1. Introduction

AML (Acute myeloid leukemia) stands out among other similar pathologies for its diversity both in terms of etiology, clinical picture, and treatment results, when this may be due to mutations in different genes or, conversely, when mutations in one gene cause clinical forms of AML of varying severity]. According to the results of a previously published study, the average age of patients diagnosed with AML was 53 years. The analysis of these data makes us think about the quality of diagnosis of AML in elderly and senile people and about the short life expectancy of elderly patients [2,5].

The pathogenesis of AML is a complex, multi-stage process, where the influence of various factors already at the initial stages of bone marrow cell formation causes significant violations of the genetic type of inheritance and epigenetic control of the development of hematopoietic stem cells (HSCs). Often, damage to the cellular environment surrounding blood vessels, immune cells, fibroblasts and other stromal structures, which are primarily responsible for both regulation and maintenance of HSC, occurs due to changes leading to an imbalance between the processes of proliferation and differentiation in the structures responsible for the formation of myeloid cells. Disruption of the interaction of cells that make up the hematopoietic "niche" of the bone marrow, formed by the stromal microenvironment, ultimately leads to a violation of the balance of their functions. In addition, the systemic disorder of the normal process of blood cell formation is supported by the action of micro-RNAs, which regulate the manifestation of pathological genes in AML and contribute to the maintenance of the malignant process without external influence [1,4].

The formation of leukemic clones in AML is caused by the processes of controlling the proliferation of bone marrow tissue, which control the genes FLT3, JAK2, KIT, MPL, NF1, PTPN11, KRAS, CBL, CBLB, SH2B3; differentiation of myeloid tissue (CEBPA, ETV6, NPM1, RARA, RUNX1); the ordering of the processes of changing protein synthesis caused by mechanisms that do not change the sequence of nucleotides in DNA (ASXL1, DNMT3A, IDH1/2, TET2) and the occurrence of mutual chromosomal rearrangements leading to the formation of chimeric genes and the subsequent vivid manifestation of related chimeric proteins, which are considered to be the main molecular events. The most well-studied of them were included in the WHO classification in 2016: AML with anomalies in t(8;21)(q22;q22), (RUNX1T1/RUNX1), AML with inv(16)(p13;q22) or t(16;16)(p13;q22), (MYH11/CBFB), AML with et(15;17)(q24;q21), t(15;17)(p22; q12), (PML/RARA) and their variants), as well as deviations in 11q23(MLL) are attributed to the group of AML with recurrent cytogenetic abnormalities. [3,6,7].

The study of the pathogenesis of tumor diseases of the blood system led to the discovery of one of the leading principles of oncogenesis in general: the cause of the formation of a malignant clone is a violation of the functioning of normal genes. As a rule, this occurs as a result of chromosomal aberrations, mutations of individual genes, or blocking of the normal regulation of gene functioning due to epigenomic events (not directly related to damage to the gene structure). Moreover, the identification of certain translocations (the exchange of chromosomal segments between two, usually non-homologous, chromosomes) and mutations allows us to confidently assume the features of the course of the disease, judge the prognosis in some cases and select adequate therapy. Inversion is a chromosomal rearrangement in which a section of the chromosome rotates by 180o. Chromosome 16 inversion — inv(16)(pl3;q22), as well as t(16;16)(pl3;q22), is found in all patients (100%) diagnosed with AML (Acute myeloid leukemia is a malignant tumor of the myeloid blood germ, in which altered white blood cells multiply rapidly. Accumulating in the bone marrow, they inhibit the growth of normal blood cells, which leads to a decrease in the number of red blood cells, platelets, and normal leukocytes) with variant M4eo (according to the FAB classification, the Franco-American-British classification is based on the predominant pathway and degree of cell differentiation) with eosinophilia and in 40% of patients with myelomonoblastic leukemia (M4); less often observed in other variants of acute myeloid leukemia (AML). These chromosomal abnormalities can only be identified on high-quality cytogenetic drugs, which are difficult to obtain from the bone marrow of patients with acute myeloid leukemia (AML). Molecular genetic techniques are an important tool for detecting chromosome 16 inversion. Leukemias associated with inv(16) or t(l6;16) are often accompanied by a specific lesion of the central nervous system, therefore modern therapeutic programs provide a number of measures to prevent such a lesion. It is clear that the timely detection of these anomalies or their molecular equivalent, the chimeric CBFB—MYH11 gene, is extremely important [3,8].

Thus, the increased interest in the pathogenetic mechanism of the development of AML, the clinic, and most importantly preventive measures and treatment, confirms the relevance of this topic.

2. Main Body

2.1. Purpose of the Study

To study the frequency of occurrence of driver–somatic mutations of the inv gene (16;9) and evaluate its role in early diagnosis, prognosis of the course, as well as control of the effectiveness of treatment of acute myeloid leukemia.

2.2. Material and Methods of Research

The peripheral blood of 145 AML patients served as the material for molecular genetic research to achieve the above goal. Patients were divided into 2 subgroups: 117 patients with AML under 60 years of age; and 28 patients with AML over 60 years of age. In the main study group of patients, the median age was 44.6±1.2 years.

The gene was studied in all patients with AML Inv (16, 9) gene mutation. Molecular genetic studies were conducted in the laboratory of the Republican Specialized Scientific and Practical Medical Center of Hematology of the Ministry of Health of the Republic of Uzbekistan (RSNPMCG of the Ministry of Health of the Republic of Uzbekistan). Diagnosis of thrombosis was carried out in accordance with currently accepted clinical guidelines.

The diagnosis of acute leukemia was established based on the detection of more than 20% of blast cells in the bone marrow punctate (in some cases in peripheral blood). If less than 20% of blast cells were found in the bone marrow, and more than 20% in peripheral blood, acute leukemia was also diagnosed.

The material was taken under sterile conditions into plastic micro-tubes with a capacity of 1.5 ml with the addition of 0.2 ml of 0.1 M EDTA solution.

The karyotype of patients with acute myeloid leukemia was studied using standard cytogenetic analysis using the GTG–banding method. The VideoTest computer system was used for cytogenetic analysis and archiving of the obtained data.

The bone marrow cells obtained by sternal puncture, as well as dividing cells of the peripheral blood of patients, were the material for a standard cytogenetic study.

In order to reliably diagnose, predict the risk of developing and clinical course of AML, molecular genetic studies were performed using real–time polymerase chain reaction.

Genotyping of Inv (16, 9) gene mutations It was carried out on the basis of the method of Tag Man probes on an amplifier Rotor-Gene Q (Quagen Germany), using the commercial test kit of Syntol LLC (Russia).

Statistical processing of the results was performed using a standard application software package OpenEpi V.9.2. Analysis of the deviation of empirical genotype frequencies from The theoretically expected Hardy-Weinberg distribution was performed using the Statistica 6.0 software package.

2.3. The Results Obtained and Their Discussion

During our study, when considering the main hematological parameters in patients with acute myeloid leukemia, the following data were obtained: the average value of hemoglobin in the blood of 145 patients with AML was 71.2±1.9 g/l, erythrocytes 2.3±0.06 million, and leukocytes – 73.6±8.4 thousand, respectively. The range of leukocytes was quite wide and ranged from 1.32 to 446x10⁹/l. Statistical data showed that hyperleukocytosis was detected in 60/145 (41.4%) patients, whose average level was 148.3±14.5 thousand. According to the literature, an increase in the content of leukocytes is an unfavorable marker of the prognosis of AML, reducing survival and increasing the frequency of relapses. Therefore, we studied the number of white blood cells in all patients with AML, and subsequently, depending on age and gender characteristics.

The average lymphocyte count in 145 patients with AML was 25.3±1.8% with a minimum of 0.76% and a maximum of 61.2%. The average monocyte level was 15±1.5%. The number of platelets ranged from 3 to 606 thousand, and the average content was 46.1± 6.9 thousand.

The average value of the ESR (erythrocyte sedimentation rate) was 49.5±1.7 mm/hour, where the minimum value was 4 mm/hour and the maximum was 80 mm/hour.

An important indicator affecting the prognosis and stratification of treatment is the number of tumor cells in peripheral blood and bone marrow. The greater the number of blast cells, the worse the prognosis.

In 109 patients out of 145, tumor cells were detected in the peripheral blood. The average value of which was 54.1±2.5%, respectively.

At the same time, the average value of blast cells in the bone marrow was 62.1± 2.3%, respectively.

Mutations in the Inv gene (16;9) were found in 4 (2.8%) of 145 patients with AML. This mutation was not registered in 141 (97.2%) out of 145. In all cases, the mutation of the above gene was of a combined nature.

When studying patients depending on the karyotype, it was found that the greatest detection of mutations in the Inv gene (16, 9) was detected in the group of patients with a normal karyotype – in 3 (5.9%) of 51 patients. In the group of patients with chromosomal aberrations, the occurrence of a mutation of the studied gene was found in 1 out of 32 patients (3.1%). Despite this, differences in the frequency of Inv gene mutation (16, 9) in patients with normal karyotype and with altered karyotype were not statistically significant (χ2=0.3; OR=0.5; p=0.6; 95%CI:0.05-5) (see Table 1).

Table 1. Differences in the frequency of Inv (16, 9) gene mutation factor in groups of patients with normal and altered karyotype

Mutations in the Inv gene (16, 9) were found in patients with morphological variants of AML M0 and M2 in 50.0% of cases. This mutation has not been registered in other morphological variants of AML.

Hyperleukocytosis was slightly more common in the group of patients without Inv gene mutation (16, 9) in 41.8% of cases compared with patients with mutations of this gene in 25.0%, respectively (χ2=0.5; OR=0.5; p=0.5; 95%CI:0.05-4.33). There was an increase in the number of white blood cells in the peripheral blood in patients with mutations in the Inv gene (16, 9) compared with the group without mutations – 78.9±57.8 versus 73.5±8.6, respectively. However, the number of blast cells in bone marrow was lower in patients without mutations in the Inv gene (16, 9) compared with the group of patients with mutations 62.6±2.3 versus 45.3±17.4, respectively, but the difference was statistically insignificant.

In the group of patients with the Inv gene mutation (16, 9), a statistically insignificant AML complication was less common (25.0%) than in patients without the mutation (53.9%) at х2=1.3; OR=0.3; p=0.3; 95%CI:0.03-2.46.

During the study, all patients with the Inv gene mutation (16, 9) (4 out of 4) achieved remission. And in patients without a mutation of this gene, remission was detected in 68.1% of cases, respectively.

Recurrence of the disease was not recorded in patients with Inv gene mutations (16, 9), and in patients without mutation, 18.4% of cases developed a recurrence of the pathology under study.

The same pattern was observed in the group of patients with resistance to chemotherapy.

According to the results of statistical analyses, it was shown that mutations in the Inv gene (16;9) were extremely rare (only in 2.6%) in young patients. A similar pattern was also observed in patients over 60 years of age, Inv mutations (16;9) were detected in 3.6% of patients, respectively (х2=0.1; OR=1.4; p=0.8; 95%CI:0.14–13.92) (see Table 2).

Table 2. Differences in the frequency of detection of inv mutation (16;9) in the groups of young and elderly patients

Nevertheless, the Inv mutation (16;9) was not detected in 97.4% of young patients and 96.4% of elderly patients.

The difference in the frequency of Inv mutations (16;9), depending on the age of patients, did not reach significant values (p <0.05).

Thus, our data showed that the presence of CBFB–MYH11 (inv (16;9)) indicates a good prognosis for AML, with long periods of remission and a high survival rate, which does not contradict the results of studies by I.A., Olkhovsky et al., 2020, as well as Kundu M., et al., 2001. [8,9].

3. Conclusions

Driver somatic mutations of the Inv genetic marker (16;9) play an important role in verifying the diagnosis and determining the prognosis of the course as well as controlling the treatment of the disease in AML patients and should be included in the patient examination algorithm.