactate dehydrogenase) levels, albumin levels, β2-MG levels, and M protein levels (P all >0.05). These results indicated that the blood levels of miR-1246 were significantly increased in MM patients with extramedullary infiltration, and were independent from age, gender, R-ISS stage, light chain type, heavy chain type, renal injury, and the levels of LDH, albumin, β2-MG and M protein.

Diagnostic value of peripheral blood miR-1246 in MM
To clarify the diagnostic value of circulating miR-1246 in MM patients, the ROC curve was made. As shown in Figure 2, miR-1246 could distinguish MM patients from healthy controls with an AUC of 0.952, the sensitivity of 87%, and the specificity of 95% [95% confidence interval (CI), 0.902-1.007; P < 0.001]. These results demonstrated a high value of circulating miR-1246 in the diagnosis of MM.

High expression of miR-1246 in peripheral blood indicated worse prognosis in MM patients
Moreover, we assessed the relationship between miR-1246 expression and the prognosis of MM patients. The univariate analysis results showed that the aged > 60 years, R-ISS stage III, two-drug regimen chemotherapy, and extramedullary infiltration were the impact factors of both PFS and OS (P < 0.05 or P < 0.001,  Figure 3). In addition, the multivariate Cox regression model analysis showed that peripheral blood miR-1246 was an independent prognostic factor for PFS (HR 2.786, 95% CI: 1.420-5.467, P = 0.003) and OS (HR 2.995, 95% CI: 1.166-7.689, P = 0.023) in MM patients, as well as R-ISS, extramedullary infiltration also had an independent prognostic impact on PFS and OS (Table 4). These above results demonstrated that miR-1246 level was an independent prognostic factor of the prognosis in MM.

Discussion
Circulating miRNA has been proposed as an attractive diagnostic marker in cancer with high specificity and sensitivity [15,23]. Numerous studies have evaluated the potential of circulating miRNAs as diagnostic biomarkers in various cancers, including breast cancer, gastric cancer, hepatocellular carcinoma, and nonsmall cell lung cancer [24,25], as well as MM [16]. Ren et al. [20] previously demonstrated that miR-1246 expression was significantly increased in MM patients. The current study further explored its value in the diagnosis of MM and the results revealed a high value of the circulating miR-1246 as a blood marker for the diagnosis of MM.
It has been demonstrated that circulating miR-1246 is a potential marker for the diagnosis of different kinds of cancers. The sensitivity of miR-1246 for the early diagnosis of breast tumors (stage I-IIa) was 80% [26]. Circulating exosomes miR-1246 was also a potential biomarker for the early diagnosis of gastric cancer (GC), which was upregulated in the    [27]. Chuma et al. [28] reported that serum miR-1246 was upregulated in hepatocellular carcinoma (HCC) patients with early recurrence, which showed a ROC curve area of 0.762, with a specificity of 77.4% and a sensitivity of 54.1% in discriminating HCC patients with early recurrence from HCC patients without early recurrence. All of the above studies revealed a higher expression pattern of miR-1246 in cancers, but Yang et al. [29] found that miR-1246 level was dramatically decreased in cervical cancer tissues, and miR-1246 level was negatively correlated with clinical stage and HPV16E6 infected status. It cannot be ruled out the difference may be caused by the various sample types. Ren et al. [20] demonstrated that miR-1246 expression was significantly increased in MM patients, which was independent from age, gender, stage, serum levels of β2-MG, albumin, calcium, creatinine, myeloma protein, hemoglobin, and the population of bone marrow plasma cells and chromosome 13 deletion, indicating that miR-1246 may be a useful marker in the diagnosis of MM. Consistently, we found that miR-1246 level was increased in the blood samples of MM as compared with the healthy individuals. In addition, circuiting miR-1246 showed a high value in distinguish MM from healthy individuals (AUC, 0.952; sensitivity, 87%; specificity, 95%).
The Extra-medullary disease (EMD) in MM is associated with poor prognosis and resistance to chemotherapy [30]. Here, we found that miR-1246 expression was significantly increased in MM patients with extramedullary infiltration, indicating a crucial value of miR-1246 in predicting the extramedullary infiltration of MM patients. Evidence has shown that miR-1246 can target CXC motif chemokine receptor type 4 (CXCR4) to inhibit cell migration and invasion in renal cell carcinoma [31] and lung cancer [32]. CXCR4 is a G proteincoupled receptor that also has been verified to be accelerated dissemination from the bone marrow [33], thus we speculate miR-1246 may promote the extramedullary infiltration of MM by targeting CXCR4.
In addition, miR-1246 has been reported to be linked to the drug resistance of cancers. High metastatic tumors induce drug resistance of tumor endothelial cells by transporting miR-1246 [34]. Targeting the miR-1246-AXIN2/GSK-3β-Wnt/β-catenin axis may help overcome chemotherapy resistance in patients with relapsed refractory leukemia [35]. Also, the expression of cerebrospinal fluid exosome miR-1246 after glioma surgery correlates with relapse rate [36]. In this study, we found that the expression level of miR-1246 in the remission group after chemotherapy was close to that of the normal control group, while increased to the level at the time of new diagnosis when recurrence. This finding suggested that the high expression of miR-1246 in MM patients may be related to chemotherapy resistance and relapse, indicating that the circulating miR-1246 level may be used to predict disease recurrence.  Also, circulating miR-1246 level is a prognosis marker of cancers. High levels of miR-1246 predicted adverse event-free survival (EFS) in patients with early breast cancer and poor PFS in patients with metastases [37]. The increased expression of miR-1246 in peripheral blood samples was associated with shorter PFS in patients with MM [20]. In this study, both of univariate and multivariate analysis showed that higher level of miR-1246 was an adverse prognostic factor for both PFS and OS.
Several limitations should be clarified in this study. As mentioned above, serum miR-1246 level was increased in several kinds of cancers and diseases, including MM, challenging only miR-1246 level to distinguish different kinds of cancers. A model should be constructed with combination of miR-1246 and clinical manifestation/information to distinguish different kinds of cancers. In addition, we didn't compare the expression levels of miR-1246 in MM and its precursor conditions, undetermined significance (MGUS) and smoldering multiple myeloma (SMM). This is meaningful and we intend to include patients with MGUS and SMM for further identification. Moreover, we didn't reveal the relationship between extramedullary disease and the amount of plasma cells, as well as the role and underlying mechanisms, such as the target genes (e.g. CXCR4), of miR-1246 in the pathogenesis of MM. Further studies focus on these issues are required in future.

Conclusion
Collectively, this study demonstrates that circulating miR-1246 level is elevated in MM patients, which can be used as a biomarker for MM diagnosis and prognosis prediction. Overall, miR-1246 is a potential liquid biopsy marker to identify MM patients with poor prognosis and help to select better combination therapy in time.

Ethics statement
The study involving human participants was approved by the Ethic Committee of the Forth Hospital of Hebei Medical University. The patients/participants provided their written informed consent to participate in this study. This research was conducted in accordance with the principles expressed in the Declaration of Helsinki.