Supervisors info:
Μαργαρίτης Αυγέρης, Αναπληρωτής Καθηγητής Ιατρική Σχολής ΕΚΠΑ (Επιβλέπων),
Ανδρέας Σκορίλας, Καθηγητής Τμήμα Βιολογίας ΕΚΠΑ,
Διαμάντης Σίδερης, Καθηγητής Τμήμα Βιολογίας ΕΚΠΑ
Summary:
Acute Lymphoblastic Leukemia represents the most frequent diagnosed form of cancer in children, constituting almost 28% of all pediatric malignancies worldwide. It is characterized from clonal expansion of lymphoid hematopoietic precursors, especially of B and T lymphocytes, which exhibit developmental arrest at varying stages of differentiation and is caused of the interaction between genetic and epigenetic factors. The diagnosis of chALL is mainly based on the detection of lymphoblast in bone marrow samples overlapping the 25% of them and the characterization of the blast according to their morphology, immunophenotype and the cytogenetic abnormalities to discriminate the patients in different risk groups and decide the most suitable treatment. Among the different forms of chALL, pre-B-ALL is the most common type of leukemia in children’s population affecting the precursor cells and is correlated to a good prognosis, while the overall survival rates surpass 90%. The promising progress of this group is directly related to the optimization of treatment protocols over the past few decades in combination with the understanding of the underlying molecular basis of the disease and the rigorous characterization of cancer cell’s genetic background. However, the survival rates vary according to the patient’s age, the profile of genetic abnormalities and the type of lymphoblast been affected, while the malignant clonal expansion of T lymphoblast has been associated with a dismissal prognosis.
Taking everything into consideration, the aim of this study is to predict the presence of minimal residual disease (MRD) promptly, evaluate patient’s response to treatment and provide a representative prognosis with clinical value, following up patients progress constantly in different stages of treatment and seeking for precising and reliable genetic biomarkers that reflect cancer’s burden. In terms of genetic locus, we decide to investigate the clinical value of CNVs in genes having an important role in hemopoiesis and cell cycle, such as IKZF1, ETV6, GATA-3 and CDKN2A, and associate the impact of contingent difference in their allele number on patient’s progress. The comparison between allele frequences was enabled by using the reference gene, LEP116, which is anticipated to present a stable transcription among healthy and nonhealthy individuals, retaining the percentage of diploid regardless of the examined carrier. In particular, the examination of CNV presence was carried out in 65 genomic DNA isolated from bone marrow cells, offered by “P. & A. Kyriakou” Children’s Hospital and treated according to BMF-ALL protocol, and the relative quantification of allele levels was based on quantitive PCR reaction.
After the levels of cycle extension was gathered, they were calibrated in comparison with the corresponding cycle values of reference gene and HeLa positive control and then, the calculated RQ levels was correlating with patient’s clinicopathologigal characteristics, using the IBM’s software SPSS. The correlation of CNV with those parameters was separated in two distinctive ways following the categorical classification of patient’s according to the allele levels in loss (Hypodiploid and Loss of Heterozygote) and diploid-amplified group, whereas the continuous study evaluated the RQ levels emerged from the reference curve. As a result of the last examination, Kruskal-Wallis test proved the positive prognostic value of ETV6 and GATA-3 losses founded in low risk group, while ETV6 was deleted with statistical significance in M1 BM response (p=0.017) and in low detected blast’s number (<1000, p=0.031) patients. Similarly, GATA-3 RQ levels were underrepresented with high statistical value in low-risk group having a M1 BM response on the 15th day (p=0.027).
On the contrary, the categorical study demonstrated the adverse clinical value of IKZF1 and CDKN2A deletions providing the high trend of relapse. More specifically, IKZF1 losses dominated in high-risk group diagnosed with M3 BM response on 15th day (p<0.001) and detective MRD levels on 33rd day (p=0.017). Meanwhile, CDKN2A allele levels had controversial results in respect to both analyses, taking into consideration that the lowest RQ levels (p=0.039) and as well as the group characterized by deletions in this gene (p=0.036) had a spike on the low-risk group with a M1 BM response. The debatable effect may have arisen from the exon gene for which primers were designed and the anticipated deregulation of this gene in the high-risk group could be explained from an alternative mechanism based on an epigenetic silence.
Concluding, the Kaplan-Meier curves of OS and PFS couldn’t reflect the statistical significance of the CNV consequences in patient’s progress in none of the examined genes. Though the limited clinical value, there is a trend of discrimination between categorical patient’s classification observed in GATA-3 and, especially in ETV6 CNV’s levels. Particularly, GATA-3 deletion showed a better clinical outcome for patient’s health in OS curve ((HR:2.661; 95%CI: 0.333-21.280;p=0.337;long-rank p=0.336), where the non-loss group’s survival probability was on the wane. In a superior way, ETV6 CNV’s evaluation displayed a highest differentiation rate and clinical efficacy in both OS (HR:30,332; 95% CI: 0.041-22,690.968;p=0.099, long-rank: p=0.099) and PFS (HR:30.636; 95%CI:0.060-15,582.910; p=0.079,long-rank p=0.078) curves, where losses in this gene are correlated with an extended survival. Taking everything into account, the absence of ETV6 and GATA-3 is related to a good prognosis while the corresponding losses of IKZF1 and CDKN2A show up an adverse effect on patient’s progress, if they are examined in comparison with gold standard markers. To achieve a better clinical significance and evaluate those markers separately, it is inevitable to increase patient population and investigate ETV6 losses singly so as to discriminate the already diagnosed as a good risk group.
Keywords:
Pediatric Acute Lymphoblastic Leukemia, Copy Number Variations (CNVs), IKZF1, ETV6, GATA-3, CDKN2A
