Ανάλυση της έκφρασης και μελέτη της γονιδιακής ρύθμισης, μέσω μορίων μικρό RNA (miRNA), αποπτωτικών γονιδίων της BCL2 οικογένειας στην παιδική λευχαιμία
Authors: Πιατοπούλου Δέσποινα 
Published: September 2021

Leukemia is a malignancy of the hematopoietic system, which consists of the infiltration of bone marrow from leukemic blasts, due to their continuous proliferation. The study of this disease is particularly important in children, as leukemia is the most common type of cancer observed during childhood, accounting for 29% of all childhood malignancies. Among leukemias, Acute Lymphoblastic leukemia (ALL) accounts for 76% of all childhood leukemic cases. During the last decades, there has been a tremendous progress concerning the diagnosis and therapy of ALL, as overall survival rates (OS) and disease-free survival rates (DFS) reach up to 90% and 80%, respectively, in developed countries. However, there are still challenges to overcome, as even with the establishment of modern therapeutic approaches, 20% of children experience relapse and poor prognosis with high mortality rates. Moreover, the therapeutic protocols of ALL stratify most patients in intermediate or high risk groups, leading often to patients’ overtreatment, which is followed by acute or late side effects. Therefore, apart from the clinically established prognostic markers, it is essential to identify new molecular markers, capable of early disease detection and precise patients’ prognosis, which will predict, as early as possible, the risk for relapse and patients’ response to treatment. The ultimate task is the development and the establishment of individualized and targeted therapeutic schemes, which will be based on the cytogenetic abnormalities and gene expression profile of each patient and will eliminate the side effects of chemotherapy treatment.Apoptosis deregulation, including the abnormal expression of its master regulators, BCL2 family genes, has been reported in various studies focused on leukemogenesis and has been correlated to patients’ outcome and resistance to chemotherapy. Thus, the discovery of the deregulated pathways and molecules involved in the upstream regulation of apoptosis and BCL2 family members, resulting in treatment failure and adverse prognosis, is of outmost importance. miRNAs represent an example of these molecules. They are a class of non-coding RNAs, 21-23bp, which regulate mainly post-transcriptionally other mRNAs. Researchers have revealed the implication of miRNAs in all aspects of the disease, from initiation and progression, to resistance to drugs and chemotherapy outcome in terms of relapse and death. Taking into account the potential of miRNAs as molecular markers of leukemia, the present Ph.D. Thesis performed, for the first time, the expression analysis and the evaluation of the clinical utility for childhood ALL of the miRNAs, miR-125b, miR-143, miR-182 and miR-195, which target BCL2 family genes, among others. The aim of the study was to reveal their potential as biomarkers in ALL differential diagnosis and in predicting patients’ treatment response and survival. MiR-125b has been the research topic of numerous studies that have focused on cancer, attributing to it either an oncogenic, or a tumor-suppressive role. It targets anti-apoptotic genes, such as BCL2, BLCW and MCL1, as well as proapoptotic genes, such as BAK1, BMF and TP53. Apart from its role in apoptosis, miR-125b promotes carcinogenesis through the induction of cellular proliferation and the inhibition of cellular differentiation. In leukemia, miR-125b can be found up - or downregulated, depending on the type of malignancy, while its abnormal expression has been linked to chemotherapy resistance. MiR-125b expression levels have been associated with the presence of specific cytogenetic abnormalities, as well as with resistance to vincristine and daunorubicin. Nevertheless, its value in differential diagnosis and in predicting disease outcome has not been estimated yet. MiR-182 plays a significant role in cellular physiology, as it is involved in the regulation of different pathways, including AKT/FOXO3A, p53, apoptosis, WNT and DNA repair. Studies in cancer have reported the deregulation of miR-182 expression, but there is not a common expression profile between different types of neoplasms. As a result, miR-182 functions either as a tumor-suppressive gene, by targeting BCL2, BCL2L12, RGS17 and CREB1, or as an oncogene, by targeting FOXO1, FΟΧO3, BRCA1, RsECK and MIM. However, there are not many studies focusing on the expression motif of this miRNA in hematological malignancies and correlating it with patients’ prognosis and outcome. MiR-143 has been reported to possess a tumor suppressive function, as it is often underexpressed in cancer, including leukemia, such as ALL, AML, CLL and Burkitt’s lymphoma. Normally, it is involved in the PI3K/Akt/mTOR pathway, inhibiting cellular proliferation and invasion, while it promotes apoptosis, by targeting ERK5, AKT, KRAS and BCL2 among others. Moreover, it regulates the epigenetic modification of genes, through the downregulation of DNMT3A expression. Despite the studies performed in leukemic samples and leukemic cell lines, which have revealed miR-143 reduced expression, its role in childhood ALL progression and in patients’ treatment response and survival, has not been reported yet. The last miRNA of this thesis was miR-195. In the majority of studies in cancer, miR-195 is downregulated and has been associated with poor prognosis. It is involved in cell cycle progression and apoptosis. Its gene targets, which support its tumor-suppressive role, include BCL2, BCLW, RAF1, CCND1, CCND3, CDK6 and E2F3, whose levels increase when miR-195 is underexpressed, inducing cell proliferation and impeding apoptosis. In leukemia, there are only a few studies which have presented roughly the expression pattern of miR-195, without however, attributing to it a specific role in disease formation and progression. To fulfill the aim of the study, a statistically significant number of bone marrow samples (272 in total) was collected and processed, from children with newly diagnosed ALL (N=125), from children of the same cohort at the end of the induction protocol (day 33) of treatment (N=83) and from children not suffering from any hematologic or other type of malignancy (N=64), which constituted the control group. The experimental procedures included the development and optimization of RNA extraction protocol from bone marrow samples, the spectrophotometric check of RNA quality and concentration, and the electrophoretic check for RNA degradation. Furthermore, polyadenylation and reverse transcription took place for the production of cDNA. To check the quality of all the aforementioned procedures, specific primers were designed and PCR reaction was optimized for the semi-quantitative evaluation of the reference gene SNOR48 and the studied miRNAs. Moreover, qReal-time PCR methodologies were developed for the quantification of the miRNAs of interest, using the fluorescent SYBR Green I assay. The expression analysis was carried out using the comparative CT, 2-ΔΔCT relative quantification method. The evaluation of the clinical significance of the studied miRNAs was performed by an extensive biostatistical analysis of the experiments’ results, using a detailed database of patients’ history, clinicopathological and follow-up data. The analysis of miR-125b revealed its significant downregulation in childhood ALL patients compared to the control group (p=0.004), and its ability to distinguish normal from leukemic BM samples (ROC curve AUC: 0.628; 95% CI 0.548–0.707; p=0.004 and univariate regression analysis OR: 0.477; 95% CI 0.288–0.790; p=0.004), independently of patients’ age and gender (OR:0.507; 95% CI 0.305–0.842; p=0.009). In addition, reduced miR-125b expression on disease diagnosis was correlated with unfavourable prognostic features, such as T-ALL and (p=0.037) and initial WBC≥50,000/μl (p=0.004). Moreover, decreased miR-125b levels on childhood ALL diagnosis were associated with significantly stronger risk for short-term relapse and poor OS of the treated patients, using Kaplan-Meier survival curves (p=0,005 and p=0.013, respectively) and univariate Cox regression analysis (HR: 4.253; 95% CI1.411–12.82; p=0.010 and HR: 3.072; 95% CI 1.209–7.806; p=0.018, respectively), while multivariate models confirmed miR-125b independent prognostic value for patients’ risk for relapse or death (HR: 3.891; 95% CI: 1.195–12.67; p=0.024 and HR: 2.963; 95% CI:1.079–8135; p=0.035, respectively). Interestingly, the downregulation of miR-125b on diagnosis was able to discriminate patients at higher risk for short-term relapse and worse survival outcome, despite the presence of favourable prognostic markers, such as B-cell immunophenotype (p=0.009 and p=0.003, respectively), initial WBC<50.000/μl (p=0.054 and p=0.047, respectively), good prednisone response on day 8 (p=0.003 and p=0.001, respectively) or M1 bone marrow response on day 15 of treatment (p<0.001 and p<0.001, respectively). Finally, patients with positive MRD on day 15 and low miR-125b levels on diagnosis presented a higher risk for relapse (p=0.010), while miR-125b downregulation on diagnosis identified a group of intermediate/high risk patients with also a higher risk for relapse or death (p=0.028 and p=0.010, respectively). At the end of the induction protocol (day 33), miR-125b was significantly upregulated compared to diagnosis in the majority (83%) of childhood ALL patients (p<0.001), while the higher values of day 33/diagnosis expression ratio were correlated with unfavourable prognostic markers, such as Τ-cell ALL (p=0.033), initial WBC≥50,000/μl (p=0.010), patients’ age <1 or ≥10 years old (p=0.025) and intermediate/high risk stratification according to ALL-IC BFM (p=0.003). In addition, increased miR-125b levels on the 33rd day were associated with significantly stronger risk for short-term relapse (p=0.016) and poor OS of the treated patients, (p=0.016), which was confirmed by univariate Cox regression analysis (HR: 4.218; 95% CI 1.174–15.16; p=0.027 and HR: 4.218; 95% CI 1.174–15.16; p=0.027, respectively). The same conclusion was extracted for patients with higher values of day 33/diagnosis expression ratio in terms of relapse and death probability using Kaplan-Meier survival curves (p=0,015 and p=0.004, respectively) and univariate Cox regression analysis (HR: 4.301; 95% CI 1.198–15.44; p=0.025 and HR: 6.789; 95% CI 1.517–30.37; p=0.012, respectively). Furthermore, the elevated values of day 33/diagnosis expression ratio were able to discriminate patients at higher risk for short-term relapse and worse survival outcome, despite the presence of favourable B-cell immunophenotype (p=0.001 and p<0.001, respectively), initial WBC<50.000/μl (p=0.051 and p=0.013, respectively), good prednisone response on day 8 (p=0.033 and p<0.001, respectively) or M1 bone marrow response on day 15 of treatment (p<0.001 and p<0.001, respectively). Finally, patients with positive MRD on day 15 and high miR-125b expression ratio levels presented increased risk for relapse (p=0.007) or death (p=0.003), while the augmented miR-125b expression ratio identified a group of intermediate/high risk patients with also higher risk for relapse or death (p=0.031 and p=0.007, respectively). Regarding miR-182, it was found to be significantly downregulated in childhood ALL patients compared to control (p<0.001) and to discriminate normal from leukemic BM samples (ROC curve 0.762; 95% CI 0.694–0.829; p<0.001 and univariate regression analysis OR: 0.221; 95% CI: 0.126 – 0.386; p<0.001), independently of patients’ age and gender (OR: 0.197; 95% CI 0.108–0.357; p<0.001). However, the expression of miR-182 on disease diagnosis did not correlate neither with patients’ clinicopathological data, nor with patients’ survival. At the end of the induction protocol (day 33), miR-182 was significantly upregulated compared to diagnosis in the vast majority (96%) of childhood ALL patients (p<0.001), while the higher values of day 33/diagnosis expression ratio were correlated with unfavourable prognostic markers, such as Τ-cell ALL (p=0.047), initial WBC≥50,000/μl (p=0.015) and intermediate/high risk stratification according to ALL-IC BFM (p=0.005). In addition, increased miR-182 levels on the 33rd day were associated with significantly stronger risk for short-term relapse (p=0.007) and poor OS of the treated patients, (p=0.044), which was confirmed by univariate Cox regression analysis for relapse (HR: 4.281; 95% CI: 1.339-13.69; p=0.014) and displayed a trend for death (HR: 2.928; 95% CI: 0.980-8.747; p=0.054).Moreover, higher expression levels of miR-182 on the 33rd day of treatment were associated to stronger risk for short-term relapse and dismal overall survival, despite the presence of favourable B-cell immunophenotype (p<0.001 and p<0.001, respectively), initial WBC<50.000/μl (p=0.027), good prednisone response on day 8 (p=0.006 and p=0.002, respectively) or M1 bone marrow response on day 15 of treatment (p<0.001 and p=0.004, respectively). Finally, patients with positive MRD on day 15 and higher miR-182 expression levels on day 33, presented increased risk for relapse (p=0.012) and a trend for death (p=0.068), while augmented miR-182 expression, also identified a group of intermediate/high risk patients with higher risk for relapse or death (p=0.012 and p=0.042, respectively). The expression analysis of miR-143 revealed its significant downregulation in childhood ALL patients compared to the control group (p<0.001), and its ability to distinguish normal from leukemic BM samples (ROC curve AUC: 0.880; 95% CI 0.831–0.928; p<0.001 and univariate regression analysis OR: 0.108; 95% CI: 0.056 – 0.207; p<0.001), independently of patients’ age and gender (OR: 0.098; 95% CI: 0.049 – 0.194; p<0.001). However, the expression of miR-143 on disease diagnosis did not correlate neither with patients’ clinicopathological data, nor with patients’ outcome. At the end of the induction protocol (day 33), miR-143 was significantly upregulated compared to diagnosis in the vast majority (91%) of childhood ALL patients (p<0.001), while its increased expression was associated with unfavourable prognostic markers, such as patients’ age <1 or ≥10 years old (p=0.023), positive MRD on the 33rd day (p=0.040) and intermediate/high risk stratification according to ALL-IC BFM (p=0.031). In addition, the higher values of miR-143 day 33/diagnosis expression ratio were also correlated with unfavourable prognostic markers, such as patients’ age <1 or ≥10 years old (p=0.046) and intermediate/high risk stratification according to ALL-IC BFM (p=0.012). Patients with overexpression of miR-143 on the 33rd day had a trend for stronger risk for disease relapse (p=0.196) and inferior OS (p=0.074), which was also shown in univariate Cox regression analysis for death (HR: 2.757; 95% CI: 0.864-8.802; p=0.087).As miR-195 is concerned, its levels were significantly downregulated in childhood ALL patients compared to the control group (p<0.001), and were able to discriminate normal from leukemic BM samples (ROC curve AUC: 0.820; 95% CI 0.761–0.880; p<0.001 and univariate regression analysis OR: 0.101; 95% CI: 0.048 – 0.212; p<0.001), independently of patients’ age and gender (OR: 0.842; 95% CI 0.740–0.959; p=0.009). However, the expression of miR-195 on disease diagnosis did not correlate neither with patients’ clinicopathological characteristics, nor with patients’ outcome. At the end of the induction protocol (day 33), miR-195 was significantly upregulated compared to diagnosis in the majority (89%) of childhood ALL patients (p<0.001), while the higher values of miR-195 day 33/diagnosis expression ratio were associated with unfavourable prognostic markers, such as patients’ age <1 or ≥10 years old (p=0.025), poor prednisone response on day 8 (p=0.007) and intermediate/high risk stratification according to ALL-IC BFM (p=0.002). Unfortunately, no statistical significant results were extracted, correlating miR-195 expression pattern on day 33 of treatment with patients’ survival data. When the evaluation of the clinical significance of each miRNA for patients’ outcome was completed, it was tested whether their combination in one common marker could offer an additional prognostic value, regarding patients’ survival. miR-125b showed a very strong and independent prognostic value as a lone marker, whereas, miR-195 did not correlate with patients’ outcome. The rest two miRNAs, namely miR-143 and miR-182, were combined to form a common marker, whose value was further examined on the 33rd day of treatment. Patients were divided in those expressing both miRNAs in low levels on day 33 and those overexpressing at least one miRNA. The overexpression of miR-143 and/or miR-182 on the 33rd day was associated with reduced disease-free (p=0.021) and overall survival (p=0.028) by Kaplan-Meier curves, which as also confirmed by univariate Cox regression analysis for relapse (HR: 4.911; 95% CI: 1.096-22.01; p=0.038) and death (HR: 4.590; 95% CI: 1.026-20.53; p=0.046). Moreover, multivariate Cox regression analysis confirmed the independent prognostic value of the combined miR-143/miR-182 marker for patients’ risk for relapse (HR: 6.907; 95% CI: 1.208-39.47; p=0.030). In addition, miR-143 and/or miR-182 overexpression was able to discriminate patients at higher risk for short-term relapse and dismal survival outcome, despite the presence of favourable B-cell immunophenotype (p=0.001 and p<0.001, respectively), good prednisone response on day 8 (p=0.027 and p<0.001, respectively), M1 bone marrow response on day 15 of treatment (p<0.001 and p=0.001, respectively) or patients’ age between 1-9 years old (p=0.041 for risk of death). Finally, patients with positive MRD on day 15 and higher miR-143/miR-182 expression levels on day 33, presented increased risk for relapse (p=0.009) or death (p=0.027), while the reduced levels of miR-143/miR-182 identified a group of intermediate/high risk patients with lower risk for relapse or death (p=0.022 and p=0.026, respectively) compared to patients presenting miR-143/miR-182 overexpression. Concluding, in the present Ph.D. Thesis, novel experimental methodologies were developed and optimized for the accurate and sensitive quantification of miRNAs in bone marrow samples from children suffering from ALL, as well as from healthy children. A statistically significant sample size of 272 samples in total was collected and processed and a detailed database of patients’ history, clincopathological and follow-up data was created for all patients. The extended biostatistical analysis revealed the significant clinical and prognostic value of miR-125b, miR-182 and of the combined marker miR-143/miR-182, and to a less extend of miR-143 and miR-195, for the differential diagnosis of ALL and the prediction of patients’ survival outcome. Finally, the study revealed the contribution of the expression pattern of the aforementioned miRNAs in ameliorating the predicting strength of the clinically established disease biomarkers, so that patients can be stratified in risk groups more accurately in order to receive the most suitable and efficient therapeutic regimen.