Detection of Aberrant Phenotypes in Acute Leukemia Patients in Relation to Some Hematological Parameters

Article information Background: Patients with acute leukemia [AL] may express aberrant antigens that are normally restricted to a different lineage. We evaluated the frequency of aberrancy in AL patients. Aim of the work: Evaluation and analysis of the incidence of aberrant phenotypes in mononuclear cells in AL patients and their relation to hematological parameters. Patients and Methods: This observational descriptive study was conducted on 150 newly diagnosed cases of AL over the period from August 2017 to March 2021. Flow cytometry was performed by Becton Dickinson [BD] FACS Calibur/BD FACS Canto flow cytometers using monoclonal antibodies against cell markers. Results: The overall frequency of aberrancy was 38.7% [58/150] in all AL patients. Aberrant antigenic expression was more frequent in acute lymphoblastic leukemia [ALL] than acute myeloid leukemia [AML], T-ALL with aberrant expression was 66.6% of all T-ALL patients, while B-ALL with aberrant expression was 45.8% of all B-ALL patients, and AML with aberrant expression was 31.1% of all AML patients. Conclusion: Aberrant phenotypes were found with considerable frequency in AL patients, and the implication of aberrant markers can help in disease monitoring, detecting measurable residual disease, and determining risk stratification and treatment intensity.


INTRODUCTION
Acute leukemia [AL] is a heterogeneous group of hematological malignancies with clonal expansion of neoplastic myeloid/lymphoid cells in blood and bone marrow [1] .
According to the blast cell lineage, AL is primarily divided into acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]. In each type of AL, blast cells express a characteristic pattern of molecules known as cluster of differentiation antigens [CD]. Classification of AL depends on the expression of lineage-specific markers on blast cells. In several cases of AL, blasts of one lineage do not exhibit the markers of normal differentiation but express unusual markers in which myeloidassociated antigens are expressed in lymphoblasts and lymphoid-associated antigens are expressed in myeloblasts. This phenomenon is called aberrant phenotypes [2] .
Aberrant expression of antigen is an abnormal expression of antigens by blasts that are normally not expressed by a particular lineage [Myeloid, B-lymphoid, and T-lymphoid] and do not fulfill the World Health Organization [WHO] criteria for mixed phenotype acute leukemia [MPAL]. This aberrant leukemiaassociated immunophenotype [LAIP] can be used to monitor measurable residual disease [MRD]. Aberrant antigens in certain leukemias may indicate the presence of a genetic event or may affect the prognosis [3] .
Flowcytometric immunophenotyping plays an important role in the identification of the lineage of AL along with the detection of aberrant antigens, which help in treatment monitoring and MRD analysis [4] .
The aim of this study was to evaluate and analyze the incidence of aberrant phenotypes in mononuclear cells in AL patients and their relation to hematological parameters.

PATIENTS AND METHODS
This prospective observational study was approved by the Research Ethical Committee. Oral and written consents were obtained from all patients after a full explanation of the study.
The present study was conducted on 150 patients suffering from AL. Patients were referred to the clinical pathology department from the pediatric and internal medicine departments, Al Hussein Hospital, Al-Azhar University, over the period from August 2017 to March 2021 at the time of initial diagnosis, before induction therapy. Patients' clinical and laboratory information was reviewed from their medical records.
Inclusion criteria: All newly diagnosed cases of AL are diagnosed based on morphology.
Exclusion criteria: Secondary leukemias evolved from myeloproliferative neoplasms and myelodysplastic syndromes, and AL cases who received treatment.
All patients were subjected to a full history, clinical examination, and laboratory investigations as complete blood picture [CBC], coagulation prolife and flow cytometry [FCM] Immunophenotyping.
Two milliliters [ml] of peripheral blood/bone marrow samples were collected from each patient on an EDTA vacutainer tube. Also, two ml of peripheral blood samples were collected from each patient on a citrated vacutainer tube for the coagulation profile.
Samples for immunophenotyping were processed within 6 hours of collection at room temperature.
Immunophenotypic analysis of leukemic cells was performed by flowcytometry using a large panel of fluorochrome-labeled monoclonal antibodies. Each test tube was labelled properly and was sequentially placed. 20-100 μl of the specimen [whole EDTA peripheral blood/bone marrow] were added to each test tube. FACS Lyse was prepared in distilled water at a 1:10 dilution. 2 ml of prepared FACS Lyse solution was added to each test tube and incubated for 10 minutes at room temperature in a dark area. The contents of the tube are centrifuged after incubation for five minutes at 2000 RPM at room temperature, and after wasting the supernatant, the residual fluid was thoroughly mixed for re-suspension of cells. This was followed by adding 2 ml of isotonic PBS to each test tube and centrifuging for 5 minutes at 2000 RPM. The supernatant was discarded again. 250 μl of BD FACS Cytofix/Cytoperm Solution were added to each tube and incubated for 20 minutes at room temperature in a dark area. This was followed by adding 2 ml of PBS to each test tube and centrifuging for 5 minutes at 2000 RPM. The supernatant was discarded again. 5 μl of the corresponding labelled monoclonal antibody were added to each test tube. The tubes were incubated in a dark area for 30 minutes at room temperature. Unbound

DISCUSSION
FCM immunophenotyping plays an important role in the diagnosis and classification of AL. It also provides prognostic as well as predictive information, aiding in modulating therapy appropriately. The apparently morphologically similar blast cells can be easily differentiated by immunophenotyping based on the expression of different CD markers. One of the important advantages of FCM immunophenotyping is its ability to analyze many cells, which improves the accuracy of leukemia diagnosis. This analysis can be completed within hours and is often sufficient [5] .
In our study, there was an adult predominance in AL; this may be because AML was more common than ALL. In accordance with these findings, others noted that the frequency of AL was higher in adults than pediatrics [6] but this was contrary to the results of other studies [7] . The frequency of aberrant expression in AML in our study was 31.1%. Other studies found that the aberrant expression ranged from 11.1% to 26.4% in AML cases [6,8] . Other studies were like ours; they reported a frequency of aberrant expression among their AML patients between 30% and 67.5% [9,10] .
In the present study, aberrant expression of B markers in AML patients showed that CD79a was the most frequently expressed marker at 16.7% [15/90] of all AML patients, followed by CD10 and CD22 at 5.6% [5/90] and 1.1% [1/90], respectively, while expression of T marker CD7 in AML patients was 12.2% [11/90]. Other studies noted different distributions of aberrant marker expression, and many of them reported that CD7 was the most common lymphoidassociated antigen in AML, followed by CD19 [11,12] . Chang et al. [13] demonstrated CD7 to be an independent prognostic factor adversely affecting disease-free survival [DFS] and Postremission survival in AML patients with a normal karyotype at diagnosis. If confirmed, CD7 expression may inform the decision to consolidate AML patients with a normal karyotype who have achieved first remission with chemotherapy, autologous, or allogenic transplantation.
Chang et al. [14] showed a correlation between CD7 expression and chromosome 5/7 anomalies but found that when all the cytogenetic groups were included, CD7 expression did not affect the prognosis.
A study carried out by El-Sissy et al. [15] suggested that CD7 expression in AML should be interpreted with cytogenetics as it may be associated with an unfavorable outcome.
In our study, 45.8% [22/48] of B-ALL patients had aberrant expression, which was like a study carried out in Sohag University Hospital by Abdullah et al. [16] who noted that the aberrancy was seen in 46% out of 15 B-ALL cases. Also, Sarma et al. [4] noted that 59.2% of cases of B-ALL had aberrant phenotypes.
Momani et al. [7] conducted a study in Jordan and noted that CD33 was the commonest aberrant marker in B-ALL patients with aberrant expression [60.5%], followed by CD13 [50%] and CD7 was expressed in only one case [3%]. These findings were also found in another study done by Alkayed et al. [17] on Jordanian children with B-ALL. In accordance with us, other studies reported that CD33 was more common than CD13 [18,19] .
In contrast to ours, other studies noted that aberrant expression of myeloid markers in T-ALL was more common than B-cell markers [20] . In controversy with all these results, including ours, others reported that there were no aberrant B-cell markers in T-ALL cases [21,22] . Similar results were reported by Vitale et al. [24] in ALL patients, who discovered that patients with aberrant myeloid expression had higher platelet counts. This investigation also revealed that the group with greater myeloid expression had higher platelet values. In contrast to our findings, Silva et al. [25] reported no statistically significant difference between ALL patients with and without aberrancy regarding platelet count.
Determining the platelet count when treating children with ALL is important. Because of the lifetime of platelets in the circulation [8-10 days], lower platelet counts in the blood of patients diagnosed with AL show the high proliferative power of the leukemic clone. In addition, because of changes in capillary fragility secondary to inflammation, fewer than 50,000 platelets/mm 3 in patients with infection can lead to hemorrhage, characterized by epistaxis, gingival bleeding, gastrointestinal bleeding, and CNS bleeding, leading to the need for transfusion therapy and supportive care for children with ALL [26] .

Conclusion:
We concluded that aberrant phenotypes were found with considerable frequency in AL patients, but further studies on larger scales are needed to confirm the correlation of aberrancy to diagnosis, prognosis, and therapeutic response.