Prognostic Value of Plasma miR-29a Evaluation in Chronic Lymphocytic Leukemia Patients

Objective: Dysregulation of microRNA expression could attenuate the course of chronic lymphocytic leukemia (CLL). Therefore, the aim of our study is to address the association between miR-29a expression and other prognostic markers in CLL patients. Methods: miR-29a expression was determined by quantitative real-time PCR in the plasma of 158 CLL patients at diagnosis beside 21 healthy controls in a prospective study. Results: The levels of miR-29a expression were found to be significantly higher in CLL patients as compared to healthy controls (P<0.001). Moreover, a significant association between high miR-29a expression and poor prognostic markers (high expression of CD38 and ZAP70, high LDH levels, Stage III Rai stage, unfavorable cytogenetic finding, time to first treatment (TTFT) and patients outcome (P<0.001 for All). Using ROC curve, we have reported that miR-29a expression levels (29a<0.76 vs >0.76) is able to discriminate severity subgroups of CLL patients. Conclusion: Up regulation of miR-29a expression at CLL diagnosis was detected. Determination of miR-29a expression concentration levels at diagnosis could be demonstrated as a prognostic biomarker in CLL patients.


Introduction
Chronic lymphocytic leukemia (CLL) is an indolent and heterogeneous neoplasm. This heterogeneity makes prognostication and therapeutic decisions is challenging. The detection of new biomarker is the focus of current research in order to improve CLL risk stratification (Aref et al., 2020;Aref et al., 2022). Recently, miRNA profiles in serum of CLL patients, have attracted attention for both risk stratification as well as target therapy (Katsaraki et al., 2021;Grenda et al., 2022). Continous efforts are carried out in current research to detect new biomarkers in order to improve risk stratification.
MicroRNAs (miRNA) are small non-coding RNAs, act as potent regulators of multiple genes expression through down regulation of translational or destabilization of target genes through binding to the 3′-untranslated region (UTR) of mRNAs. miRNAs are associated with the development of several diseases and the their dysregulation is a cardinal feature of cancer. miRNAs play important roles in several biological processes, such as cell proliferation, apoptosis, differentiation and cell cycle. Thus, abnormal expression or dysfunction of miRNAs are associated with the development of diseases, including cancer (Zhao et al.,
Small RNAs are frequently dysregulated in cancer, circulate in body fluids in a stable form, representing interesting candidates for non-invasive biomarkers. Soluble miRNAs can be identified as cell-free entities or as a composition in extracellular vesicles (EVs). They are secreted by many cell types, including cancer cells, and can be assessed in the peripheral blood, making them an ideal source of tumor biomarkers (Anelli et al., 2021).
In recent years, miR-29 has emerged as a critical miRNA in various cancers, and it has been shown to regulate multiple oncogenic processes. Although miR-29a has been thoroughly documented as a tumor suppressor in the majority of studies, some controversy remains with conflicting reports of miR-29 as an oncogene ( Kwon et al 2019;Raeisi et al., 2020). Recently, Nguyen et al., (2022) and Cao (2023) stated that miR-29a-3p is a promising biomarker and prospective therapeutic target for the diagnosis and prognosis of colorectal cancer (CRC).
The aim of our study is to evaluate the expression levels and the prognostic relevance of miR-29a expression in CLL patients

Materials and Methods
The current study included 158 newly diagnosed patients with CLL (100 male; 58 female) with mean age 61.39 ± 10.07 year at Mansoura university oncology center, Egypt ( 2019-2022) he study protocol was approved by the IRB of Mansoura Faculty of Medicine. The CLL diagnosis was based on morphological assessment, Lymphocytes ≥ 5.0X10 9 /L lymphocytes, Immunophenotyping results (CD5/CD19; CD23, FMC7,s IgM, CD200, kappa/Lambda restriction). For All included patients both clinical (age, sex, Rai staging) as well as laboratory prognostic markers (Lymphocytes doubling time, ZAP70%, CD38%, Beta-2 microglobulin, Serum LDH, FISH cytogenetic findings for 17pdel, 11qdel, 13qdel, Trisomy 12). Time to first treatment (time from diagnosis till the patients required therapy) was recorded for every patient included in this study.

Plasma RNA extraction from CLL patients
From each CLL patient included in this study 5 ml EDTA blood sample was obtained at time of the diagnosis. Fresh plasma samles were separated by centrifugation at 3,000 rpm/ minute for 10 minutes. The separated patients plasma samples were stored in aliquots at -80℃. RNAs extraction were obtained using the miRNA isolation kits (Qiagen, Inc). The quality of miRNA was assessed by Nano drop (Thermo Fisher Scientific, Inc., USA) at wave length 230, 260, and 280; The ratio of 260/230 equal 1.8-2.1 which pointed for high purity of RNA

Complementary DNA (cDNA) Synthesis
According to the manufacturer's instructions, cDNA was synthesized using a Taqman microRNA Reverse Transcriptase(RT) kit (Applied Biosystems, USA). In brief, for synthesis of cDNA we use 2~20 ng of total RNA. The RT reaction mixture contained 0.15 μL of 100 mM dNTP mix (100 mM each dATP, dGTP, dCTP, and dTTP at a neutral pH), 1 μL of 50 U/μL RT, 1.5 μL of 10x RT buffer, 0.19 μL of 20 U/μL RNase inhibitor, and the total volume of the reaction was adjusted to 15 μL with nuclease-free water. The run program for cDNA synthesis experiment was done as follows: 16℃ for 30 min then 42℃ for 30 min, and 85℃ for 5 min. RT reactions were performed using a 96 test thermal cycler (Arkitik, Thermo Scientific, USA).

miRNA analysis using qPCR
According to manufacturer instruction, TaqMan miRNA small RNA assay (Applied Biosystems, USA) was used to detect and quantify miRNA expression using miRNA-specific primers. In brief, 2 μL of cDNA was added to 10 μL of probe qPCR mix and 8 μL of nuclease water. The following TaqMan small RNA assay (Applied Biosystems, USA) primers were used: hsa-miR-29a and hsa-miR-16-5p. All analyzed miRNAs are of human origin. qPCR reactions were performed using DT prime 4 real time PCR (DNA Technology, Russia). The experiments were done in duplicate. Data analysis were done by using the 2-delta Ct method . The has-miR-16-5p, was used as an endogenous control.

Statistical analysis
Statistical analysis was performed using Statistical package for Social Science (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.)was used for statistical analysis. The quantitative data are presented as median and range in non-parametric parameters, or mean and SD in parametric parameters, while non-quantitative data was presented as percentage. Comparing miRNA expression results in CLL patients and controls was done using Mann-Whitney-U test. Correlations studies between continuous data were done by Spearman rank test. In order to compare clinical as well as demographic data we used the χ 2 and Fisher's exact tests. The optimal cut off value that predict shorter time to the first treatment (TTFT) was identified by ROC curve. COX regression analysis was applied for prediction of TTFT. Kaplan-Meier curve was used to address the impact of miR-29a expression levels on TTFT in CLL patients. The statistical significant level was considered when p value less than 0.05.

Expression pattern of miR-29a in CLL patients as compared to healthy subjects
The CLL patients group was subdivided into two subgroups based on miRNA levels (miR-29a <0.76 vs >0.76 ). Comparing patient's characteristics and laboratory parameters among CLL subgroups revealed that WBCS, smudge, LDH were significantly elevated in miR-29a >0.76 group compared to miR-29a <0.76 group. On the other hand TTFT was significant reduced in miR-29a >0.76 group compared to miR-29a <0.76 group. There was significant increase in the frequency of ZAP70+, CD38+ and high ß2-microglobulin in subgroup of CLL patients have miR-29a >0.76 group compared to those with subgroup of patients with miRNA29a <0.76 . There was significant difference as regard Rai staging, pattern of BM lymph, and FISH findings among groups (Table 2).
In order to address the optimal cut off value that could predict shorter TTFT, we used ROC analysis. The results Prognostic Value of Serum miR-29a Evaluation in Chronic Lymphocytic Leukemia Patients smudge cells percentage, LDH, miR-29a, ZAP70, CD38, ß2-microglobulin, Rai staging as covariates. In multivariate analysis, miRNA29a, positive CD38, Beta2 microglobulin and high Rai staging were significant risk factors for shorter TTFT (Table 3).

Survival analysis
Using Kaplan Meier curve, we have evaluated the impact of miR-29a expression levels on TTFT in CLL patients. The analysis revealed that CLL patients with high miRNA 29a (>0.79 ) expression showed shorter TTFT as compared to those with low miRNA 29a expression (< 0.79) (P <0.001) (Figure 3).

Discussion
miRNAs play various roles in pathogenesis and evolution of cancer as well as initiation, tumor growth, and metastasis. Moreover, miRNAs may be used as diagnostic biomarker and as therapeutic target An optimal biomarker must have several characteristics (Califf, 2018): allow early disease diagnosis, accurate minimal residual disease identification, and to be specific to disease , and non-invasive. Previous studies indicated that miR-29a can act as either oncogene or tumor suppressor gene. The expression patterns of miR-29a in CLL patients is controversial (Jiang et al., 2014;Anelli et al., 2021).
In this study our findings revealed that miR-29a was significantly increased in CLL patients as compared to healthy controls. This finding is consistent with that reported by Raeisi et al., (2020) and Li et al (2011). In contrast, Calin et al., ( 2005) found that miR-29a was down regulated in CLL patients. In a previous study, miR-29c was reported to be elevated in AML patients and was found to be correlated with a higher frequency of disease relapse (Butrym et al., 2016).
A recent report found that all miR-29 family members were consistently decreased in the CLL microenvironment and correlated in a significant manner with shorter overall indicated that the best discriminating miR-29a expression value was 0.79. The area under the curve (AUC) was 0.742 (p=<0.001) (Figure 2).
Cox regression analysis was carried out in order to identify factors that could predict time to the first treatment. This was done using age, gender, WBCs,   (Sharma et al., 2020 ).
High miR-29a expression was significantly associated with CLL poor prognostic markers namely high WBCs count, low smudge cells count, high LDH levels , short TTFT, ZAP70+, CD38+ and high ß2-microglobulin, and advanced Rai stage. These findings could be attributed to significant association between up-regulation of miRNA29a and p53 deletions (Grenda et al., 2022).
ROC analysis was conducted to identify the optimal Cut off levels for prediction of shorter TTFT. The analysis revealed that the best cutoff value for miR-29a was 0.79. The area under the curve (AUC) was 0.742 (p<0.001). Furthermore, Cox regression analysis was conducted to address the independent factor that could predict CLL patients TTFT, using age, gender, WBCs count, smudge cells count, LDH concentration levels, miR29a expression, Positive ZAP70%, CD38%, ß2-macroglobulin, Rai staging as covariates. In multivariate analysis, miRNA29a, positive CD38, ß2microglobulin and high grade Rai stage were significant risk factors for shorter TTFT. Previous report stated that there is significant association between miR-29a expression levels and p53 deletion in CLL patients (Grenda et al., 2022). Moreover, a recent report by Sharma et al., (2020) showed that a miRNA-miR-29 dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by B-cell receptor activity.
In conclusion, Up regulation of miR-29a expression at CLL diagnosis was detected. Determination of miR-29a expression levels at diagnosis could be demonstrated as a prognostic biomarker in CLL.   Figure 3. Impact of miR-29a Expression Levels on TTFT in CLL Patients. CLL patients with high miR-29a expression showed shorter TTFT as compared to those with low miR-29a expression .