|Year : 2018 | Volume
| Issue : 2 | Page : 69-75
Prognostic value of renin gene expression in acute myeloid leukemia
Nahela A Shalaby1, Deena S Eissa1, Naglaa M Hassan2, Nevin M.Al Azhary2, Amany A Saleh3
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical Pathology, National Cancer Institute, Cairo University, Cairo, Egypt
3 Medical Biochemistry Department, Faculty of Medicine, Menoufia University, Egypt
|Date of Submission||17-Oct-2017|
|Date of Acceptance||30-Nov-2018|
|Date of Web Publication||7-Aug-2018|
Nevin M.Al Azhary
National Cancer Institute, Cairo
Source of Support: None, Conflict of Interest: None
Background The majority of acute myeloid leukemia (AML) patients respond to initial chemotherapy, but only a minority of cases achieves long-term survival. Renin, as a part of the renin–angiotensin system, is directly linked to the activities of hematopoietic cytokines during normal hematopoiesis and in myeloproliferative neoplasms. In some hematological malignancies, blast cells have been observed to express renin, which is not found in normal marrow.
Aim The aim of this was to study the expression of the renin gene as a prognostic marker in AML.
Patients and methods Renin expression was measured in 45 AML patients and five controls by using quantitative real-time reverse-transcriptase PCR.
Results Renin expression was detected in 93.3% of AML patients at diagnosis and 100% at relapse. Relapsed AML patients showed higher renin gene expression levels compared to those detected at complete remission and at diagnosis (P=0.000–0.001), respectively. None of our control subjects were positive for renin gene expression. At diagnosis, AML patients with haemoglobin less than 10 g/dl showed higher renin levels compared to those with haemoglobin of at least 10 g/dl. We used median renin gene expression levels to divide AML patients into high and low groups at diagnosis. At diagnosis, the group with higher renin expression showed higher total leukocyte count (TLC) than did the group with lower renin expression. At relapse, the higher renin expression group showed a higher peripheral blood (PB) blast percentage than did the lower renin expression group.
Conclusion Renin expression can predict outcomes in AML patients and could be used as a therapeutic target.
Keywords: acute myeloid leukemia, prognostic factors, qRT PCR, renin gene
|How to cite this article:|
Shalaby NA, Eissa DS, Hassan NM, Azhary NM, Saleh AA. Prognostic value of renin gene expression in acute myeloid leukemia. Egypt J Haematol 2018;43:69-75
|How to cite this URL:|
Shalaby NA, Eissa DS, Hassan NM, Azhary NM, Saleh AA. Prognostic value of renin gene expression in acute myeloid leukemia. Egypt J Haematol [serial online] 2018 [cited 2018 Nov 12];43:69-75. Available from: http://www.ehj.eg.net/text.asp?2018/43/2/69/238767
| Introduction|| |
Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes . AML results from the clonal transformation of hematopoietic precursors through the acquisition of chromosomal rearrangements and multiple gene mutations . Standard induction chemotherapy leads to complete remission (CRs) in 40 to more than 90% of cases, depending on patient age and genetic alterations ,.
In the past few years, research in molecular biology has been instrumental in deciphering the pathogenesis of AML . However, recent large clinical trials have highlighted the need for new innovative strategies in the diagnosis and prognosis of AML because outcomes, particularly for older patients, have not substantially changed in the past three decades .
Renin, as a part of the renin–angiotensin system (RAS), is directly linked to activities of the erythropoietin, thrombopoietin and other hematopoietic cytokines during normal hematopoiesis and in myeloproliferative neoplasms. It is effective even at the stage of primitive embryonic hematopoiesis . The genes for all RAS components have been cloned; gene expression studies could verify their mRNA expression and regulation in many tissues . The structure of the human renin gene is similar to that of human pepsinogen, a closely related aspartyl protease enzyme; this may suggest that renin and pepsinogen have a common evolutionary origin . The human renin gene was isolated from a Charon 4A human genomic library; it spans about 11.7 kb and is assigned to chromosome 1q42 . Blot-hybridization analyses of the isolated gene clone and the total cellular DNA after digestion with restriction enzymes revealed that human renin is encoded by a single gene .
The RAS has been locally identified in most organs. The RAS starts with the conversion of angiotensinogen into angiotensin I (Ang I) through the effects of renin. Ang I is then converted by angiotensin-converting enzymes to angiotensin II (Ang II), which acts on angiotensin receptors (AT): AT1 and AT2 . An intrinsic local RAS has been reported in bone marrow and regulates both the physiological (cell growth, proliferation and differentiation) and pathological (neoplastic hematopoietic cell proliferation) production of blood cells in an autocrine/paracrine manner . Local renin has been suggested to influence leukemic cell production within the bone marrow microenvironment . Blast cells have been observed in some diseases, such as AML, chronic myeloid leukemia and acute lymphoid leukemia, to express renin, but normal bone marrow does not display this expression. The highest frequency of renin expression was observed in the AML patients (47.2% of cases). Renin expression has been reported to disappear during CR of AML and to re-appear with disease relapse . The prognostic value of renin mutations has been investigated in much AML research; however, the clinical significance of its expressions in AML cases remains poorly understood.
| Patients and methods|| |
This study was carried out on 45 bone marrow aspiration (BMA) specimens from AML patients who presented to the Adult Medical Oncology Department, National Cancer Institute, Cairo University and five healthy age-matched and sex-matched controls [collected from donors of bone marrow transplantation (BMT)]. The AML patients were classified as: 15 patients at initial diagnosis; the same 15 patients re-assessed at CR; and 15 patients at relapse (two of whom were our patients and 13 were new cases presenting with relapsed AML). Diagnosis was established after clinical, morphological, cytochemical, flow cytometric and cytogenetic analyses. All cases met AML diagnosis standards. Written informed consent was approved by the Institutional Review Board Ethical Committee of the National Cancer Institute, which follows the rules of Helsinki Institutional Review Board.
Inclusion criterias are as follows:
- Patients proven to have AML.
- Either sex was eligible.
- Age 18–70 years.
- Egyptian patients.
Exclusion criterias are as follows:
- Pediatric age group.
Sample collection and RNA extraction
Bone marrow samples (1 ml) from patients and controls (both control and patient samples were the same type (BM) so that sample quality was consistent) were collected on EDTA and treated with erythrocyte lysis solution; leukocytes were collected and stored in buffer RLT (1×107 leukocytes) at −80°C until used for complete RNA extraction. Total RNA was extracted from BM leukocytes using a QIAamp RNA extraction blood Mini kit (Qiagen; Valencia, CA, USA) according to the manufacturer’s instructions. RNA was converted to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies; Foster City, CA, USA) according to the manufacturer’s instructions and stored at −20°C until used.
Quantitative assessment of gene expression levels was performed using a TaqMan gene expression assay (Applied Biosystems), as recommended by the manufacturer. The Step One Real-Time PCR System (Applied Biosystems) was used for real-time analysis ([Figure 1]). Relative expression of the renin gene was analyzed by the comparative Ct method (2−ΔΔCt), using glyceraldehyde-3-phosphate dehydrogenase as the endogenous control. Data were expressed as the fold change in renin gene expression in patients normalized to the expression levels of the endogenous control and relative to the healthy controls, sequence of primers and probes were shown in [Table 1].
Data management and analysis were performed using SPSS, version 20 (SPSS Inc., Chicago, IL, USA). Qualitative data were presented as number and percentages, while quantitative data were presented as mean, SD and ranges for parametric data and median with interquartile ranges for nonparametric data. The two groups’ qualitative data were compared using a χ2-test; a Fisher exact test was used instead of a χ2-test when the expected count in any cell was less than 5. The two groups’ qualitative data with parametric distributions were compared using an independent t-test, while data with nonparametric distributions were compared using the Mann–Whitney U-test. A Wilcoxon rank test was used to compare the two paired groups’ quantitative data with nonparametric distribution. A Kruskall–Wallis test was used to compare more than two groups regarding quantitative data with a nonparametric distribution. Spearman’s correlation coefficients were used to assess the correlation between two quantitative parameters in the same group. All hypotheses tests were conducted at the α level of 0.05, with a 95% confidence level.
| Results|| |
The present study was carried out on 45 BM aspirate specimens. The AML patients were classified as: 15 patients at initial diagnosis; the same 15 patients re-assessed at CR; and 15 patients at relapse (two of whom were our patients and 13 were new cases presenting with relapsed AML).
Cases at diagnosis and CR included eight (53%) males and seven (47%) females, with a male to female ratio of 1.1 : 1. Ages ranged from 18 to 54 years with a mean±SD of 33.8±9.31 years. The relapsed cases included nine (60%) males and six (40%) females, with a male to female ratio of 1.5 : 1. Their ages ranged from 18 to 61 years with a mean±SD of 38.5±14.06 years, cytogenetic and molecular analysis in AML patients at diagnosis are shown in [Figure 2] and [Figure 3].
|Figure 2 Cytogenetic analysis in acute myeloid leukemia patients at diagnosis.|
Click here to view
|Figure 3 Molecular analysis in acute myeloid leukemia patients at diagnosis.|
Click here to view
Five age-matched and sexes-matched healthy control subjects were chosen from BM donors as candidates for allogenic BM transplantation. They included three (60%) males and two (40%) females, with a male to female ratio of 1.5 : 1. Ages ranged from 18 to 55 years with mean±SD of 33.4±15.16 years. Informed consent was obtained from each patient and control subject before enrollment in the study.
Renin gene expression
The median level of renin gene expression in the control group was one-fold change. In comparison to this, out of the 45 AML patients, 42 (93.3%) patients exhibited high renin gene expression. None of our control subjects were positive for renin gene expression.
Renin gene expression was high in at the time of AML diagnosis and relapse, with significant high expression between renin at relapse and renin at diagnosis (P=0.001) and significant high expression between relapse and CR (day 28) (P=0.000).
Relationship of renin gene expression to the demographic and clinicopathological characteristics of acute myeloid leukemia patients
AML patients with lower haemoglobin (Hb) (<10 g/dl) showed significantly higher renin gene expression levels (P<0.05) than did those with higher Hb (≥10 g/dl). The expression of renin gene was negatively correlated with age (P<0.05). However, no significant differences were detected between renin gene expression levels and other demographic or lab data ([Table 2] and [Table 3]).
|Table 2 Relationship of renin gene expression to qualitative demographic and clinicopathological characteristics of acute myeloid leukemia patients at diagnosis, day 28 complete remission and relapse|
Click here to view
|Table 3 Correlation study between renin gene expression and quantitative data of acute myeloid leukemia patients at diagnosis, day 28 complete remission and relapse|
Click here to view
According to the statistical tests, we chose the median level of renin gene expression as our cut-off. The cut-off was 8.8 at diagnosis, 0.9 at CR and 7.3 at relapse ([Table 4]).
|Table 4 Classification of acute myeloid leukemia patients according to median renin gene expression at diagnosis, complete remission and relapse|
Click here to view
Comparison of low and high renin expression groups in relation to the studied age and hematological data of acute myeloid leukemia patients
AML patients with higher renin expression (≥8.8) showed higher total leukocyte count (TLC) than those with lower expression (P<0.05). However, no significant differences were detected between high and low renin gene expression groups and age, Hb, platelet (Plt), peripheral blood (PB) blasts% and BM blasts% (P>0.05).
At complete remission
There were no significant differences detected between the expression of the renin gene and age, TLC, Hb, Plt count, PB blasts% and BM blasts% (P>0.05).
AML patients with higher renin expression (≥7.3) showed higher PB blasts% than those with lower expression (P<0.05). However, no significant differences were detected between high and low renin gene expression groups and age, TLC, Hb, Plt and BM blasts% (P>0.05).
Comparison of low and high renin expression groups in relation to the other studied demographic and clinicopathological groups of acute myeloid leukemia patients
At diagnosis, the high and low renin expression groups showed no significant differences regarding sex, hepatomegaly, splenomegaly, lymphadenopathy, TLC, Hb, Plt, FAB classification and cytogenetic and molecular analyses (P>0.05). At CR and relapse, the high and low renin gene expression groups showed no significant differences regarding the demographic or lab data groups (P>0.05) shown in [Table 5].
|Table 5 Comparison of low and high renin expression groups in relation to the studied qualitative demographic and clinicopathological groups of acute myeloid leukemia patients at diagnosis, complete remission and relapse|
Click here to view
| Discussion|| |
AML is a heterogeneous hematopoietic stem cell disorder. Many recurrent chromosomal structural variations and mutations contribute to AML pathogenesis ,. Only a limited number of studies has addressed the molecular abnormalities acquired in the relapse of AML, but important discoveries have been made in the genetics and molecular biology associated with therapy-resistant diseases. We still need more details about the extent to which genetic alterations are present at diagnosis and the variations that occur during treatment that allow leukemic cells to survive . The identification of such markers would provide prognostic value in predicting the outcome of AML.
It has been suggested that the presence of the RAS in the BM might affect cellular proliferation and differentiation in physiological and pathological states . Accordingly, renin expression (the initializing enzyme of the RAS) is presumed to have a role in AML development and can be used as an aberrant marker of AML prognosis .
The present work evaluated renin gene expression in blast cells obtained from 45 BM specimens taken from AML patients (15 at diagnosis, 15 at CR and 15 at relapse) and five healthy BM donors using qRT-PCR.
Renin gene expression was detected in 93.3% of our studied AML patients at diagnosis. However, renin gene expression has been reported in 41 , 47.2  and 77%  of studied AML patients at diagnosis. These differences may be due to the different methodologies used, for example, Iglesia et al.  used a SYBER Green I kit in qRT-PCR. None of our control subjects were positive for renin gene expression. Also, Castrop et al.  and Haznedaroglu et al.  reported that the renin gene is expressed in the myeloid blasts of AML patients, but not in the BM of healthy donors.
In our study, AML patients showed higher renin gene expression levels at diagnosis compared to the levels detected at CR (day 28). Moreover, renin gene expression was detected in 100% of studied AML patients at relapse. Relapsed AML patients showed higher renin gene expression levels compared to the levels detected at CR, which were comparable to the levels detected at diagnosis.
These findings are in accordance with Iglesia et al. , Iglesia et al.  and Castrop et al. , who reported renin gene expression at diagnosis followed by its disappearance during hematological remission and reappearance at relapse. They demonstrated that renin gene expression was related to disease activity.
At diagnosis, AML patients with Hb less than 10 g/dl showed higher renin gene expression levels compared to those with Hb of at least 10 g/dl. Moreover, renin gene expression levels were negatively correlated with the patients’ ages. No significant differences could be detected between renin gene expression and the other studied demographic and clinicopathologic characteristics of AML patients at diagnosis, CR or relapse.
The results of Iglesia et al.  agree with our results in denoting no relationship between renin gene expression and the FAB subtypes or the cytogenetic risk markers detected in AML patients at diagnosis. In contrast to Wulf et al. , we did not find a higher incidence of renin positive cases in AML with monocytic component (M4 and M5 FAB types).
In this study, we used the median renin expression levels to divide AML patients into high and low expression groups at diagnosis, CR and relapse. At diagnosis, the higher renin expression group showed higher TLC than did the lower renin expression group. At relapse, the higher renin expression group showed higher PB blast percentage than did the lower renin expression group. On the contrary, no significant differences were detected between the high and low renin expression groups at different disease stages. To the best of our knowledge, no previous research has divided AML patients into renin expression groups or addressed the relationships of high and low renin expression groups with the studied parameters.
| Conclusion|| |
Our study has provided robust evidence for the differential expression of renin gene through the various stages of AML. Renin expression is higher at initial diagnosis and relapse and lower at CR; renin gene expression is absent in healthy BM. These results suggest an important role for renin gene expression in the pathobiology and progression of AML, indicating the possibility of novel therapeutic agents that can substantially improve outcomes in patients with AML.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zhou JD, Yao DM, Li XX, Zhang TJ, Zhang W, Ma JC et al.
KRAS overexpression independent of RAS mutations confers an adverse prognosis in cytogenetically normal acute myeloid leukemia. Oncotarget
Rubnitz JE, Gibson B, Smith FO. Acute myeloid leukemia. Haematol Oncol Clin North Am
Sanz M, Iacoboni G, Montesinos P, Venditti A. Emerging strategies for the treatment of older patients with acute myeloid leukemia. Ann Hematol
Almeida A, Ramos F. Acute myeloid leukemia in the older adults. Leuk Res Rep
Estey E, Döhner H. Acute myeloid leukaemia. Lancet
O’Donnell MR, Abboud CN, Altman J, Appelbaum FR, Coutre SE, Damon LE et al.
Acute myeloid leukemia. J Natl Compr Canc Netw
Beyazit Y, Purnak T, Guven GS, Haznedaroglu IC. Local bone marrow renin–angiotensin system and atherosclerosis. Cardiol Res Pract
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin
Lu H, Cassis LA, Vander Kooi CW, Daugherty A. Structure and functions of angiotensinogen. Hypertens Res
Brian ZR, Jing L, Su L. Ethnic background and genetic variation in the evaluation of cancer risk: a systematic review. PLoS One
Fyhrquist F, Saijonmaa O. Renin-angiotensin system revisited. J Intern Med
Sayitoglu M, Haznedaroğlu IC, Hatirnaz O, Erbilgin Y, Aksu S, Koca E et al.
Effects of imatinib mesylate on renin-angiotensin system (RAS) activity during the clinical course of chronic myeloid leukaemia. J Int Med Res
Koca E, Haznedaroglu IC, Acar K et al.
Renin-angiotensin system expression in the K562 human erythroleukaemic cell line. J Renin Angiotensin Aldosterone Syst
Uz B, Tatonyan SC, Sayitoglu M, Erbilgin Y, Ng OH, Buyukasik Y et al.
Local hematopoietic renin-angiotensin system in myeloid versus lymphoid hematological neoplastic disorders. J Renin Angiotensin Aldosterone Syst
Rodríguez-Lázaro D, Cook N, Hernández M. Real-time PCR in food science: PCR diagnostics. Curr Issues Mol Biol
Montgomery H, Humphries SE, Leung PS. Renin-angiotensin systems: the new frontier. Int J Biochem Cell Biol
Tatonyan SC, UZ B, Sayitoglu M et al.
Renin–angiotensin system (RAS) expressions in myeloid leukemic cell lines. Int J Hematol Oncol
Bachas C, Schuurhuis G, Zwaan C, van den Heuvel-Eibrink M, den Boer M, de Bont E et al.
Gene expression profiles associated with pediatric relapsed AML. PLoS One
Deng A, Ning Q, Zhou L, Liang Y. SIRT2 is an unfavorable prognostic biomarker in patients with acute myeloid leukemia. Sci Rep
Hackl H, Astanina K, Wieser R. Molecular and genetic alterations associated with therapy resistance and relapse of acute myeloid leukemia. J Hematol Oncol
Iglesia S, Casares M, López-Jorge CE. Renin expression in acute leukaemia. J Renin Angiotensin Aldosterone Syst
Beyazit Y, Aksu S, Haznedaroglu IC, Kekilli M, Misirlioglu M, Tuncer S et al.
Overexpression of the local bone marrow renin-angiotensin system in acute myeloid. J Natl Med Assoc
Iñigo Sde L, Casares MT, Jorge CE, Leiza SM, Santana GS, Bravo de Laguna SJ et al.
Relevance of renin expression by real-time PCR in acute myeloid leukemia. Leuk Lymphoma
Castrop H, Höcherl K, Kurtz A, Schweda F, Todorov V, Wagner C. Physiology of kidney renin. Physiol Rev
Haznedaroglu IC, Malkan UY. Local bone marrow renin-angiotensin system in the genesis of leukemia and other malignancies. Eur Rev Med Pharmacol Sci
Wulf GG, Jahns-Streubel G, Nobiling R, Strutz F, Hemmerlein B, Hiddemann W et al.
Renin in acute myeloid leukaemia blasts. Br J Haematol
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]