Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 34  |  Issue : 1  |  Page : 291--296

Role of homeobox-A9 gene expression in patients with de novo acute myeloid leukemia


Samia H Kandel1, Iman A Ahmedy1, Safaa I Tayel2, Azza R Mohamed3,  
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Clinical Pathology Department, Ashmoun General Hospital, Menoufia, Egypt

Correspondence Address:
Azza R Mohamed
Ashmoun, Menoufia 32717
Egypt

Abstract

Objective To study the clinical significance and the prognostic value of homeobox-A9 (HOXA9) gene in patients with newly diagnosed acute myeloid leukemia (AML). Background HOX genes are transcription factors. In humans, the 39 HOX genes are organized into four genomic regions (the HOXA, B, C and D clusters), located on four chromosomes (chromosomes 7, 17, 12, and 2, respectively). They play important roles during embryogenesis. The characteristic expression of HOX genes can also be detected in different adult tissues. Patients and methods This prospective case–control study was conducted on 60 patients with newly diagnosed AML and 40 healthy patients serving as a control group. They underwent a full history, clinical examination, complete blood count, bone marrow examination, and quantification of HOXA9 in blood by quantitative real-time PCR assay. Results From the results of this study, we found that HOXA9 was higher in patients with AML than healthy controls (P < 0.001). The highest incidence was found in AML M7 and M5, in patients with AML with older age, with hepatomegaly and splenomegaly, in patients with AML with unfavorable cytogenetic criteria, and in patients with AML who did not achieve complete remission. Conclusion HOXA9 is higher in patients with AML than controls. High HOXA9 expression has a negative prognostic effect on patients with AML and poorer response to chemotherapy. Moreover, it represents an independent prognostic factor mediating chemotherapy.



How to cite this article:
Kandel SH, Ahmedy IA, Tayel SI, Mohamed AR. Role of homeobox-A9 gene expression in patients with de novo acute myeloid leukemia.Menoufia Med J 2021;34:291-296


How to cite this URL:
Kandel SH, Ahmedy IA, Tayel SI, Mohamed AR. Role of homeobox-A9 gene expression in patients with de novo acute myeloid leukemia. Menoufia Med J [serial online] 2021 [cited 2021 Dec 7 ];34:291-296
Available from: http://www.mmj.eg.net/text.asp?2021/34/1/291/312000


Full Text



 Introduction



Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by the accumulation of acquired somatic genetic alterations in hematopoietic progenitor cells that alter normal mechanisms of self-renewal, proliferation, and differentiation [1]. AML is characterized by differentiation arrest and accumulation of myeloid blasts in the bone marrow (BM) that lead to insufficiency of normal hematopoiesis [2],[3]. Approximately 70–80% of patients with de novo AML achieve an initial complete remission (CR) after chemotherapy, but long-term disease-free survival remains as low as 30–50% [4]. Homeobox (HOX) genes are members of the transcription factor. In humans, the 39 HOX genes are organized into four genomic regions (the HOXA, B, C, and D clusters) located on four chromosomes and play a crucial role in embryonic development [5],[6],[7]. Many studies had elucidated the role of homeobox-A9 (HOXA9) in solid cancers such as breast, ovarian, colon, prostatic, kidney and lung [8], but few of them had addressed its role in hematological malignancies [9]. We aimed in this study to shed light on the role and the prognostic value of quantitative real-time (QR)-PCR expression of HOXA9 in patients with newly diagnosed AML.

 Patients and methods



This prospective case–control study was conducted in the Clinical Pathology Department in collaboration with the Medical Biochemistry and Molecular Biology DEPARTMENTS, Faculty of Medicine, Menoufia University, from January 2017 to October 2018. It included 100 studied participants who were categorized into the following groups: group I included 60 patients with newly diagnosed AML (36 males and 24 females, with age ranged from 18 to 81 years). Patients with AML were documented by clinical picture, blood picture, and BM examinations. Group II included 40 (21 males and 19 females with age ranged from 17 to 85 years) control participants who were age-matched and sex-matched with the AML patient group. Informed written consent was taken from every participant in the study and was approved by the Research Ethics Committee in Menoufia Faculty of Medicine. It was conducted according to the Helsinki Declaration. All patients underwent the following: full history taking, general examination, and laboratory investigations.

Exclusion criteria included patients with any other hematologic malignancies or hematologic disorder and patients or families who refused or discontinued the study. Favorable criteria for AML include young age (<10 years), no lymphadenopathy or central nervous system infiltration, leukocyte counts up to 50×103, whereas unfavorable criteria include older age, extramedullary involvement, increased lactate dehydrogenase, and hyperleukocytosis, increased BM blasts, which is associated with a bad outcome. For blood sampling and laboratory investigations, venous blood samples (5 ml) were withdrawn on EDTA tubes. The following investigations were done: complete blood count [XN-1000 (19723); Sysmex, Kobe, Japan], BM assessment (Egypt Company), and HOXA9 gene expression by (Cairo, Egypt) QR-PCR assay. Measurement of HOXA9 gene expression was quantitatively. Quantitative assay of HOXA9 gene expression in whole blood using reverse transcriptase PCR QR-PCR technique was done as follows: total RNA isolation from whole blood using kits supplied by Pure Link RNA Mini kit (Life Technologies, Carlsbad, California, USA), followed by ensuring the quality and purity of RNA. Extracted RNA was stored in −80°C till the time of use. The first step was use of PCR for cDNA synthesis (reverse transcription step) using cDNA reverse transcription kits (Applied Biosystem), using Applied Biosystems 2720 thermal cycler (Singapore). The second step was use of PCR for cDNA amplification (real-time PCR step) The cDNA was used in SYBR green-based QR-PCR for quantification of by SensiFAST SYBR Lo-ROX kit; Bioline, Memphis, Tennessee, USA, using the following designed primers Waltham, Massachusetts, USA (Thermo Fisher Scientific, Invitrogen, USA): HOXA9 gene primers, forward 5'-ATCGATCCCAATAACCCAGCA-3'and reverse 5'-TGGTGTTTTGTATAGGGGCAC-3'. ABL 1 gene primers were used as a reference housekeeping gene, forward 5'-TTCAGCGGCCAGTAGCATCTGACTT-3' and reverse 5'-TGTGATTATAGCCTA AGACCCGGAGCTTTT-3'. Applied Biosystems 7500 software version 2.0.1 was used for data analysis. Relative quantification of gene expression was performed using the comparative ΔΔCt method. The target mRNA is normalized to an endogenous reference gene (ABL1 gene) and relative to a control. Each run was accomplished using melting curve analysis to confirm amplification specificity and primer-dimers absence.

Statistical analysis

Data collected were tabulated and analyzed by the statistical package of the social science (SPSS, version 20; SPSS Inc., Chicago, Illinois, USA) on an IBM personal computer. The χ2-test was used when comparing qualitative data. Odds ratios and 95% confidence intervals were calculated to assess the risk conferred by a particular allele and genotype. The Student t-test was used to test the difference between normally distributed quantitative data among the studied groups. The Mann–Whitney U-test was used to test the difference between not normally distributed quantitative data among the studied groups. Kruskal–Wallis test, a nonparametric test of significance, was used for comparison between more than two groups not normally distributed having quantitative variables. Spearman correlation (r) is a test used to measure the association between two not normally distributed quantitative variables or one quantitative and one qualitative ordinal variable. The receiver operating the characteristic (ROC) curve is a graphic representation of the relationship between sensitivity and specificity at different cutoff points for the diagnostic test. It was used in this study to measure the cutoff point at which HOXA9 predicts acute myeloblastic leukemia. All tests were two tailed, and statistical significance was assumed at a P value equal or less than 0.05.

 Results



This study involved 60 patients with AML, comprising 36 (60%) male and 24 (40%) female, with age ranging between 18 and 81 years old (mean ± SD 50.22±16.12 and median of 50 years). The study also included 40 age-matched and sex-matched healthy individuals as a control group, comprising 21 (52.5%) males and 19 (47.5%) females, with age ranging from 17 to 85 years, with a mean of 42.08±17.28 years. The cutoff value of HOXA9 expression is considered positive when more than 3.40. Moreover, 39/60 (65%) AML cases were HOXA9 positive, whereas 21/60 (35%) were HOXA9 negative. However, 35/40 (87.5%) control group individuals were HOXA9 negative, whereas 5/40 (12.5%) were HOXA9 positive. We revealed that AML group showed a statistically significant higher HOXA9 expression level than the control group (P < 0.001; [Table 1]).{Table 1}

The highest incidence of HOXA9 was found in different (French–American–British) FAB subtypes of acute myeloid leukemia in the following descending order: M7>M5>M1>M6>M4>M2>M3.

Regarding demographic data, HOXA9 expression correlated with older age (P < 0.001). However, it did not correlate with sex [Table 2]. Regarding clinical parameters, HOXA9 expression showed positive significant expression with hepatosplenomegaly (HSM) (P < 0.001). However, central nervous system infiltration and lymphadenopathy did not show any significant statistical relationship with HOXA9 expression.{Table 2}

Regarding the hematologic level, HOXA9 was correlated with low hemoglobin percentage (P < 0.001) and low platelet counts (P < 0.001; [Table 3]). However, it did not correlate with leukocyte counts and percentages of BM blasts [Table 2] and [Table 3]. Regarding the relationship between HOXA9 and the cytogenetic information (normal, unfavorable and favorable karyotypes), it showed positive significant expression with unfavorable cytogenetic AML, for example, t(9:11) positive (P < 0.001) [Table 4].{Table 3}{Table 4}

In this study, 28 patients achieved CR, whereas 32 patients did not achieve it. This study showed that patients with AML with positive HOXA9 expression had lower rates of CR (P < 0.001; [Table 4]. The ROC curve [Figure 1] showed that HOXA9 expression at cutoff of 3.40 or more can discriminate AML cases from healthy control with sensitivity of 63.33% and specificity of 87.50% [Table 5]. Furthermore, univariate analysis revealed that age (years), liver or spleen, and HOXA9 are significant factors in achieving CR, and after multivariate analysis, it was found that high HOXA9 expression was found to be independent factor for prediction of CR [Table 6].{Figure 1}{Table 5}{Table 6}

 Discussion



AML represents ∼80% of acute leukemia cases in adults and 15–20% cases in children. It is the most frequent leukemia in neonates. It is slightly more common in men and in Whites [10]. Several reports have demonstrated that HOX genes are not only potent regulators of embryonic development but also play significant roles in the regulation of many processes in adult organisms [11]. The aberrant expression of HOX genes has been reported in most patients with leukemia. Many studies have elucidated the role of HOX genes in solid cancers such as breast, ovarian, colon, prostatic, kidney and lung [8] but few of them have addressed its role in hematological malignancies [9]. We aimed in the present work to shed light on the clinical significance and the prognostic value of QR-PCR expression of HOXA9 in patients with newly diagnosed AML. We investigated its relation with various clinical, laboratory, and standard prognostic factors.

This study included 60 patients with de novo AML and 40 age-matched and sex-matched healthy control group. Diagnosis of AML was based on blast immunophenotyping, FAB recommendation, and cytogenetic studies. The patients were followed up for 6 months after induction chemotherapy. This study revealed that HOXA9 expression was statistically higher in patients with AML than the control group (P < 0.001). This agreed with Zhào et al. [12] and Li et al. [9] who detected positive HOXA9 expression in patients with AML. It was found that during hematopoiesis, the highest expression of HOXA9 gene occurs in the stem and early hematopoietic progenitor cells. With maturation, its expression gradually decreases, and it is minimal in differentiated hematopoietic cells [6]. Moreover, HOXA9 has been most intensively studied as it is overexpressed in more than 50% of AML [13]. Moreover, Kingsley et al. [14] found out that HOXA9 dysfunction has been implicated in acute myeloid leukemia, and expression of the gene has been shown to differ markedly between erythrocyte lineages of different stages of development [14]. HOXA9 is expressed on chromosome 7 and the nucleoporin (NUP98) gene is expressed on chromosome 11. However, a gene translocation which may occurs in humans moves NUP98 onto chromosome 7, where it fuses with HOXA9 to form the NUP98-HOXA9 oncogene [15]. This oncogene has been widely implicated in AML, and expression of this oncogene is the single most highly correlating factor for poor AML prognosis [16]. The oncogene has been found to increase proliferative rates of hematopoietic stem cells while impairing their differentiation. A significant relation was found between HOXA9 and different FAB subtypes (P < 0.001). The highest incidence of HOXA9 was found in AML M7 and M5. Zhao et al. [12] showed decrease HOXA9 expression in M2 and M3. Moreover, Goa et al. [17] detected that between AML Ml and M3, AML M3 showed the lowest level of HOXA9 gene expression. M3 subgroup is characterized by the presence of the PML-RARa fusion gene, which generates an aberrant retinoic acid receptor unresponsive to the physiological levels of HOXA9 [18]; thus, these specific molecular genetic aberrations, rather than differentiation per se, underlie the observed differences in HOX gene expression in AML M3. Regarding demographic data, HOXA9 expression level in patients with AML correlated with older age (P < 0.001). Moreover, a positive significant correlation was found between age and HOXA9 in patients with AML. Age was also found to be an independent factor affecting CR achievement in our univariate analysis in AML cases particularly; these findings were in accordance with Zhao et al. [12] who detected positive HOXA9 expression with age older than 60 years. However, Gao et al. [17] found no significant difference between HOXA9 expression and age.

No significant difference was found between sex and HOXA9 expression in AML. These findings were in accordance with Zhào et al. [12]. Regarding clinical parameters, we demonstrated that HOXA9 expression level correlated positively with HSM in patients with AML (P < 0.001). Additionally, HSM was also an independent factor affecting CR achievement in our univariate analysis. However, Gao et al. [17] postulated no significant correlation between HOXA9 expression and clinical parameters in AML cases. Regarding hematological parameters, a significant negative correlation was found between HOXA9 and hemoglobin level (HB) (P < 0.001) and platelet count (P < 0.001). HOXA overexpression inhibits erythropoiesis and megakaryopoiesis, resulting in decrease HB level and platelet count [19]. No significant relation was found between HOXA9 expression level and total leukocytic count and BM blasts, which agreed with Zhao et al. [12] but disagreed with Gao et al. [17], who reported that total leukocytic count was significantly correlated with HOXA9 expression. The present study showed that low HOXA9 expression level was correlated with favorable cytogenetic AML. This agrees with Andreeff et al. [13], who reported similar result in their study. This study showed that patients with AML with positive HOXA9 expression had lower rates of CR (P < 0.001) as compared with HOXA9 nonexpressing patients. Li et al. [20] reported similar results in his study. It was explained that co-expression of HOXA9 suppresses Meis1-mediated apoptosis in a variety of human leukemia cell types, which make leukemia cells resistant to chemotherapy [21]. Furthermore, from the multivariate analysis, we revealed that HOXA9 high expression is not considered an independent prognostic factor predicting CR achievement in patients with AML (P = 0.086).

ROC curve showed that HOXA9 expression at cutoff 3.40 or more can discriminate AML cases from healthy control with sensitivity of 63.33% and specificity of 87.50%, Furthermore, univariate analysis revealed that age (years), hepatomegaly, splenomegaly, and HOXA9 are significant factors in achieving CR, and after multivariate analysis, it was found that high HOXA9 was not found to be an independent factor for prediction of CR.

HOXA9 expression is associated with poor prognostic factors like old age, extramedullary dissemination, and FAB M1 and M7 subtypes. This indicates that HOXA9 expression might be a factor of poor prognosis. Starkova et al. [22] found out that the overexpression of certain HOX genes and their cofactors are known as poor prognostic markers in patients with leukemia.

 Conclusion



HOXA9 displays a negative prognostic effect on patients with AML. Moreover, it represents an independent prognostic factor in predicting CR achievement.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Fröhling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malignancies, pathogenetic and clinical implications. J Clin Oncol 2005; 23:6285–6295.
2Estey E, Dohner H. Acute myeloid leukemia. Lancet 2006; 368:1894–1907.
3Shahab S, Shamsi TS, Ahmed N. Prognostic involvement of nucleophosmin mutations in acute myeloid leukemia. Asian Pac J Cancer Prev 2013; 14:5615–5620.
4Erkut N, Menteşe A, Özbaş HM, Ermantaş N, Sümer A, Örem A, et al. The prognostic significance of soluble urokinase plasminogen activator receptor in acute myeloid leukemia. Turk J Hematol 2016; 33:135–140.
5Cillo C, Cantile M, Faiella A, Boncinelli E. Homeobox genes in normal and malignant cells. J Cell Physiol. 2001; 188:161–9.
6Abramovich C, Humphries RK. Hox regulation of normal and leukemic hematopoietic stem cells. Curr Opin Hematol 2005; 12:210–216.
7Spencer DH, Young MA, Lamprecht TL, Helton NM, Fulton R, O'Laughlin M, et al. Epigenomic analysis of the HOX gene loci reveals mechanisms that may control canonical expression patterns in AML and normal hematopoietic cells. Leukemia. 2015; 29:1279–89.
8Bhatlekar S, Fields JZ, Boman BM. HOX genes and their role in the development of human cancers. J Mol Med (Berl) 2014; 92:811–823.
9Li L, Zhao CT, Cui BL, Wu SL, Liu XD, Su Z, et al. Expression of HOXB4, PRDM16 and HOXA9 in patients with acute myeloid leukemia and its clinical significance. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2016; 24:326–331.
10Lichtman MA, Kaushansky K, Kipps TJ, Prchal JT, Levi MM, editors. Williams manual of hematology. 8th edition USA: Mcgraw–Hill Companies. 2011. pp. 289–306.
11Alharbi, RA, Pettengell R, Pandha HS, Morgan R. The role of HOX genes in normal hematopoiesis and acute leukemia. Leukemia 2013; 27:1000–1008.
12Zhao P, Tan L, Ruan J, Wei XP, Zheng Y, Zheng LX, et al. Aberrant expression of HOXA5 and HOXA9 in AML. Asian Pac J Cancer Prev 2015; 16:3941–3944.
13Andreeff M, Ruvolo V, Gadgil S, Zeng C, Coombes K, Chen W, et al. HOX expression patterns identify a common signature for favorable AML. Leukemia 2008; 22:2041–2047.
14Kingsley PD, Greenfest-Allen E, Frame JM, Bushnell TP, Malik J, McGrath KE. Ontogeny of erythroid gene expression. Blood 2013; 121:e5–e13.
15Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, Toyama K, et al. Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet. 1996; 12:154–8.
16Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G. Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia. Mol Cell Biol. 2001; 21:224–34.
17Gao L, Sun J, Liu F. Higher expression levels of the HOXA9 gene closely associated with MLL-PTD and EZH2 mutations, predict inferior outcome in acute myeloid leukemia. Onco Targets Ther 2016; 9:711–722.
18Roche J, Zeng C, Barón A, Gadgil S, Gemmill RM, Tigaud I, et al. HOX expression in AML identifies a distinct subset of patients with intermediate cytogenetics. Leukemia 2004; 18:1059–1063.
19Crooks GM, Fuller J, Petersen D, Izadi P, Malik P, Pattengale PK, et al. Constitutive HOXA5 expression inhibits erythropoiesis and increases myelopoiesis from human hematopoietic progenitors. Blood 1999; 94:519–528.
20Li DP, Li ZY, Sang W, Cheng H, Pan XY, Xu KL. HOXA9 gene expression in acute myeloid leukemia. Cell Biochem Biophys 2013; 67:935–938.
21Wermuth PJ, Buchberg AM. Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HOXA9 in murine and human cell lines. Blood 2005; 105:1222–1230.
22Starkova J, Zamostna B, Mejstrikova E, Krejci R, Drabkin HA, Trka J. HOX gene expression in phenotypic and genotypic subgroups and low HOXA gene expression as an adverse prognostic factor in pediatric ALL. Pediatr Blood Cancer 2010; 55:1072–1082.