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ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 4  |  Page : 1238-1243

Expression signature of long noncoding RNA growth-arrest-specific 5 during induction therapy in adult B-cell acute lymphoblastic leukemia


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Medical Oncology, National Cancer Institute, Cairo University, Cairo, Egypt

Date of Submission02-Sep-2021
Date of Decision13-Oct-2021
Date of Acceptance18-Oct-2021
Date of Web Publication24-Dec-2021

Correspondence Address:
Hanan H Elsheity
Msc, Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_158_21

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  Abstract 


Background
Several studies have highlighted long noncoding RNA growth-arrest-specific 5 (GAS5) as a key player in various normal and pathological conditions. Overall, GAS5 acts as a tumor suppressor, whose downregulation is directly connected to tumor proliferation, tumor progression, and therapy-related resistance across different types of tumors. To date, its importance in acute leukemia has not been widely investigated.
Objectives
To investigate the changes in the expression levels of long noncoding RNA GAS5 during induction chemotherapy of adult patients with B-cell acute lymphoblastic leukemia (B-ALL).
Patients and methods
Peripheral blood samples were obtained before starting therapy from 35 adult patients with B-ALL and then on day 15 and day 33 of induction therapy. Twenty control samples were collected from healthy donors. GAS5 expression was estimated using reverse-transcription quantitative PCR technique.
Results
Pretreatment GAS5 expression of B-ALL patients was significantly decreased when compared with that of healthy controls (P = 0.002). Analysis demonstrated that GAS5 expression was significantly increased on day 15 as compared with its level at diagnosis (P < 0.001). On day 33, GAS5 expression was significantly increased in comparison with its level on day 15 (P < 0.001). Moreover, our results showed that low GAS5-expression levels at diagnosis were significantly associated with initial leukocyte count more than 30 × 103/μl and BCR–ABL1-positive B-ALL (P = 0.01, P = 0.015, respectively).
Conclusions
We reported a downregulation of circulating GAS5 in B-ALL patients, which showed elevation after initiation of induction therapy. Downregulation of GAS5 was associated with unfavorable prognostic factors, denoting its prognostic potential in B-ALL.

Keywords: B-cell acute lymphoblastic leukemia, induction chemotherapy, long noncoding RNA growth-arrest-specific 5, reverse-transcription quantitative PCR


How to cite this article:
Khalifa KA, Abdlfattah RM, Elsheity HH, Shehata AM. Expression signature of long noncoding RNA growth-arrest-specific 5 during induction therapy in adult B-cell acute lymphoblastic leukemia. Menoufia Med J 2021;34:1238-43

How to cite this URL:
Khalifa KA, Abdlfattah RM, Elsheity HH, Shehata AM. Expression signature of long noncoding RNA growth-arrest-specific 5 during induction therapy in adult B-cell acute lymphoblastic leukemia. Menoufia Med J [serial online] 2021 [cited 2024 Mar 29];34:1238-43. Available from: http://www.mmj.eg.net/text.asp?2021/34/4/1238/333227




  Introduction Top


Acute lymphoblastic leukemia (ALL) is a heterogeneous hematological malignancy that originates from lymphoid precursors, more often from B-cell or T-cell lineages than from a natural-killer cell precursor. ALL shows a bimodal incidence, with a peak in childhood and another in older adults; nearly 54.2% of patients are less than 20 years of age, while 21.7% are aged 55 or above [1].

B-cell acute lymphoblastic leukemia (B-ALL) is a neoplasm described as excess proliferation of malignant B-cell precursors within the bone marrow. In adults, ALL is the second most common acute leukemia with B-ALL that accounts for ~75% of cases and T-ALL comprises the remaining cases [2],[3].

In contrast to most cases of pediatric ALL that is curable, adult ALL is considered as a disease with a historically dismal outcome, with restricted therapy options and low cure rates due to adverse genetic factors and chemotherapy intolerance [4],[5]. However, recently major improvement in our knowledge of molecular basis and pathophysiology of ALL has led to proper stratification and identification of subgroups at high risk, for example, Philadelphia-like ALL. Moreover, the detection of minimal residual disease has become a substantial tool in patient's stratification and in selecting the appropriate treatment strategies [6].

New progress in genome-sequencing technology has revealed the dark regions of the genome, namely the noncoding RNAs (ncRNAs). These ncRNAs are a diverse group that has essential functions and regulatory roles. Long noncoding RNAs (lncRNAs) constitute a subgroup of ncRNAs that have size larger than 200 nucleotides [7],[8].

Growing evidence has showed that lncRNAs are essential regulators of considerable biological activities such as transcription, translation, cell cycle, and apoptosis. Furthermore, current studies have reported lncRNAs as critical players in cancer initiation and progression [9],[10]. Thus, lncRNAs have been a hot topic of research.

One of the most important topics related to lncRNA research are its usage as diagnostic and prognostic molecular biomarkers in diverse types of cancer. As biomarkers, they are very promising and suitable for many reasons. First, circulating lncRNAs can be identified in samples, for example, peripheral blood, saliva, and urine that can be noninvasively obtained from patients. Second, lncRNAs have the property of being highly tissue specific. Third, their stability in body fluids. They can be analyzed in samples by using a variety of molecular techniques, such as reverse-transcription quantitative PCR (RT-qPCR), microarray, and RNA sequencing [11],[12],[13].

The newly emerged lncRNA growth-arrest-specific 5 (GAS5) has attracted the attention of researchers in recent years as it plays a crucial role as an antioncogene in many cancers such as gastric cancer, cervical cancer, and melanoma [14],[15],[16].

Although GAS5 has been implicated in a variety of solid tumors, data about its expression in hematological tumors remain unsatisfactory. To date, its importance in acute leukemia has not been widely investigated. The present study aimed to estimate the changes in the plasma levels of GAS5 during remission–induction chemotherapy of B-ALL patients. We have measured GAS5 expression at three checkpoints, including at diagnosis, day 15 and day 33.


  Patients and methods Top


Peripheral blood samples were obtained from 35 adult patients with newly diagnosed B-ALL (21 males and 14 females with a median age of 32 years, range: 19–69 years) who were admitted to the Clinical Hematology Department, Nasser Institute for Therapy and Research, Egypt, from December 2019 to December 2020. The diagnosis was confirmed by morphology, immunophenotyping, and cytogenetics. As regards therapy protocols, patients less than or equal to 40 years received Dana-Farber Cancer Institute Protocol [17], while patients aged 40–60 years received Holzer protocol [18]. For pH-positive ALL cases, imatinib was added to the protocol [19]. Blood samples were obtained initially before starting chemotherapy or radiotherapy and then on day 15 and day 33 from starting chemotherapy. Twenty control samples were collected from healthy donors. The study protocol was approved by Ethics Research Committee of Menoufia University and informed consent from each patient and control was taken.

Analysis of lncRNA GAS5-expression levels was performed using RT-qPCR technique. Two milliliters of venous blood were collected and transferred into dipotassium EDTA tube, centrifuged for 10 min at 4000 rpm, and the supernatant plasma was preserved in Eppendorf tubes at −20°C until analysis. The total RNA was purified from supernatant plasma using miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to standard manufacturer's protocol. The isolated RNA was checked for concentration and purity by NanophotometerN60 (IMPLEN, München, Germany).

Complementary DNA was synthesized using RT2 First Strand Kit (Qiagen). For each sample, the genomic DNA-elimination mix (7 μl RNA, 2 μl of buffer GE, and 1 μl of nuclease-free H2O) was prepared then and incubated at 42°C for 5 min. The reverse-transcription mix (4 μl of buffer BC3, 1 μl of control P2, 2 μl of RE3 reverse-transcriptase mix, and 3 μl of nuclease-free H2O) was prepared and mixed with the genomic DNA-elimination mix. The previous mixture was incubated at 37°C for 60 min and finally the reaction was stopped by incubation at 95°C for 5 min. The reactions were stored at −20°C, until being used for PCR protocol.

PCR-amplification mix was supplied by Qiagen and included a mixture of 3 μl of complementary DNA, 12.5 μl of RT2 SYBR Green Master Mix, 1 μl of RT2 lncRNA qPCR primer assay, and 8.5 μl of nuclease-free H2O to obtain a final volume of 25 μl. Relative gene expression was examined using ABI 7500 real-time PCR detection system (Applied Biosystems, Foster City, California, USA). Each sample was analyzed in duplicate. The sequences of primers specific for GAS5 were forward, 5′-AGCTGGAAGTTGAAATGG-3′; reverse, 5′-CAAGCCGACTCTCCATACC-3′. GAPDH was used as a reference gene for standardizing the expression of targeted GAS5 lncRNA. GAPDH-specific primers were forward, 5′-GAAGGTGAAGGTCGGAGTC-3′; reverse, 5′-GAAGATGGTGATGGGATTTC-3′. Cycling conditions of RT-qPCR were stated at 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and at 60°C for 1 min. The relative expression level of GAS5 was calculated by the 2-ΔΔCt method [ΔΔCt = ΔCt (sample)ΔCt (control)]; ΔCT = CT (target gene)−CT (reference gene); Δ = delta; Ct = threshold cycle.

Statistical analysis

SPSS software (IBM SPSS statistics, version 22.0; IBM Corp., Armonk, New York, USA) was used as statistical software for data analysis. Quantitative data are expressed as mean ± SD and median (minimum–maximum), while qualitative data as the number of cases and percentages. The association between clinical and laboratory parameters of ALL patients and GAS5-expression level was assessed using Student's t test. Significance of the obtained results was judged at 5%.


  Results Top


Our study included 35 adult B-ALL patients with their ages 34.23 ± 13.82 years. The clinicopathological features of B-ALL patients at diagnosis are outlined in [Table 1].
Table 1: The clinicopathological characteristics of B-cell acute lymphoblastic leukemia patients at diagnosis

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The levels of GAS5 expression were measured at three checkpoints, including at diagnosis, day 15 and day 33. GAS5-expression levels were 2.03 ± 0.49; 5.06 ± 0.79; and 14.29 ± 5.0, respectively.

The RT-qPCR analysis revealed that GAS5 expression was significantly higher on day 15 in comparison with its level initially at diagnosis (P < 0.001). On day 33, GAS5-expression level was significantly increased as compared with its level on day 15 (P < 0.001). Moreover, the expression level of GAS5 at diagnosis of B-ALL patients was significantly low when compared with that of healthy controls (2.52 ± 0.63) (P = 0.002) [Figure 1].
Figure 1: GAS5-expression levels of B-ALL patients and normal controls. B-ALL, B-cell acute lymphoblastic leukemia; GAS5, growth-arrest-specific 5.

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We investigated the plasma levels of GAS5 at diagnosis in association with clinical and laboratory parameters that were considered as prognostic factors. No association was observed between age and the levels of GAS5 expression, while initial leukocyte count more than 30 × 103/μl was significantly associated with lower GAS5 levels at diagnosis (P = 0.01, [Figure 2]).
Figure 2: Relation between GAS5 expression and initial leukocyte count. GAS5, growth-arrest-specific 5.

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An interesting finding was also observed during data analysis, a significant association was demonstrated between low GAS5-expression levels and BCR–ABL1-positive B-ALL (P = 0.015, [Figure 3]).
Figure 3: Relation between GAS5 expression and BCR–ABL1 expression. GAS5, growth-arrest-specific 5.

Click here to view



  Discussion Top


Comprehensive gene-expression study has added substantial knowledge to the molecular aspects of B-ALL, especially the recognition of definite high-risk subcategories [20]. Furthermore, progress in RNA sequencing has characterized ncRNAs as key participants in cancer development [21]. LncRNAs have been identified as central regulatory elements that may drive many important cancer phenotypes [22]. A growing body of research shows that lncRNAs may be involved in the pathophysiology and progression of B-ALL. Therefore, their utilization as molecular biomarkers and/or novel therapeutic targets is very promising [23].

Initially, lncRNA GAS5 was characterized as a tumor suppressor gene that had a role in cell-growth arrest and apoptosis [24]. Later, it was found that lncRNA GAS5 could directly interact with the DNA-binding domain of the glucocorticoid (GC) receptor. This prevented receptor binding to the GC-responsive element in target genes, blocking their transcription [25]. Because of its action on GC-responsive element, lncRNA GAS5 was considered to act as a repressor of the GC receptor activity [26]. Lucafo et al. [27] found that poor responders had higher lncRNA GAS5 levels in peripheral blood mononuclear cells (PBMC) obtained from blood donors treated with GCs compared with good responders. Consequently, they suggested that deregulated expression of GAS5 might affect response to GC therapy and they provided an assumption that altered expression of endogenous GAS5 was a GC-mediated event.

This is the first study that has investigated the changes in the plasma levels of GAS5 during remission–induction chemotherapy of adult B-ALL patients. We have reported that the plasma levels of GAS5 expression were significantly higher on day 15 in comparison with its levels at diagnosis.

We have also recognized that GAS5 expression was significantly increased on day 33 compared with its expression levels on day 15. Furthermore, the initial expression level of GAS5 of B-ALL patients was significantly lower when compared with that of healthy controls.

Few data are available about GAS5 expression in acute leukemia and especially in ALL. Gasic et al. [28] investigated 29 children with ALL and demonstrated that GAS5 expression was higher on day 15 of the remission–induction therapy as compared with its level at diagnosis. In contrast to our results, they reported that GAS5-expression level was decreased on day 33 as compared with day 15. This might be explained by the following points: Gasic and colleagues used isolated PBMCs to estimate expression levels of GAS5 while we used plasma samples, our study included only adult patients with B-ALL, while their study that included pediatric patients with most cases were B-ALL, and therapy protocols used in both studies were different.

In addition, they stated that four B-ALL patients out of 29 showed increased level of GAS5 expression between day 15 and day 33.

Furthermore, they elucidated their results as follows: GAS5-expression level was low at ALL diagnosis, increased on day 15 under the effect of GC therapy, and then decreased on day 33 when GC dose was significantly reduced. This hypothesis should be tested as it was reported that GAS5-expression levels were affected when PBMCs were treated with a combination of rapamycin and GCs [29]. This means that GAS5-expression levels can be affected by the coadministration of GCs with other drugs, and this is the case during remission–induction protocol of ALL therapy.

We also analyzed the association between GAS5 expression at diagnosis and established prognostic factors and demonstrated interesting findings; low levels of GAS5 expression at diagnosis were significantly associated with two unfavorable prognostic factors, including initial leukocyte count of more than 30 × 103/μl and BCR–ABL1-positive ALL.

Some limitations still exist in our study. First, the relatively small sample size, so further studies on a larger cohort of patients are required to support our findings. Second, the definite prognostic role of the dysregulated lncRNA GAS5 and its effect on response to therapy has yet to be identified. Subsequently, longer follow-up period is recommended to precisely clarify the prognostic merit of GAS5 in B-ALL. Third, GAS5-expression levels were analyzed using plasma samples only, comparison and validation of the results using PBMC samples should be done in subsequent studies.


  Conclusion Top


Our study demonstrated that the initial expression level of GAS5 at diagnosis of B-ALL was significantly decreased when compared with that of healthy controls, then an increase was observed on day 15 and day 33. Furthermore, low levels of GAS5 expression at diagnosis were significantly associated with two unfavorable prognostic factors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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