Abstract

Objective: Acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) are neoplastic blood disorders in which the cancerous white blood cells accumulate, resulting in a significant morbidity and mortality. Human leukocyte antigen (HLA) association is observed as one of the factors in the development of leukemia. The objective of the present study was to analyze the allele frequency of HLA Class I (HLA-A, HLA-B, and HLA-C) and Class II (HLA-DRB1 and HLA-DQB1) in Indian acute leukemia patients and to compare them with the frequencies in healthy, unrelated Indian individuals. Materials and Methods: We included 500 Indian leukemic patients (AML = 324 and ALL = 176) and 1000 unrelated, healthy, Indian individuals as controls. The HLA typing was performed using polymerase chain reaction with sequence-specific oligonucleotide probes. Results: On univariate analysis, allele frequencies of HLA-AFN*0111 and HLA-DRB1FN*0111 were lower in patients with ALL (P = 0.0181 and P = 0.0025, respectively). Whereas of HLA-AFN*0111, HLA-DRB1FN*0111, and HLA-BFN*0151, these frequencies were relatively lower in patients with acute leukemia (AML + ALL) (P = 0.0382, P = 0.0093 and P = 0.0384, respectively) and HLA-CFNx0101 (P = 0.0304) in AML when compared with control individuals. In contrast, the HLA-BFN*0139 and HLA-CFN*0107 allele frequency was higher in acute leukemia (P = 0.00372 and P = 0.0463, respectively) and in AML (P = 0.0010 and P = 0.0178, respectively) than that in controls. On multivariate analysis, BFNx0139 showed positive associations with acute leukemia (P = 0.006) and AML (P = 0.002). HLA-AFN*0111 and-DRB1FN*0111 showed a negative association with acute leukemia (P = 0.009 and P < 0 class="i" xss=removed>P = 0.013 and P < 0 class="b" xss=removed>Conclusions: The HLA-BFN*0139 has a positive association with AML and acute leukemia, whereas HLA-AFN*0111 and HLA-DRB1FN*0111 alleles have negative association with ALL and HLA-BFN*0151 along with these two alleles with acute leukemia. No positive association was observed with ALL. HLA-CFN*0101 frequency was lower in AML patients than that in controls.

Introduction

Leukemia is a type of cancer that targets the leukocytes. The cell lineage affected by the cancer determines the kind of leukemia and the affect can be sudden or “acute” or can develop slowly or “chronic.”[1] Acute myeloid leukemia (AML) is a malignant neoplasm of myeloid cells originating in and infiltrating the bone marrow. Acute lymphoid leukemia (ALL) originates in lymphoid cells.[2]

The human major histocompatibility complex (MHC) is called the human leukocyte antigen (HLA) system as these molecules can be detected on the surface of leukocytes using specific alloantibodies.[3] The HLA complex of genes is found on the short arm of chromosome 6. They play an important role in incompatibility matching between donor and recipient for transplantations.[4] The HLA loci include Class I (HLA-A, -B, and -C) and Class II (HLA-DR, -DQ, and -DP) antigens. The HLA genes are the most commonly known polymorphic genes. The frequencies of HLA antigens differ among different populations.[5] The HLA has a property of nonrandom association of HLA alleles across the genome, which is known as linkage disequilibrium.[6] The role of MHC and its associations have been studied in over fifty different diseases, and many of them have viral origination.[7]

The first MHC association in mouse leukemia was reported by Lilly in 1964; since then, the studies on HLA association with leukemia began.[8] In 1967, the first HLA study in ALL was reported with an increased frequency of HLA-A2.[9] In 1970, the first HLA haplotype association in ALL was reported with HLA-A2B12.[10] The association of HLA-A2 is still not confirmed in any of the studies. Seremetis et al. reported one of the most powerful associations on AML patients examining DRB loci.[11] The largest HLA association study reported in 1987, done on International Bone Marrow Transplant Registry data, showed HLA-Cw3 and Cw4 associations with AML and ALL.[12] Another study in 1979 showed HLA-Cw7 association with ALL.[13] All these studies done to find the HLA disease association were serological ones. A recent study done on Korean population claimed the HLA-C3 association with AML.[14] As stated earlier, HLA is highly polymorphic and its distribution varies with population. The HLA associations with acute leukemia have not been studied in Indian patients, so it is important to study this disease in the Indian population. The objective of the present study was to analyze the allele frequency of HLA Class I (HLA-A, HLA-B, and HLA-C) and Class II (HLA-DRB1 and HLA-DQB1) in Indian acute leukemia patients. This is possibly the first HLA leukemia association study in the Indian population.

Materials and Methods

Patients

This prospective observational study was carried out at a clinical laboratory in the national capital region of India. The study included data collected from patients diagnosed with ALL and acute AML (no further classification on AML and ALL was studied), who visited the clinical laboratory during the study period of January 2013 to December 2019 for the tests and mostly were BMT (bone marrow transplant) candidates. The data were collected from 500 Indian acute leukemia patients (males = 328, females = 172) regarding their age, gender, year of diagnosis, type of disease, and laboratory reports such as peripheral blood counts and medical history. Out of the total 500 samples, AML samples were of 304 (males = 203, females = 121) and ALL samples collected were of 176 (males = 125, females = 51). It was ensured that the patients are not repeated in case they have visited the laboratory more than once during the study period. The study was approved by the Institutional Ethics Committee for Health Related Research.

Control group

The control group consisted of 1000 (males = 663, females = 337) healthy unrelated donors and volunteers from a bone marrow transplant registry (Genebandhu),[15] which is also located in the national capital region of India. All the individuals were of Indian origin. The controls were age- and gender-matched to the patient groups.

Human leukocyte antigen typing

The HLA tissue typing was performed at the clinical laboratory situated in the national capital region. The laboratory has ISO15189 accreditation to carry out HLA typing from the National Accreditation Board of Testing and Calibration Laboratories (NABL). DNA was extracted by NucleospinR DNA extraction kit (MACHEREY-NAGEL, GmbH and Co. KG, Düren, Germany). HLA-A, -B, -C and -DRB1 and -DQB1 typing was performed with polymerase chain reaction with sequence-specific oligonucleotide probe (PCR-SSOP) technique using Lifecodes HLA SSO typing kits (Immucor, Stanford, CT, USA) on Luminex platform.

Statistical analysis

The allele frequencies were calculated by direct count using Microsoft Excel 2007. Comparison between the two observed frequencies was made using Chi-square analysis including Yates correction where the cell frequency was <20>

Results

A total of 500 leukemic patients were studied; all the 500 patients were tested on HLA-A, -B, and -DRB1 loci and 470 were tested on -C and -DQB1 loci. In the AML group, a total of 324 patients were studied; all the 324 patients were tested on HLA-A, -B, and -DR loci and 306 were tested on -C and -DQB1 loci. In the ALL group, a total of 176 patients were studied and all the 176 patients were tested on HLA-A, -B, and -DR loci and 164 were tested on -C and -DQB1 loci. A total of 1000 unrelated healthy individuals as controls were studied at HLA-A, -B, -C, -DRB1, and -DQB1 loci. The allele frequencies were calculated instead of gene frequencies.[16]

Distribution of human leukocyte antigen frequencies between acute leukemia patients and controls

The frequency of HLA-A, -B, -C, -DRB1, and -DQB1 alleles in patients with acute leukemia and control is summarized in [Table 1] and [Table 2]. Significant differences were observed in the allelic distribution of patients with acute leukemia compared vis a vis controls upon studying HLA Class I (-A, -B, and -C) and HLA Class II (-DRB1 and -DQB1) loci. A significant negative association of the HLA-AFNx0111, HLA-BFNx0151, and HLA-DRB1FNx0111 alleles with acute leukemia [26.4% vs. 31.6%, P = 0.0382; 13.6% vs. 17.8%, P = 0.0384; and 16.2% vs. 21.9%, P = 0.0093, respectively; [Table 1] and [Table 2] was observed. In contrast, the frequency of HLA-BFNx0139 and HLA-CFNx0107 allele was significantly higher in patients than that in controls (1.8% vs. 0.3%, P = 0.00372: 49.15% vs. 43.6%, P = 0.0463).

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