Serum estrogen and its soluble receptor levels in Egyptian patients with acute leukemia: case-control study

Acute leukemias are malignant neoplastic diseases that arise from either lymphoid [ALL] or myeloid [AML] cell lines that are distinguished by the proliferation of BM non-functional immature cells and subsequently released into the bloodstream. ALL is prevalent malignancy in young, while AML in older. Diagnosis is usually routinely performed through peripheral blood count and smear then confirmed by BM aspirate. It is remarkable to notice that leukemia can be manifested at high, low, and even at normal leucocyte count. While treatment results have improved steadily over the last decades in younger and adults, limited changes have been in survival among subjects of age > 60 years. Aim of the work is to measure the serum estrogen [E2] and its soluble receptor [ER] levels in acute leukemia patients and extrapolate its possible clinical significance. This study included 40 [20 females and 20 males] healthy volunteers clinically free from any disease, 40 [20 females and 20 males] AML patients, and 40 [20 females and 20 males] ALL. To all subjects, serum E2 and its soluble ER level were investigated by ELISA. Serum E2 [pg/ml] level was lower in AML and ALL female and male patients groups than control group. Serum ER [ng/ml] level was lower in AML and ALL female and male patients groups than control group. Estimation of serum E2 and its soluble ER level is of edifying diagnostic value. Determination of serum E2 and its soluble ER level in AML and ALL patients is of value in deciding treatment therapeutic target protocol.


Background
Acute leukemia is a bone marrow [BM] malignant disease in which the normal hematopoietic marrow cells replaced by an early proliferative myeloid or lymphoid precursors [1][2][3]. Acute leukemias are characterized upon their differentiation into lymphoid or myeloid lineages. In acute lymphoblastic leukemia (ALL), the abnormal proliferation in immature lymphocytes or lymphoid progenitor cells [4].
Two major types of ALL are known B-ALL and T-ALL. AML implicates the myeloid series from which neutrophils, eosinophils, basophils, monocytes, and megakaryocytes are derived [5,6]. Each leukemia type has its own morphological cytochemical and immunological differences, different prognostic markers, and lines of treatment. Prognostic factors could predict treatment outcome in acute leukemia patients, either complete remission after chemotherapy or disease resistance to conventional protocols [7]. A single prognostic factor cannot reliably predict prognosis, but it must be correlated with all available information [8].
Estrogen negatively regulates BM cells proliferation, which turn into progenitors of myeloid and lymphoid. Moreover, significant bone marrow hematopoiesis alteration is present in estrogen insufficiency and in ER knockout mice [9]. Estrogen functions through alpha ER-α and beta ER-β receptor [10]. Estrogen receptor gene is present on chromosome 6 long arm, which is often altered in hematopoietic neoplasms [11,12]. In healthy controls, ER-α is unmethylated compared to acute leukemia patients; hence, it can act as an epigenetic biomarker of leukemia [12]. A previous study found that estrogen suppresses the stem cells differentiation into myeloid and lymphoid. In addition, serum estrogen [E2] has a negative effect which might be by ER-β on immune system [13]. In vitro studies on leukemia have revealed various cytotoxic impact of clomiphen, a wellknown ER antagonist in breast cancer patients [14,15].

Aim of the work
Measure the serum E2 and its soluble ER levels in acute leukemia patients and extrapolate its possible clinical significance as diagnostic markers.

Methods
Subjects submitted work were grouped into the following: group I, 40 healthy [20  At the protocol end and restoration BM cellularity, aspiration of BM was taken. Less than 5% BM blasts were considered complete remission and 2nd induction cycle was taken by those who did not reach complete remission.

Statistical analysis
Data assessment by SPSS program V20.0, the K-S test was used to check normality. Results (Table 2).

Liver function parameters in AML and ALL patients groups and control subjects
The results showed that the mean value of AST levels [U/l] in ALL patients is significantly higher than in control group [P 2 = 0.007]. Moreover Table 3 showed that ALT levels [U/l] in AML and ALL patients were significantly higher than in control group [P 1 ≤ 0.001, P 2 ≤ 0.001]. While, serum albumin concentration [mg/dl] in AML and ALL patients were significantly lower than in control group [P 1 = 0.024, P 2 = 0.007] ( Table 3).

Kidney function parameters in AML and ALL patients and control group
The results showed that in AML and ALL patients, the mean values of serum urea concentration [mg/dl] [P 1 ≤ 0.001, P 2 ≤ 0.001] and serum creatinine concentration [mg/dl] [P 1 = 0.034, P 2 ≤ 0.001] were significantly higher than in control group. Also, the mean value of serum creatinine concentration [mg/dl] in ALL patients group was significantly higher than in those with AML [P 3 = 0.008] ( Table 4).

Mean values of WBC count [× 10 3 /μl], PLT count [× 10 3 / μl], and hemoglobin conc. [g/dl] in AML and ALL patients and control group
The results showed that WBCs count mean value in AML and ALL patients were higher than in control group [P 1 = 0.014, P 2 ≤ 0.001], while Hb conc. and PLT count in both patients groups were lower than in control group [P 1 ≤ 0.001, P 2 ≤ 0.001] ( Table 5).
Correlation of serum ER and E2 levels with different biochemical and hematological parameters in AML and ALL male/female group As presented in Table 6 (Table 7 and Fig. 1B).

Discussion
The role of estrogen receptor has been well established in cancer breast. Several studies have explored its role in     [24][25][26][27] for which anti-estrogens were tried in an attempt to cure these malignancies. Yet the estrogen and its soluble estrogen receptor clinical significance in acute leukemia patients have not been investigated. Our work hypothesizes that determining the E2 and soluble ER levels could provide valuable information in treating acute leukemia patients.
The non-steroidal anti-estrogens [AE] are a vast class of artificial compounds that are derived from triphenylethylene as tamoxifen. They are estrogen antagonists whose cellular effects are not merely by estrogenic blockade [28].
In cells of breast cancer, tamoxifen induces in vitro TGF-B1 and phospholipases expression activates cellular. It can arrest the BC cell cycle in G phase [29]. It has anti-angiogenic action is not interceded via ER [30].
Anti-estrogens [AE] exert oxidative stress, influencing calcium signaling [31], and conduct the action of variant receptors away of ER. Moreover, AEs induce apoptosis via caspase activity [32,33] and antagonize drug resistance [28]. Hayon et al. [28] investigated the ER distinctive effects of anti-estrogens on ALL cell lines. Their findings revealed that anti-estrogens have growth inhibitory effects and by means of apoptosis and opposing of drug impedance.
These effects were confirmed when AEs were but together with other cytotoxic drugs. They added that cell cycle progression block may occur in leukemic cells ER deficient.
In the current work, we assessed the soluble ER in patients with acute leukemia. The mean soluble ER was lower in patients compared to the control in both AML and ALL patients. The low serum soluble ER in patients could reflect a low ER expression on leukemic cells.
Our findings especially in ALL patients whose ER levels were lower than AML agrees with, Hayon et al. [28] who reported that lymphoblastic cells do not express estrogen receptors and the anti-estrogens role in their study which involved apoptosis induction was ER independent. They found that nafoxidine, another antiestrogen, proved to be more potent than tamoxifen or clomiphene.
The difference between the three anti-estrogens could be due to affinity binding difference to anti-estrogen binding sites. We could attribute the low level of ER in acute leukemias to possible methylation and consequently gene silencing. This has been revealed in previous studies which demonstrated that ERs expression could be controlled by genetic and epigenetic mechanisms in human cancers [34,35].
Yao et al. [12], studied CPG promoter methylation of estrogen receptors in leukemia. They used RT-PCR and MSP-PCR in leukemia cell lines and direct DNA sequencing. They reported that only ER α was specifically methylated and inactivated nearly in all acute leukemia patients while no methylation in control group which agrees with our findings where serum ER was elevated in the control upon comparing with the leukemic patients. This highlights that silencing of the gene expressing ER by methylation can be important in pathogenesis of leukemia or it is partially depending on the carcinogenic insult that induced the neoplastic disease [12].
Cytosine methylation inactivates genes participate in neoplasia or tumor suppressor genes. The degree of hyper methylation is due to DNA methyltransferase upregulation.
In the current work, the range of serum level of soluble serum ER was variable and large and this could be explained by that not all AML subgroups do express the ER equally. We could postulate that the AML patient's different behavior is due to either the different leukemic subtypes or to the state of their cholesterol metabolism. Yom-Tov et al. [15] and de Medina et al. [36] stated that anti-estrogen can function as a ligand for anti-estrogen binding microsomal site, generating cell death through cholesterol metabolism regulation.
In addition, the degree of methylation in AML patients all subtypes is not the same. Toyota et al. [37] studied the aberrant methylation profile in AML. They deduced that hyper methylation of some genes associated with reduced levels of their expression and they found that age inversely correlated with the number of methylated genes. This agrees with our study, as we reported an inverse significant correlation between the mean serum ER level and age, yet it was only for male patients, we could not establish this correlation in females whether AML or ALL.
This elucidate relation between methylation and age is significant in older patients retain little genes methylated and that of AML biology in elderly is totally unlike AML young patients. This agrees with Qingli et al. who found a negative association between age and ER this reflects that AML biology in adults is different from that in elderly or the different triggering factors that led to AML.
As regards the serum ER levels was lower in M4 and M5 male patients compared to other subtypes in males yet the difference was not statistically significant.
This could be interpreted by the small sample size or different cell of origin in M4 and M5. Yet, in female patients, the mean level of serum ER in M4 and M5 patients was higher than the other subtypes. This indicates that the different levels of serum ER with specific subtypes highlight different methylation levels.
Moreover, the serum ER lower level in ALL compared to AML patients could be attributed to the occurrence of hyper ethylating phenotypes in ALL than in AML.
This agrees with Toyota et al. [37] whose preliminary data suggested the occurrence of hyper methylation phenotypes in ALL reflecting different gene expression profiles, implying the presence of specific carcinogenic insults such as radiation exposure or previous cytotoxic chemotherapeutic drugs.
In the current work, significant elevated ALT and AST levels were reported in patients versus the control. This was notable in ALL compared to AML patients reflecting that the leukemic impact is more prominent on the liver in ALL patients. The same findings were reported in renal function tests which were more elevated in ALL than AML patients.
Soluble ER can be a biological marker of leukemia. ER α A the isoform in comparison to other isoforms of ER was specifically and highly methylated in leukemic patients and was no methylation in controls [12]. Li et al. [38] added that the different levels of methylated ER reflect different exposure to carcinogenic insults.
In the current work, the estrogen level was elevated in the control than the patients and that was statistically significant both in AML and ALL males and females patients.
In the current work, the mean serum albumin was positively correlated with ER in AML male patients, and this was not the same in AML or ALL female patients.
In the current work, AML male patients had a lower ER level than females. This agrees with previous studies who found a higher ER methylation and subsequent lower level of ER among males. These findings reflect an association between ER levels with sex [37,38].
The significant decline in serum of ER levels and E2 concentration in male and female patients groups with acute leukemias compared to their corresponding normal controls propose the capability of applying any one of these variables in acute leukemia diagnosis to distinguish the patients with acute leukemias from normal controls. This led us to compare the diagnostic power of these indices to decide which of decisive diagnostic value. This comparability also concerned with identification of the precision specificity and sensitivity for each parameter and their corresponding cut-off value. This comparability was achieved through ROC curve plotting in such a way that the greatest plot below the ROC curves consistent with superior diagnostic test.
Serum ER either in male or female patients showed the greater area below the curve [0.926 and 0.880, respectively] followed by E2 [0.870 and 0.808, respectively].
These results indicate that serum ER is superior to serum E2 for diagnosis of male and female acute leukemia patients. Despite serum ER and E2 having been detected in acute leukemia patients, to our knowledge, this is the first work to compare diagnostic significance for serum ER with those of serum E2 with estimation of the precise cut-off value, specificity, and sensitivity of each parameter in acute leukemia patients.