الفهرس 
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Abstract
Evidence suggests that p53 plays a pivotal role in normal and leukemic hematopoiesis and is central in a complex web of AML-related signaling pathways. AML genetic and epigenetic aberrations encompass the p53 gene, protein, and regulated network, among others. The vast majority of cases of de novo AML have unaltered TP53 alleles; data from The Cancer Gene Atlas (TCGA) including adult patients with AML documented that " ~ "8% of AML cases harbor TP53 mutations. In light of the discouraging outcomes after treatment with aggressive chemotherapy in patients harboring TP53 mutations. TP53 mutations is included in the adverse-risk category in the ELN risk-stratification system.
In the light of this, this work aimed to detect p53 mutations in newly diagnosed AML patients and to identify their morphological, clinical and cytogenetic characteristics.
To achieve this aim, the present work was carried on 60 de novo AML patients. They were recruited from the Hematology oncology Unit, Ain Shams University hospitals and outpatients, during the period from October 2021 to October 2022. They were 36 (60 %) males and 24 (40%) females with male to female (M: F) ratio of 1.5:1. Their age ranged from 19 to 70 years old with a Mean ± SD of 32.83 ± 11.96. Diagnosis was based on standard morphologic, cytochemical and immunophenotyping criteria.
Informed consent was obtained from patients to use their samples in this study. Patients were evaluated at day 28 of therapy to assess therapeutic response.
All Patients were subjected to the following: Complete history taking and thorough clinical examination, Laboratory investigations, including: Complete blood count using XN-1000 (Sysmex, Japan), examination of Leishman‘s stained peripheral blood films, bone marrow aspiration and examination of Leishman‘s stained bone marrow smears, cytochemical studies using myeloperoxidase stain. Immunophenotyping of bone marrow or peripheral blood samples using the acute leukemia panel using NAVIOS 2 Laser 6 Color FCM (Beckman Coulter, USA) to detect the FAB category.
Fluorescence in situ hybridization analysis (FISH) cytogenetic analysis using the following probes:
• LSI dual color dual fusion RUNX1-RUNX1T1 {for detection of t(8;21) (q22;q22), +8and +21)
• LSI dual color single fusion PML-RARA for detection of t(15;17) (q22;q12)
• LSI dual color single fusion BCR-ABL for detection of t(9;22) (q34;q11)
• LSI CBFβ break apart rearrangement for detection of inv(16) (p13;q22)
• LSI MLL break apart rearrangement for 11q23 rearrangements
• LSI RARA (17q21) dual color break apart rearrangement for detection of 17q rearrangement
• LSI 5q31 with LSI 5p15.2 control for detection of deletion 5q and monosomy 5
• LSI 7q31 with centromeric control for detection of deletion 7q and monosomy 7
• LSI 17p13.1 for detection of p53 deletion
Two age matched healthy volunteers were used as controls; to check the intensity of signals of the used probes.
All patients were further subjected to testing for TP53 Pro72Arg SNP by real time polymerase chain reaction.
Follow up was implemented for patients on day 28 by CBC and bone marrow examination, for assessment of response to therapy.
Immunophenotyping (IPT) was done for all AML patients.
FISH was done for all patients and revealed the following:
• 16 patients (26.7%) were negative for all common aberrations using the routine panel of FISH probes and the rest
• 12 patients (20%) patients had t(15;17) (q22;q21)
• 11 patients (18.3%) had t(8;21) (q22;q22)
• 5 patients (8.3%) patients had t(9;11)
• 7 patients (11.7%) patients had t(16;16) / inv16 (p13;q22)
• 3 patients (5%) had 17q21 rearrangement
• 2 patients (3.3%) had del 17p13
• 1 patient (1.7%) had t (8;21) (q22;q22)+ del 17p13 both
• 1 patient (1.7%) had del 17p13, -7
• 1 patient (1.7%) had monosomy 5 (del(5q31))
• 1 patient (1.7%) had t (9;22) (q34;q11)
Karyotyping was done for all patients and revealed the following:
• 21 patients (35%) had normal karyotyping
• 12 patients (20%) patients had t(15;17)
• 9 patients (15%) patients had t(8;21) (q22;q22) while the other two found by FISH yielded normal karyotyping
• 4 patients (3.3%) patients had t(9;11)11q23 rearrangements and the other one found by FISH yielded normal karyotyping
• 6 patients (6.7%) patients had t(16;16) / inv16 (p13;q22) while the other one found by FISH yielded normal karyotyping
• 1 patient (1.7%) had 17q21 rearrangements while the other two found by FISH one yielded normal karyotyping and the other failed karyotyping
• 2 patients (3.3%) patients had del 17p13
• 1 patient (1.7%) had t(9;22) (q34;q11)
• 4 patients (6.7%) had failed karyotyping
In studied patients 13 patients (21.7%) had NPM mutation, 12 patients (20%) had FLT3-ITD mutation and 4/60(6.7%) had combined genetic mutation of both NPM and FLT3-ITD.
In the studied patients 7 patients (11.7%) had p53 mutation of which 5 patients were heterozygous and 2 were homozygous.
In the studied patients 14 patients (23.3%) had unfavorable and intermediate prognosis according to the ELN 2022 risk stratification while 32 patients (53.3%) had favorable prognosis.
In the studied patients, 6 (10%) of the patients were resistant to therapy, 13(21.7%) went through incomplete remission and 41(68.35%) had complete remission.
Statistical analysis of our research showed that male gender and lower TLC were significantly associated with good outcome with (P-value ≤ 0.05).
In our work, 17q rearrangement showed significant association with bad outcome with (P-value ≤ 0.05) and t(8;21) that was found to be highly significant in association with good outcome with (P-value< 0.01).
In the current work, FLT3ITD mutation was found to be highly significant in association with bad outcome with (P-value< 0.01). NPM mutation was significant in association with good outcome with (P-value ≤ 0.05).
Importantly, P53 mutation was significantly associated with advancing age, male gender and higher TLC with (P-value ≤ 0.05).
11q23 rearrangement showed significant association with P53 mutation with (P-value ≤ 0.05) and t(8;21)+del 17p, del 17p& -7 and del 17p only was found to be highly significant in association with p53 mutation with (P-value< 0.01).
Normal karyotyping showed significant negative correlation with P53 mutation with (P-value ≤ 0.05).
P53 mutation was found to be highly significant in association with ELN 2022 risk stratification (Unfavorable) with (P-value< 0.01).
P53 mutation was found to be highly significant in association with bad outcome with (P-value< 0.01).
In conclusion, most de novo AML patients do not display genetic alterations in the TP53 gene. Nevertheless, individuals with such alterations are typically older, have higher total leukocyte counts, and are associated with unfavorable cytogenetic aberrations, and poorer outcomes. The existence of TP53 mutations can be indicative of a less favorable response to traditional therapeutic protocols.