Cytogenetic Characteristics of de novo Acute Myeloid Leukemia in Southern Vietnam

Background: The cytogenetic characteristics are important factors for risk stratification at diagnosis of acute myeloid leukemia (AML); however, cytogenetic profile of Vietnamese patients with AML remains undetermined. In this study, we present the chromosomal data of de novo AML patients in Southern Vietnam. Methods: We performed cytogenetic testing for 336 AML patients using G banding. If the patients had suspected abnormalities, fluorescence in situ hybridization with probes of inv(3)(q21q26)/t(3;3)(q21;q26), 5q31, 7q31, t(8;21)(q21.3;q22), 11q23, t(15;17)(q24;q21), inv(16)(p13q22)/t(16;16)(p13;q22)were analyzed. Patients without above aberrations or with normal karyotype were tested by fluorescence in situ hybridization using probe 11q23. Results: We found that the median age was 39 years. According to French – American – British classification, AML-M2 is the most frequent type with 35.1%. Chromosomal abnormalities were detected in 208 cases, accounting for 61.9%. Among structural abnormalities, t(15;17) was the most common (19.6%), followed by t(8;21) and inv (16)/t(16;16) in 10.1% and 6.2%, respectively. In perspective of chromosomal numerical abnornmalities, loss of sex chromosomes are the most common (7.7%), followed by +8 in 6.8%, -7/del(7q) in 4.4%, +21 in 3.9% and -5/del (5q) in 2.1%. The prevalence of addditional cytogenetic aberrations accompanying with t(8;21) and inv(16)/t(16;16) were 82.4% and 52.4%, repectively. None of +8 cases was associated with t(8;21). Regarding cytogenetic risk assessment according to European Leukemia Net 2017, there were 121 (36%) patients in favorable-risk, 180 (53.6%) in intermediate-risk and 35 (10.4%) in adverse-risk group. Conclusion: In conclusion, this is the first comprehensive cytogenetic profile of Vietnamese patients diagnosed with de novo AML, which helps clinical doctors in prognostic classification for AML patients in Southern Vietnam.


Introduction
in Southeast Asia including Malaysia, Singapore, and Thailand (Enjeti et al., 2004;Niparuck et al., 2009;Meng et al., 2013). The results of the above studies show that the common chromosomal abnormalities in AML are almost present in the reports, but the prevalence varies by geographic region and ethnicity. Vietnam is an agricultural country and having experienced a long period of war, the accumulation of toxic substances such as herbicides, lethal weapons and defoliants for a long time can affect genetic changes. There are currently no chromosomal abnormalities data in newly diagnosed AML patients in Southern Vietnam reported, so we performed this study to clarify whether the cytogenetic characteristics are different from its counterpart in other countries around the world.

Patients
A total of 336 Vietnamese de novo AML patients were recruited from the Blood Transfusion and Hematology Hospital and Cho Ray Hospital at Ho Chi Minh City, Vietnam from 2015 to 2021. AML diagnosis was made based on morphological examination of bone marrow aspiration according to the French -American -British (FAB) classification with eight subtypes from M0 to M7. Cytogenetic risk stratification was based on European Leukemia Net (ELN) 2017. Written informed consents for chromosomal analyses were obtained from enrolled patients. This study was approved by the Ethics Committees of the University of Medicine and Pharmacy at Ho Chi Minh City (number 440/ĐHYD-HĐĐĐ).

Conventional cytogenetic analysis
Two mL of bone marrow aspiration or 4 mL of peripheral blood samples (if immature cells accounted for at least 10% of peripheral blood cells and white blood cell count was at least 10 x 10 9 /L) in 300 units of lithium heparin were obtained. Chromosomal culture procedure was performed within one hour. 2 x 10 7 cells were cultured in 10 mL of RPMI-1640 medium containing 10% fetal bovine serum (Sigma life science, USA), 1% antibiotic (Gibco, USA) and 200 µL Phytohemagglutininlymphocyte-conditioned medium, then incubated at 37 o C, 5% CO 2 for 24 hours for bone marrow samples and 48 hours for blood samples. After cell division was stopped using demecolcine solution (Sigma life science, USA), the cell membrane was destroyed with 0.075M KCL hypotonic solution and fixed with Carnoy solution (Methanol: Acetic acid = 1: 3). Cell residues were washed and chromosome slides were prepared. The slides were baked at 60°C for 4 hours, treated with 0.1% trypsin for an appropriate time, stained with Giemsa and analyzed under an optical microscope. For each patient, an average of 20 cells were analyzed using Ikaros software (MetaSystems, Germany). Karyotypes were encoded according to the International System for Human Cytogenetic Nomenclature (ISCN) 2013 criteria. Cases with three or more clonal abnormalities were defined as complex karyotype.

Cytogenetic characteristics
All 336 patients in this study had cytogenetic profile analyzed. It was showed that 208 patients had abnormal chromosomes, accounting for 61.9%. Regarding the cytogenetic risk stratification according to ELN 2017, there were 121 (36%) patients classified as favorable-risk, 180 (53.6%) patients as intermediate-risk and 35 patients as (10.4%) adverse-risk group.
In addition, there were 23 patients carrying +8, among whom the rates of favorable-, intermediate-and adverserisk groups were 26.1%, 47.8% and 26.1%, respectively. Six patients were in favorable-risk group, including 5 patients with inv(16) and 1 patient with t(15;17); there was no case associated with t(8;21). In the intermediaterisk group, there were 9 patients with +8 alone and 2 patients with one another abnormality (+14 or +21.) All 6 patients in the adverse-risk group had complex karyotype, including 1 patient with inv (3)(q21q26).

Discussion
This is the first study to describe the cytogenetic profile of AML patients in Southern Vietnam. Among 336 AML cases, there were 162 male patients (48.2%) and 174 female patients (51.8%), with a female: male ratio of 1.07. Females accounted for a higher proportion in our study, similar to the results of Niparuck P et al. study (52.8%) (Niparuck et al., 2009), although other reports showed that more males acquired AML (Byrd et al., 2002;Enjeti et al., 2004;Sierra et al., 2006;Cheng et al., 2009;Meng et al., 2013). The higher risk of leukemia in men may be due to work-related and environmental factors such as pesticides, tobacco and radiation. The fact that Vietnam and Thailand are agricultural countries, where women participate in occupational activities similar to what men do, can explain the higher risk of this disease among women in these two countries. Regarding morphological classification according to FAB, AML-M2 accounted for the highest percentage (35.1%), followed by AML mono (M4/M5) (31.2%) and M3 (19.6%) and the lowest being M6 (1.5%) and M7 (2.4%). AML-M2 had the highest    (Byrd et al., 2002;Enjeti et al., 2004;Cheng et al., 2009;Udupa et al., 2020), however, several studies reported that M4/M5 comprised the highest rate (Abuhelwa et al., 2017;Elnaggar et al., 2022).
Using conventional cytogenetic analysis with G-band staining, we detected 61.9% out of 336 AML patients carrying chromosomal abnormalities, similar to previous reports with rates ranging from 40 to 60% (Byrd et al., 2002;Enjeti et al., 2004;Wakui et al., 2008;Cheng et al., 2009;Khoubila et al., 2019;Elnaggar et al., 2022). The t(15;17) translocation was the structural abnormality with the highest incidence, higher than other reports in the world (Byrd et al., 2002;Enjeti et al., 2004;Cheng et al., 2009;Grimwade et al., 2010;Khoubila et al., 2019;Elnaggar et al., 2022), especially in Malaysia (2.3%) and in Morocco (3.7%). This difference may be due to race, exposure factors and molecular genetic techniques used (Cheng et al., 2009). All patients with confirmed or suspected AML-M3 based on cell morphology and cell surface markers were examined by 3 tests including chromosomal analysis, FISH with t(15;17) dual color-dual fusion probe and reverse transcription polymerase chain reaction for PML-RARA detection, in order not to miss cases of AML-M3 with cryptic translocations, insertions or poor quality chromosomes.
The rate of complex chromosomes in the high-risk group also varies from study to study. There were 4.2% cases with complex chromosomes in this study, higher than the results in Egypt (Elnaggar et al., 2022) and India (Udupa et al., 2020), but lower than many other studies (Byrd et al., 2002;Enjeti et al., 2004;Wakui et al., 2008;Cheng et al., 2009;Grimwade et al., 2010;Meng et al., 2013).
In perspective of risk stratification based on the ELN 2017 criteria, it is showed that the percentages of favorable-, intermediate-and adverse-risk groups were 36%, 53.6% and 10.4%, respectively. In our study, the ratio of the favorable-risk group was higher than its counterpart in the Egypt (Elnaggar et al., 2022) and Morocco (Khoubila et al., 2019) but lower than in India (Udupa et al., 2020). The favorable-risk group in India was much more common because t(8;21) and inv(16)/t(16;16) were more prevalent, while t(15;17) in our study comprised higher percentage than in other studies. This difference emphasizes the variation of cytogenetic characteristics by geographical location.
In conclusion, this study is the first comprehensive analysis of conventional cytogenetics in Vietnamese patients with de novo AML. The cytogenetic profile in our study had some differences in prevalence of chromosomal abnormalities when compared with that of other countries. Our data would help the clinical doctors in prognostic classification for AML patients in Southern Vietnam.

Author Contribution Statement
All authors contributed equally in this study.