الفهرس 
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Abstract
C
ytogenetic analysis of metaphase and interphase cells is a key component to the evaluation of all patients with newly diagnosed or relapsed acute myeloid leukemia (AML). The malignant cells in most patients with AML have non-random, acquired clonal chromosomal abnormalities. In some cases, specific cytogenetic abnormalities are closely, and sometimes uniquely, associated with morphologically and clinically distinct subsets of the disease.
Evidence suggests that de novo and secondary AML occur via similar cytogenetic and genetic pathways, several of which involve aneuploidy, the loss or gain of chromosomes. Aneuploidy of specific chromosomes, recurrent translocations and rearrangements have been detected in different AML subgroups showing diagnostic, prognostic, and therapeutic importance.
In the light of this, this work aimed to detect different numerical chromosomal aberrations in AML patients (trisomy 8, trisomy 13, trisomy 21, monosomy 5 and monosomy 7) in addition to routine cytogenetic profile using Fluorescence in situ Hybridization (FISH), and to assess their relation with other prognostic factors and therapeutic response.
To achieve this aim, the present work was carried on 37 AML patients. They were recruited from the Hematology oncology Unit, Ain Shams University hospitals and outpatients, during the period from December 2012 to January 2014. They were 24 (64.9%) males and 13 (35.1%) females, with a male to female ratio of (1.8:1). Their ages ranged from 19-70 years, with a mean of (48.4±15.2) years. Diagnosis was based on standard morphologic, cytochemical and immunophenotyping criteria. Patients were subdivided into 2 groups: Twenty two newly diagnosed patients (group I) and fifteen patients in relapse (group II).
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 LH750 (Beckman coulter), 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 EPICS XL coulter flow cytometer to detect the FAB category. Fluorescence in situ hybridization analysis (FISH) cytogenetic analysis using the following probes:
- Centromeric (CEP) 13 for detection of trisomy 13 (+13).
- LSI dual color dual fusion RUNX1-RUNX1T1 {for detection of t(8;21) (q22;q22), +8 and +21}.
- LSI dual color single fusion PML-RARA for detection of t(15;17) (q22;q21).
- LSI dual color single fusion BCR-ABL for detection of t(9;22) (q34;q11).
- LSI CBFB break apart rearrangement for detection of inv(16) (p13,q22).
- LSI 5q31 with 5p15.2 for detection of deletion 5q and monosomy 5.
- LSI 7q31 with centromeric control for detection of deletion 7q and monosomy7. Two age-matched healthy volunteers were used as controls; to check the intensity of signals of the used probes.
By applying several FISH probes on slides prepared by culture of PB and/or BM samples, well spread interphases and metaphases analyses yielded normal cytogenetic profile in 15 (40.5%) patients. Numerical aberrations were detected in 13 out of the 37 (35.1%) patients, among which +8 was the most common one, being detected in 5 (13.5%) patients, all were newly diagnosed patients, loss of chromosomes 5 (-5) was the second most common followed by -7, the former was detected in 3 (8.1%) patients while the latter in 2 (5.4%) patients, all of them were in relapse. +21 was observed in 2 (5.4%) patients, both of them were in relapse, +13 in 1 (2.7%) patient (in relapse), while structural aberrations were detected in the form of t(8;21), t(15;17), t(9;22), inv 16, 11q23 rearrangement. All patients, who were positive for +8, t(8;21); t(15;17) and inv(16) were newly diagnosed cases.
Follow up of all patients was done at day 28 after induction therapy by clinical examination, assessment of PB and BM blasts count, revealed 24/37 (64.9%) patients with complete remission and 13/37 (35.1%) patients with incomplete remission.
Two (5.4%) out of the 15 (40.5%) patients with normal cytogenetic profile failed to achieve remission, they may hide cryptic aberrations or somatic mutations which elucidate the disturbance of cellular growth, proliferation, and differentiation processes in hematopoietic progenitor cells. Acquired gene mutations with prognostic relevance are highly recommended to be identified as mutations of FLT3, CEBPA, IDH1/2 and NPM1.
Statistical analysis of patients’ outcome with prognostic markers among group I (Newly diagnosed patients) revealed significant association (p<0.05) of CR with TLC < 50 x109/L (p=0.000) and IPT patterns of M2 and M4 (p=0.042). On the other hand, age, gender, hepatomegaly, splenomegaly, Hb and platelet count showed non-significant statistical difference between the patients who achieved complete remission and those with incomplete remission (p>0.05).
Statistical analysis of patients’ outcome with prognostic markers among group II (Patients who were selected in relapse) revealed significant association (p<0.05) of CR with TLC < 50 x109/L (p=0.003) and Hb level < 10 g/dl (p=0.038). On the other hand, age, gender, hepatomegaly, splenomegaly, platelet, FAB subtype and associated numerical aberrations showed non-significant statistical difference between the patients who achieved complete remission and those with incomplete remission (p>0.05), except for +21, which showed high significant correlation, FAB subtypes (p=0.002); (being negative for M2 & M4), and platelet count <100 (p=0.008); associating its clinical outcome.
In conclusion, the most common numerical chromosomal aberrations encountered in AML is the chromosomal gain in the form of trisomy 8, in newly diagnosed patients with favorable outcome. On the other hand, chromosomal loss is the most common numerical aberrations in relapsed patients in the form of -5 as the most common one related to poor outcome, followed by -7, +21, while +13 is the least frequent one. Initial TLC, Hb and immunophenotyping represent the most significant standard prognostic factors in relation to patients outcome. Moreover, numerical aberrations in addition to recurrent structural aberrations showed significant association with patients’ outcome and may be used as prognostic indicators for therapeutic response.