Diagnostic and prognostic significance of miRNA-511 and miRNA-16 expressions in adult patients with B-acute lymphoblastic leukemia

Manal El Sorady; Abeer Elhadidi; Omar Gallab; Nermeen Eldabah; Mohamed Halaby Elbahoty

doi:10.4103/ejh.ejh_41_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 47 | Issue : 3 | Page : 204-209

Diagnostic and prognostic significance of miRNA-511 and miRNA-16 expressions in adult patients with B-acute lymphoblastic leukemia

Manal El Sorady¹, Abeer Elhadidi², Omar Gallab¹, Nermeen Eldabah², Mohamed Halaby Elbahoty¹
¹ Department of Internal Medicine, Alexandria University, Alexandria, Egypt
² Department of Clinical and Chemical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission	10-Apr-2021
Date of Acceptance	05-May-2021
Date of Web Publication	03-Jan-2023

Correspondence Address:
Mohamed Halaby Elbahoty
Hematology Unit, Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_41_21

Abstract

Background Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations in a single lymphoid progenitor cell. MicroRNAs (miRNAs) are critical regulators of gene expression, tumor suppression, and oncogenesis. Aim To evaluate miRNA-511 and miRNA-16 expression in Egyptian adult patients with B-ALL. Patients and methods A total of 37 newly diagnosed adult patients with B-ALL admitted to Alexandria Main University Hospital in 2019 were included. Complete blood count, bone marrow aspiration, immunophenotyping, BCR-ABL testing, karyotyping, miRNA extraction using miRNeasy Mini followed by cDNA synthesis AQ6 (RQ-PCR combines cDNA synthesis from RNA templates using miScript II RT kit), and finally real-time PCR for miRNA-511 and miRNA-16 expression were among the investigations that were conducted. Results Mean age of patients with ALL was 30.65 ± 10.39 years, with male to female ratio of 1.4 : 1. Cytogenetic findings showed that only three patients had favorable risk, and the rest were either intermediate risk (19) or high risk (15). Among the high-risk group, there were 11 patients with Philadelphia chromosome (BCR-ABL 190) positive. Regarding the expression of miRNAs, most patients showed overexpression of both miRNA-16 and miRNA-511. MiRNA-511 was overexpressed in 81.1% (30) patients; among these patients, 43.3% (13) had adverse cytogenetic findings. MiRNA-16 was overexpressed in 70.3% (26) of patients, and half of them (13) had adverse cytogenetic findings. receiver operating characteristic curves showed diagnostic significance in B-ALL for miRNA-16, with sensitivity of 75.7% and specificity of 80%, and for miRNA-511, sensitivity was 89.2% and specificity was 90% (P<0.05). Conclusion MiRNA-16 and miRNA-511 were significantly overexpressed in adult patients with B-ALL. They have a role in diagnosis but a weak role in patient prognosis.

Keywords: acute lymphoblastic leukemia, microRNA, microRNA-16, microRNA-511

How to cite this article:
El Sorady M, Elhadidi A, Gallab O, Eldabah N, Elbahoty MH. Diagnostic and prognostic significance of miRNA-511 and miRNA-16 expressions in adult patients with B-acute lymphoblastic leukemia. Egypt J Haematol 2022;47:204-9

How to cite this URL:
El Sorady M, Elhadidi A, Gallab O, Eldabah N, Elbahoty MH. Diagnostic and prognostic significance of miRNA-511 and miRNA-16 expressions in adult patients with B-acute lymphoblastic leukemia. Egypt J Haematol [serial online] 2022 [cited 2023 Mar 30];47:204-9. Available from: http://www.ehj.eg.net/text.asp?2022/47/3/204/366865

Introduction

Acute lymphoblastic leukemia (ALL) is a malignant hematological disease that results from multistep somatic mutations in a single lymphoid progenitor, leading to bone marrow failure [1]. Despite the majority of patients with ALL enter in remission following induction therapy, a high relapse rate occurs [2]. Better understanding of the signaling pathways in leukemogenesis will help in subclassification of the disease [2,3]. MicroRNAs (miRNAs) are an abundant class of small, noncoding, single-stranded RNAs of ~22 nucleotides, of which many are evolutionarily conserved [4]. They are critical regulators of gene expression. They have a significant role in leukemia development and tumorigenesis through overexpression of ‘oncomiRs’ or genetic loss of tumor-suppressor miRNAs.

Aim

The aim was to evaluate miRNA-511 and miRNA-16 expression, correlate both miRNAs expression with poor prognostic factors, and assess the treatment outcome in relation to expression of both miRNAs.

Patients and methods

A case–control study design was used. A total of 37 newly diagnosed adult patients with B-ALL admitted to Alexandria Main University Hospital Internal Medicine Department were enrolled in the study and compared with 10 healthy controls. Informed consent was obtained from all participants in the study and all procedures were approved by Faculty of Medicine Alexandria University ETHICS COMMITTEE IRB NO: 00012098 & FWA NO: 00018699. Full history taking and physical examination were done. Routine investigations were performed including complete blood count, bone marrow aspiration, flow cytometry, and BCR-ABL PCR testing. Karyotyping analysis of a minimum of 20 metaphases for each case was performed using software CytoVision, version 7.5 (Applied Imaging, Agilent, UK). MiRNA extraction was done from peripheral blood or bone marrow mononuclear cells using miRNeasy Mini Kit (QIAGEN, cat. no. 217004, Maryland, USA). RQ-PCR combines cDNA synthesis from RNA templates with PCR to provide a rapid, sensitive method for analyzing gene expression. cDNA synthesis was performed using miScript II RT Kit (QIAGEN, cat. no. 218161) for miRNA reverse transcription according to the manufacturer’s protocol. Afterward, strata gene MX3000P real-time PCR system (Agilent, Germany) was performed to assess the expression of miRNA-511 and miRNA-16 using miScript (SYBER Green PCR Kit, cat. number 218073, QIAGEN Sciences Inc, 19300 Germantown Rd, Germantown, MD 20874) and miScript Primer Assay for miRNA-511-5P, cat. no. MS00006993 and miRNA-16-1-3P, cat. no. MS00008806. The relative quantification of miRNAs was expressed as relative gene expression, using the comparative CT method (Delta–delta Ct method), which was normalized with respect to SNORD 68 (cat. no. MS003712). Close follow-up was done for a period of 1 year for patients during the course of their chemotherapy and were followed up regarding outcome, infections, and mortality.

Written consent form was obtained from each patient after explaining the purpose of the study.

Results

Mean age of patients with B-ALL was 30.65 ± 10.39 years, with male to female ratio of 1.4 : 1. Patients were categorized according to the risk stratification by cytogenetics and Ph chromosome status. Common presenting symptoms and signs among patients were anemia, hepatosplenomegaly, lymphadenopathy, and fever. As for complete blood count, all patients were anemic with thrombocytopenia and leukocytosis. Cytogenetic findings showed only 8.1% (three) of the patients had favorable risk (hyperdiploidy), and the rest were between intermediate risk (51.3%) (19) and high risk (40.5%) (15). Overall, 73% (11) of the patients in the high-risk group were Philadelphia (BCR-ABL 190) positive. MiRNA-511 was underexpressed in only two (5.4%) patients and overexpressed in 30 (81.1%) patients; among them, 43.3% (13) had adverse cytogenetic findings. Meanwhile, miRNA-16 was underexpressed in five (13.5%) patients and overexpressed in 70.3% (26) patients; half of them (13) had adverse cytogenetic findings. Most of the patients completed chemotherapy, except 18.9% (seven), who died early within the follow-up; all these patients had overexpression of miRNA-511, but five were overexpressed for miRNA-16, and the other two patients were normal. Among those who received treatment, 43.2% (16) were in complete remission and 37.8% (14) were either refractory or relapsed during the course of treatment. Receiver operating characteristic (ROC) curves showed statistical significance for both miRNA-16 and miRNA-511 in diagnosis of B-ALL, with miRNA-16 having sensitivity of 75.7% and specificity of 80%, whereas miRNA-511 had sensitivity of 89.2% and specificity of 90% (P<0.05). Regarding survival analysis, there was no statistically significant difference between those who were under, normal, or overexpressed for miRNA-511 and miRNA-16 (P value for Log rank=0.229 and 0.981, respectively). Among the studied group, nine (24%) patients developed COVID-19 viral infection, and of those, three (33%) patients died, forming 21% of all deaths. The three patients were either refractory or relapsed and treated mostly as palliative cases ([Table 1][Table 2][Table 3]).

Table 1: Demographic characteristics and cytogenetic results of studied patients.

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Table 2: Association between risk stratification with microRNA-16 and microRNA-511 and BCR-ABL transcript status among B-acute lymphoblastic leukemia patients

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Table 3: Association between white blood cells count with microRNAs expression among studied B-acute lymphoblastic leukemia patients

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Area under curve=90.5%, P value less than 0.001 ([Figure 1]).

Figure 1: ROC curve showing diagnostic ability of microRNA-511. ROC, receiver operating characteristic.

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At cutoff point of 1.387, sensitivity was 89.2% and specificity was 90%.

Area under curve=77.8%, P value of 0.007 ([Figure 2]).

Figure 2: ROC curve showing diagnostic ability of microRNA-16. ROC, receiver operating characteristic.

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At cutoff point of 1.205, sensitivity was 75.7% and specificity was 80%.

Discussion

More than 1000 miRNAs are present in the human genome. They are the key molecular components, targeting most protein-coding transcripts in nearly all physiological and pathological processes [5]. Since 1993, investigators are interested to study the role of miRNAs in the pathogenesis of various diseases. There is a lot of promise for using miRNAs as prognostic and diagnostic biomarkers because of variations in miRNA expression profiles.

The most abundant miRNAs in most of the cells is miR16, which seems to play a key role in cell proliferation and apoptosis [6]. We assessed the miRNA-16 expression in 37 adult patients with B-ALL. It was overexpressed in most cases compared with control group like previous studies [7].

ROC curve showed statistical significance for miRNA-16 in diagnosis of B-ALL, with sensitivity of 75.7% and specificity of 80% (P<0.05). Our results showed wide variation among patients, and this was in co-ordinance with Kaddar et al. [8], who evaluated the miRNA-16 expression in 93 children having ALL. They found no specific expression profile for miRNA-16 in ALL cases and 18 lymphoid cell lines. Moreover, B-ALL showed more diversity in results than T-ALL. Our results showed no statistical correlation for miRNA-16 expression with other prognostic factors, such as cytogenetic stratification and leukocytosis (P=0.174 and 0.491, respectively). These findings were similar to Kaddar and colleagues (P=0.079 and 0.06, respectively) in B-ALL group but was significant in total ALL group. It was shown in our study that there was no significant association between survival analysis and expression of miRNA-16; however, in Kaddar et al. [8], disease-free survival (DFS) study revealed that patients with B-ALL had significantly different DFS between groups defined by quartiles (log-rank test, P=0.038). Very few relapses were observed for patients with low miRNA-16 levels, and the shortest DFS was found for miRNA-16 high expression above quartile 75. This could point that miRNA-16 might be an independent prognostic marker. However, the overall survival rate in the entire ALL group and in B-cell ALLs showed no significant differences [8], like results of the study by Nair et al. [9].

Accordingly, the high expression of miRNA-16 was associated with poor prognosis in B-ALL. This is in contrast to the studies conducted on chronic B-cell neoplasms. Tuncer et al. [10] reported that underexpression of miRNA16-1 was observed in 88.3% of the patients with DLBCL. Moreover, it was significantly decreased in CLL [6]. It was reported that the low level of miRNA-16 in CLL was correlated with the high level of the anti-apoptotic protein BCL-2 expression [10]. MiRNA-16 has been reported to act as a tumor-suppressive miRNA in many cancer types, and multiple apoptosis-related genes are targeted by miRNA-16, including BCL-2, cyclin D1, and cyclin E [11]. The loss of miRNA-16 and overexpression of its targets can lead to acute myeloid leukemia and more infrequently to B-cell lymphoma [12].

There are other nonhematological malignancies that had low miRNA-16 expression, such as nonsmall cell lung carcinoma, liver cancer, breast cancer, ovarian cancer, prostate cancer, stomach cancer, pituitary adenoma, and osteosarcoma. Ectopic expression of miRNA-16 targets BCL-2-induced apoptosis in some cell lines and suppressed tumorigenesis in animal models of leukemia and solid tumors [6]. Furthermore, no correlation was found in ALL between miR-16 expression levels and the expression of its targets (BCL-2, CDK6, cyclin E and D1) [8]. However, the high expression of miRNA-16 is association with poor prognosis in B-ALL cases. This suggests that miRNA-16 may behave as tumor suppressors and could trigger apoptosis [13]. It regulates several genes expression such as MAP7 and PRDM4 at both the transcript and protein levels. Therefore, expression of MAP7 protein was significantly abolished by pre-miRNA-16, thus inhibiting cell proliferation; meanwhile, anti-miRNA-16 significantly increased the expression levels of MAP7 protein in cells leading to stimulation of cell proliferation [14].

On the contrary, miRNA-16 targets many functions synergistically. MiR-16 family triggers an accumulation of cells in G0/G1 by silencing multiple cell cycle genes.e expression of Cyclin D1 (CCND1) was regulated by miR-16 family directly, and miR-16 induced G1 arrest in A549 cells partially by CCND1. Furthermore, several other cell cycle genes were revealed to be regulated by miR-16 family, including Cyclin D3 (CCND3), Cyclin E1(CCNE1) and CDK6. Coordinate regulation of these and other cell cycle regulators suggests highly orchestrated cell cycle control by the miRNA-16 family of miRNAs [15]. The definite role of miRNA-16 in the pathophysiology of B-ALL is still mysterious. The deregulation of miRNAs might be due to several factors such as genetic alterations either chromosomal or epigenetic changes and interaction of the different genes. There is also the influence of interleukins and immunoglobulins on miRNAs deregulation of miRNAs [16].

As for miRNA-511, ROC curve of the current study showed statistical significance for miRNA-511 in diagnosis of B-ALL with 89.2% sensitivity and 90% specificity (P<0.05). Similarly, Luna-Aguirre et al. [7] assessed 77 circulating miRNA expressions in B-ALL and found that hsa-miR-511 was overexpressed. Their miRNA-511 ROC analysis showed that in plasma at ΔCt cutoff of 9.458, it distinguished B-ALL from normal controls, with an area under the curve value of 1, and 100% of sensitivity and specificity respectively. In addition, the study by Nair et al. [9] conducted on patients with Ph-negative ALL reported that miRNA-511 was upregulated in B-ALL BM samples but downregulated in T-ALL BM samples. These data emphasized the role of miRNA-511 as diagnostic biomarker for B-ALL with high sensitivity and specificity. Regarding the correlation for miRNA-511 expression with other prognostic factors, such as cytogenetic stratification and leukocytosis, no statistical significance was reported in our study (P=0.729 and 1, respectively).

In our study, there was no significant association between survival analysis and expression of miRNA-511. On the contrary, Cox proportional hazard regression analysis was carried by Nair et al. [9] on 100 ALL samples for correlating the miRNAs expression signatures with patients’ survival, and they showed that of 397 miRNAs identified with ALL, only 11 of them were significantly correlated with the overall survival, not including miRNA-511.

Previous studies declared that miRNA-511 had possible anti-leukemic roles via anti-angiogenesis, anti-proliferative, and pro-differentiation effects [17]. This is in contrast to other studies describing the miRNA-511 as a novel potent modulator of immune response. The more overexpressed miRNA-511 decreases antigen presentation through Toll-like receptor 4 of dendritic cells, which reduces Dendritic Cell-SIGN level. This leads to suppression of leukemic cells recognition escaping antibody and T-cell-mediated immunity. More studies are needed for the functional role of miRNA-511 in B-ALL and to evaluate its use as indicator of therapy, and in the assessment of biological and/or therapeutic targets for patients with B-ALL [18].

Few cases of ALL with COVID-19 have been reported during the pandemic, and the course has been mild [19]. There is little experience in patients with newly diagnosed ALL. Providers may have concern about initiating multiagent chemotherapy in these patients, particularly corticosteroids, which are an essential part of induction regimens [20].

In the era of COVID 19, among the studied group, nine (24%) patients developed COVID-19 disease, where diagnosis was confirmed by symptoms, chest computed tomography findings, and positive PCR swabs. These patients were managed in hospitals according to the Egyptian guidelines that include symptomatic treatment and oxygen support, chemotherapy was stopped, high risk patients were anticoagulated, and broad-spectrum antibiotics with sometimes antiviral including Tamiflu and remdesivir were used. Foà et al. [19] stated that it becomes increasingly important that patients with Ph+ALL are identified at diagnosis and advised for a chemotherapy-free induction. Dasatinib (and steroids) alone achieved 20–25% of patients can become molecularly negative.

COVID-19’s mortality rate is difficult to quantify as it is variable. Mortality rises among older people. The effect of underlying immunosuppression is controversial. Initial reports from China suggested that patients with cancer had a higher risk of severe events as compared with patients without cancer (39 vs. 8%). Other data, based on posttransplantation cohorts, showed that unlike common viruses, coronaviruses do not cause more severe disease in immunosuppressed patients as the host innate immune response appears to be the main driver of lung tissue damage during infection [21].

Conclusion

Specific miRNAs like miRNA-511 and miRNA-16 offer acceptable level of sensitivity and specificity in B-ALL diagnosis. Despite, they were insignificant in identifying disease progression and outcomes of B-ALL patients. Both were significantly overexpressed in adult B-ALL, and also both miRNAs have strong positive statistically significant correlation. Cytogenetic and BCR-ABL were significantly associated with risk stratification. Although both miRNAs have diagnostic ability for B-ALL, we cannot depend on them as they are very expensive in developing countries like Egypt.

The main limitation of the study was the small sample size, which leads to increased type II error, so further studies with larger sample is required to study prognostic abilities of both miRNAs and their effect on survival.

Financial support and sponsorship

Nil.

Conflicts of interest

The author has no conflicts of interest to declare.

References

1.	Larson RA Acute Lymphoblastic Leukemia. In: Kaushansky K, Lichtman MA, Prchal JT, Levi MM, Press OW, Burns LJ, et al., editors. Williams Hematology, 9e. New York, NY: McGraw-Hill Education; 2015.
2.	Schrappe M, Hunger SP, Pui CH, Saha V, Gaynon PS, Baruchel A, et al. Outcomes after induction failure in childhood acute lymphoblastic leukemia. N Engl J Med. 2012;366(15):1371–81. doi: 10.1056/NEJMoa1110169. PMID: 22494120; PMCID: PMC3374496.
3.	Chiarini F, Lonetti A, Evangelisti C, Buontempo F, Orsini E, Evangelisti C, et al. Advances in understanding the acute lymphoblastic leukemia bone marrow microenvironment: From biology to therapeutic targeting. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2016;1863(3):449.
4.	Xiong J Emerging roles of microRNA-22 in human disease and normal physiology. Curr Mol Med 2012; 12:247–258.
5.	Ha M, Kim VN Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 2014; 15:509.
6.	Ramaiah MJ Functions and epigenetic aspects of miR-15/16: Possible future cancer therapeutics. Gene Rep 2018; 6:12.
7.	Luna-Aguirre CM, de la Luz Martinez-Fierro M, Mar-Aguilar F, Garza-Veloz I, Trevino-Alvarado V, Rojas-Martinez A, et al. Circulating microRNA expression profile in B-cell acute lymphoblastic leukemia. Cancer Biomark 2015; 15:299–310.
8.	Kaddar T, Chien WW, Bertrand Y, Pages MP, Rouault JP, Salles G, et al. Prognostic value of miR-16 expression in childhood acute lymphoblastic leukemia relationships to normal and malignant lymphocyte proliferation. Leuk Res 2009; 33:1217–1223.
9.	Nair RA, Verma VK, Beevi SS, Rawoof A, Alexander LE, Prasad ER, et al. MicroRNA signatures in blood or bone marrow distinguish subtypes of pediatric acute lymphoblastic leukemia. Transl Oncol 2020; 13:100800.
10.	Tuncer SB, Akdeniz D, Celik B, Kilic S, Sukruoglu O, Avsar M, et al. The expression levels of miRNA-15a and miRNA- 16–1 in circulating tumor cells of patients with diffuse large B-cell lymphoma. Mol Biol Rep 2019; 46:975–980.
11.	Liang H, Fu Z, Jiang X, Wang N, Wang F, Wang X, et al. miR-16 promotes the apoptosis of human cancer cells by targeting FEAT. BMC Cancer 2015; 15:448.
12.	Lovat F, Fassan M, Sacchi D, Ranganathan P, Palamarchuk A, Bill M, et al. Knockout of both miR-15/16 loci induces acute myeloid leukemia. Proc Natl Acad Sci 2018; 115:13069.
13.	Coronado RE Chapter 15-therapeutic effect of extracellular vesicles derived from adult/perinatal human mesenchymal stem cells. In: Atala A, Cetrulo KJ, Taghizadeh RR, Murphy SV, Cetrulo CL, editors. Perinatal stem cells. Boston: Academic Press 2018. p. 201–215.
14.	Yan X, Liang H, Deng T, Zhu K, Zhang S, Wang N, et al. The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells. Mol Cancer 2013; 12:92.
15.	Linsley PS, Schelter J, Burchard J, Kibukawa M, Martin MM, Bartz SR, et al. Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression. Mol Cell Biol 2007; 27:2240.
16.	Braga TV, Evangelista FCG, Gomes LC, Araújo SSDS, Carvalho MDG, Sabino ADP Evaluation of MiR-15a and MiR-16-1 as prognostic biomarkers in chronic lymphocytic leukemia. Biomed Pharmacother 2017; 92:864–869.
17.	Fleischmann KK, Pagel P, von Frowein J, Magg T, Roscher AA, Schmid I The leukemogenic fusion gene MLL-AF9 alters microRNA expression pattern and inhibits monoblastic differentiation via miR-511 repression. J Exp Clin Cancer Res 2016; 35:9.
18.	Tserel L, Runnel T, Kisand K, Pihlap M, Bakhoff L, Kolde R, et al. MicroRNA expression profiles of human blood monocyte-derived dendritic cells and macrophages reveal miR-511 as putative positive regulator of toll-like receptor 4. J Biol Chem 2011; 286:26487–26495.
19.	Foà R, Vitale A, Vignetti M, Meloni G, Guarini A, De Propris MS, et al. Dasatinib as first-line treatment for adult patients with Philadelphia chromosome–positive acute lymphoblastic leukemia. Blood. 2011;118(25):6521-8.
20.	Phillips L, Pavisic J, Kaur D, Dorrello NV, Broglie L, Hijiya N Successful management of SARS-CoV-2 acute respiratory distress syndrome and newly diagnosed acute lymphoblastic leukemia. Blood Adv 2020; 4:4358–4361.
21.	Gavillet M, Carr Klappert J, Spertini O, Blum S Acute leukemia in the time of COVID-19. Leuk Res 2020; 92:106353.