Introduction
Acute myeloid leukaemia (AML) is a rare haematologic malignancy but is the most common subtype of acute leukaemia in adults and has the shortest survival rate (5-year survival = 24%) [
1].The age-adjusted incidence of AML is 4.3 per 100,000 annually, and the median age of diagnosis is 68 years in the United States. With the emergence of an ageing population, the incidence of acute myeloid leukaemia will further increase with poor prognosis in elderly individuals [
2]. Accurate evaluation of prognosis is critical for the management of AML. Currently, according to ELN risk stratification, AML is classified into three prognostic risk groups: favourable, intermediate, and adverse groups [
3]. These are based on both cytogenetics and molecular biomarkers that predict different responses to standard therapeutics and survival. Meanwhile, clinical factors, increased age, and poor performance status are both associated with lower rates of complete remission (CR) and decreased overall survival (OS) [
4]. Multivariate model analyses suggest that other variables, such as platelet count, serum creatinine, or albumin, account for most of the increased risk of treatment-related mortality (TRM) seen in AML patients [
5]. However, these factors have not been added to the current system that differentiates the prognostic subsets in AML patients. For a specific AML patient, it can be difficult to make an accurate evaluation of the prognosis. Therefore, it is of great clinical significance to explore the comprehensive and prognostic factors that reflect the systemic status of the host, and the biological behaviour of leukaemia cells may provide novel and effective therapeutic ideas for AML patients.
Trace elements play a key role in human metabolism. Zinc (Zn) and copper (Cu) are two essential trace elements that are important cofactors for many enzymes and play a critical role in maintaining the integrity and stability of DNA. As two transition metals, Cu and Zn are associated with a variety of biological reduction and oxidation (redox) processes. Increasing data have indicated the possible coincidence and association of these trace element disturbances in the pathogenesis of cancer by dysregulation of the redox enzyme activity [
6,
7]. In B-cell chronic lymphocytic leukaemia(CLL), excess Cu has been considered to be a potent oxidant and has a potential role in carcinogenesis and prognostic value [
8,
9]. Copper is an acute phase reactant that increases in response to infection, injury, and chronic inflammatory conditions. Zinc has been particularly linked to immune processes, inflammation, and the metabolism of oxidative radicals. Recently, a systematic review regarding the association between serum Zn levels and outcomes in hospitalized patients showed that Zn deficiency was associated with longer stay and even death risk, although there were some limitations in the studies [
10]. Therefore, the levels of these trace elements are related to the state of the body itself and the prognosis of malignancy. The serum levels of Zn and Se were significantly decreased and Cu levels were significantly increased in the serum of patients with AML [
11,
12], and one earlier study showed that the levels of these micronutrients might be helpful in the prediction of response to induction chemotherapy in patients treated for AML [
13]. However, we do not know the predictive value of these trace elements for long-term survival in AML patients. Better than the corresponding serum Cu and serum Zn levels, one study indicated that the serum copper to zinc ratio (SCZR) can be an indicator of the extent and prognosis of malignancy [
14].
In this prospective case–control study, we evaluated the association of serum copper level (SCL) and SCZR at diagnosis with survival among younger patients with de novo AML from northeast China.
Discussion
To our knowledge, this is the first and largest study to date that has investigated the influence of SCL and SCZR at diagnosis on survival in specific patients with AML. Patients with acute myeloid leukaemia are also in an acute phase at the time of initial diagnosis. A common feature of these acute conditions is an increase in the Cu to Zn ratio (CZr) [
18]. Hence, in addition to measuring the serum copper and zinc concentrations themselves, paying attention to the ratio between Cu and Zn may have its special significance. Some studies have also revealed that these dysregulation of CZR may have clinical significance, as a prognostic and/or predictive biomarker of treatment response in cancers, and its clinical application may guide the selection of optimal treatment strategies in the future [
19]. In haematological malignancies, one study indicated that elevated serum Cu levels were associated with either relapse or disease progression, whereas normal Cu levels were linked with remission or stable disease. Furthermore, they found an association between high SCL and several adverse prognostic markers in CLL [
20], which was later confirmed by another study and indicated that serum Cu could be a valuable prognostic marker in B-cell chronic lymphocytic leukemia [
9]. However, the association of SCL and SCZR with the survival of AML patients is unclear. The outcome of AML is heterogeneous, with both patient-related and disease-related factors contributing to an individual patient’s long-term survival. Accurate prognostic assessment of AML is imperative but not fixed. A recent study showed the impact of a multimetal score on AML patient survival, which indicated that these metals might indicate prognostic value when applied to a specific population of front-line AML patients [
21]. In our study, in addition to cytogenetic stratification, serum Cu levels were associated with DFS and OS, and SCZR was only associated with OS in these younger de novo adult AML patients. Moreover, SCZR was associated with the ELN risk group and CR1, and the association indicated the prognostic value of these markers in newly diagnosed AML patients.
Essential elements, Cu and Zn, are involved in oxidative stress in cells, and the alterations of these trace elements are significant and can play a role in the occurrence, development, and even prognosis of cancers [
8,
9,
11,
12,
22]. Studies from different countries have shown the levels of these trace elements with regional characteristics. In contrast to our results, data from MD Anderson in the United States suggest that serum zinc levels are not significantly reduced in patients with acute myeloid leukaemia [
21]. However, most investigations reported an increased Cu concentration and a decreased Zn concentration in AML patients, and a meta-analysis analysed the data and confirmed this phenomenon [
11,
23,
24]. However, no data were available about the serum Cu and Zn status in patients with AML from Northeast China. In this study, we enrolled 105 newly diagnosed adult AML patients and first confirmed the results in these AML patients from our region. In 82 controls, the median serum Zn level was 0.861 mg/L, similar to recent results from our centre [
25], but was significantly lower than the median levels reported in other studies in China, such as among healthy residents of Jinan, China (Zn level, 1.32 ± 0.49 mg/L) [
26]. Compared to healthy controls, the serum levels of Zn in AML patients were significantly decreased, while the serum levels of Cu were significantly increased (Table
1). Meanwhile, the SCZR showed a significant difference between AML patients and healthy controls. However, the clinical significance of these changes is not clear.
In this study, serum Cu levels were positively related to the blast percentage of bone marrow and negatively related to
CEBPAdm. Deficiency or overload of Cu is associated with various human diseases. Excessive free Cu can disturb Zn homeostasis, which may compromise the antioxidant defence mechanism, thereby increasing oxidative stress [
27]. In this study, the inflammatory parameter CRP, which is also known as a positive acute phase protein, was mostly related to the serum Cu level. This, in turn, indicated that the increase in Cu levels might be the result of inflammatory conditions in AML patients. Ceruloplasmin (Cp), a six-copper-containing protein synthesized and secreted mainly by the liver, is a major Cu carrier in the plasma. It has been shown that Cp may increase during the acute phase reaction together with other acute phase proteins following tissue trauma or acute infection. IL-6, a proinflammatory cytokine, seems to be specifically involved in the synthesis of Cp through the transcription factor FOXO1 [
28]. In this study, CRP, whose production is stimulated by IL-6 and closely related to the Cu level, indirectly supported this evidence. Moreover, in AML, a gene encoding Cp was upregulated [
29]. These data might explain why the high Cu level was the result of the high expression of Cp in patients with AML. In addition to CRP, a high level of Cu was related to a lower ALB level and a high blast percentage in bone marrow but was neither related to cytogenetic risk stratification nor did it affect the CR rate in AML patients. These results indicated that the adverse impact of Cu on the clinical outcomes of AML patients was due to the imbalance of homeostasis itself and might be related to its important role in angiogenesis and leukaemia cell growth [
30].
In this study, serum Zn levels were lower among 75.8% of the AML patients. The serum concentrations of zinc and copper appear to be less sensitive to nutritional changes except during periods of severe deficiency or supplementation [
31].This indicated that most AML patients were under a severe condition of Zn deficiency in the circulation, although this was not associated with the clinical outcomes of AML patients. Zinc is essential for a variety of enzymes and transcription factors that regulate DNA damage repair, DNA replication, cell apoptosis, and response to oxidative stress, and may upregulate the expression of tumor suppressor proteins such as p53, and zinc deficiency is also associated with immune dysfunction [
32,
33]. In a randomized, controlled study of children and adolescents receiving chemotherapy for leaukemia, zinc supplementation reduced the number of infectious bouts and positively affected children's nutritional status and weight gain [
34]. In another study, administration of 150 mg of zinc gluconate daily facilitated a reduction in the severity and duration of oropharyngeal mucositis in leukaemia patients [
35]. The functions of the thymus and circulating T cells are highly sensitive to chemoradiotherapy, and Zn plays an important role in thymic function and immune homeostasis. A study of the role of zinc as a chemoprotectant found that after autologous stem cell transplantation high-dose oral zinc resulted in increased T-cell receptor excision rings and CD4 + naive lymphocytes and prevented torque tenovirus reactivation [
36]. Zn possesses antioxidant and anti-inflammatory properties. The antioxidant properties of zinc are manifested in increased activation of antioxidant proteins and enzymes, such as glutathione and catalase. Studies have shown that zinc supplementation has an inhibitory effect on immune activation, and zinc deficiency further aggravates the activation of the NF-κB system in the case of severe infection. In vitro studies have shown that zinc reduces the activation of NF-κB and its target genes, such as TNF-α and IL-1β, and increases the gene expression of A20 and PPAR-α, two zinc finger proteins with anti-inflammatory properties [
37].
Compared to single trace elements, the positive value of SCZR was meaningful, and the ratio suggests that patients were in a condition of oxidative stress [
27]. While oxidative stress plays an important role in the occurrence and development of leukaemia and is closely related to the treatment and prognosis of leukaemia [
38]. Meanwhile, the SCZR was an indicator of the nutritional status of Zn [
39]. Zn deficiency should be highly suspected in individuals with high SCZR. It is assumed that 75%–85% (9–14 μmol/L) of Zn in the body is bound to albumin, which is the major part of the Zn exchange pool. In this study, SCZR was closely related to ALB and CRP levels, which were reported to be associated with adverse outcomes in patients with newly diagnosed AML undergoing intensive induction [
40,
41]. Previous studies revealed the validity of the SCZR for the severity of nutritional status, inflammation, oxidative stress, immune dysfunction, and infection associated with Zn deficiency [
42]. One study suggested that an increased SCZR, especially serum Cu concentration, was associated with an increased risk of incident infections in middle-aged and older men in Eastern Finland [
43]. In our study, we observed a higher incidence of nonrelapse death, where infection was one of the crucial causes. Furthermore, we observed that Cu levels were associated with infection severity during induction chemotherapy and the association of SCZR with PNI, which was related to the nutrition status of the AML patients. This indicated that SCL and SCZR could serve as biomarkers in the general evaluation of nutrition status, oxidative stress, and stability of the internal environment. Meanwhile, SCZR was positively related to ELN risk stratification, and bone marrow blast percentage was negatively related to
CEBPA dm. These results indicated that SCL and SCZR could present both patient-related factors and leukaemia-related factors that were associated with the prognosis of AML. Therefore, they may serve as important predictive biomarkers for the clinical outcomes of AML patients undergoing intensive chemotherapy.
The strengths of our study include the prospective case-cohort study with newly diagnosed AML patients, and all patients received standardized 3 + 7 induction chemotherapy and high-dose cytarabine-based consolidation treatment. In addition, we used inductively coupled plasma spectrometry methods for the measurement of serum element levels, which is considered to be the most reliable method for trace element determination. Moreover, the current first-determined associations of SCL and SCZR with clinical outcomes of AML may provide more information about the biological effect of these trace elements on AML prognosis; at the same time, regulating and targeting the homeostasis of trace elements may have important therapeutic implications [
19].
There were several limitations in the current study. First, the sample size of the patients was relatively small. This may lead to limited power for multivariable modelling in assessing the independent prognostic values of cytogenetics, SCL, and SCZR simultaneously. The prognostic values of SCL and SCZR and DFS or OS need to be confirmed with more studies with larger sample sizes in prospective clinical trials. Second, this is a hospital case–control cohort study, and there may exist some choice bias with the population enrolled. Randomized controlled trial studies are needed to determine whether regulation of these trace elements can improve the prognosis of AML patients.
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