Hepcidin in Children and Adults with Acute Leukemia or Undergoing Hematopoietic Cell Transplantation: A Systematic Review

Simple Summary In this systematic review, we summarized the observational studies on hepcidin in patients treated for acute leukemia or undergoing hematopoietic cell transplantation (AL/HCT). Thanks to the rigorous methodology used, we were able to trace the available literature conscientiously and draw the following conclusions: (1) in both children and adults with AL and qualified for HCT, hepcidin levels are high regardless of the phase of the disease or iron resources;, (2) AL therapy, and HCT in particular, may affect hepcidin levels, but the data, especially for children, are fragmented; (3) pre-HCT hepcidin levels may help predict post-HCT outcomes; (4) there is a need to standardize the determination of hepcidin levels in the clinical setting. We find a very large discrepancy in the reported mean and median hepcidin levels, both in healthy subjects and in AL. This significantly hinders the interpretation and comparison of the results. Abstract Objectives: The association between hepcidin and acute leukemia (AL) or hematopoietic cell transplantation (HCT) in children and adults remains obscure. We aimed to assess this potential relationship through a systematic review of observational studies. Methods: An electronic search of three databases, including PubMed, Scopus, and Web of Science Core Collection, was performed up to 31 March 2022. Two independent reviewers assessed the search results according to predetermined inclusion and exclusion criteria, following PRISMA guidelines. Results: Of the 3607 titles identified, 13 studies published between 2008 and 2021 met the inclusion criteria. Most studies included a moderate number of participants and controls and used enzyme-linked immunosorbent assay (ELISA) to determine serum hepcidin levels. The principal findings: (1) serum hepcidin levels in patients with AL or undergoing HCT are increased compared to controls, regardless of the patient’s age and the phase of disease treatment; (2) AL therapy and HCT significantly influence serum hepcidin levels; (3) serum hepcidin may predict a worse outcome in patients with AL and post-HCT. Conclusions: This systematic review provides an overview of observational studies that deal with the association of hepcidin with AL and HCT. Although disturbances in iron metabolism are common in AL and HCT, and hepcidin seems to play a cardinal role in their modulation, more extensive research is needed.


Introduction
Acute leukemias (AL) are belligerently progressive neoplasms of the bone marrow, characterized by clonal expansion of immature and highly undifferentiated hematopoietic cells [1,2]. In conformity with the 2020 Global Cancer Statistics study, leukemias were diagnosed worldwide in over 474,000 patients with over 311,000 mortalities [3]. Despite substantial progress in AL patient management, these diseases remain a grievous clinical concern in pediatric and adult groups [4,5].
It is becoming increasingly clear that iron overload is an exceptionally influential component of the pathophysiology of AL, and, as recent studies show, this over-abundance is a fundamental factor that may negatively affect the outcome of patients [6,7]. Although iron overload is observed in some patients with AL at diagnosis [8,9], the leading causes of this phenomenon are frequent blood transfusions [10,11] during chemotherapy treatment [12][13][14][15]. Notably, many investigations revealed a close relationship between iron overload-primarily defined as hyperferritinemia-and poor prognosis in patients undergoing hematopoietic cell transplantation (HCT) [8,16,17]. This was also confirmed in a meta-analysis of 25 studies with 4545 patients undergoing HCT, which demonstrated that pre-transplantation hyperferritinemia has a negative prognostic role and is associated with decreased overall survival and progression-free survival, as well as a higher incidence of non-relapse mortality and bloodstream infections [18].
Due to the extreme toxicity of iron in the human body, an immeasurably precise mechanism operates to control its levels inside and outside of cells. Many proteins are involved in this machinery, but hepcidin is of crucial importance. Produced by the hepatocytes, this small 25-amino acids protein inhibits iron absorption and releases from tissue resources by degrading ferroportin, the sole known cellular iron exporter [19]. Considering the paramount role of hepcidin in iron metabolism and the disturbances noted in AL and after HCT, it is understandable that researchers were looking for the role of hepcidin in the course of diseases.
Studies published to date, also by our team, suggest that hepcidin levels are high in both children and adults with AL [20,21]. Most studies, however, focus on assessing hepcidin levels in HCT patients as a predictor of adverse events such as infections, acute graft-versus-host disease (aGVHD), and poor overall survival [22][23][24]. That notwithstanding, the disorganized data on the relationship between hepcidin, AL, and HCT may cause an earnest misinterpretation of the role of hepcidin in the pathophysiology and prognosis of the disease process or the post-transplant patient response.
To clarify this issue, we performed a systematic review of studies reporting the association between hepcidin levels, AL, and HCT. As far as we are aware, our study is the first that exclusively focuses on the assessment of hepcidin in these patient groups.

Search Methodology
We conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Supplementary Table S1) [25] and prospectively registered the review on PROSPERO (identifier: CRD42022323952).
We comprehensively searched the PubMed, Scopus, and Web of Science Core Collection electronic databases to identify studies published until 31 March 2022 (date of the last search), with detailed search terms for: "hepcidin", "leukemia", and "hematopoietic stem cell transplantation". The full PubMed search strategy is shown in Supplementary Table S2 and was appropriately translated for the other two databases. There were no restrictions on language or publication type. We also hand-searched the bibliographies of all the included studies and relevant review articles to identify any remaining studies. Duplicate studies were manually deleted using Zotero, version 6.0.4 (Corporation for Digital Scholarship, Vienna, VA, USA).

Study Selection
Two reviewers (A.S. and M.Ł.) made the initial selection of the studies based on titles and abstracts. Next, we obtained the full texts of the studies that seemed to fulfill the inclusion criteria for evaluation. Any discrepancies were resolved by consensus, referring back to the studies, in consultation with a third reviewer (J.S.).

Inclusion Criteria
Studies were deemed eligible if they met the following criteria: (1) were observational (case-control, cohort, or cross-sectional studies) [26] and (2) included original data relevant to measuring hepcidin levels in pediatric and adult AL patients or those undergoing HCT. Stages of disease or treatment modalities were not the criteria for excluding the study from the systematic review. We did not set a minimum number of patients as a criterion for a study's inclusion; however, case reports were not included in the systematic review.

Exclusion Criteria
Studies were excluded if they included patients with diseases other than AL-unless patients with AL constituted only a part of the study group. When AL patients comprised only a portion of the study group, we included such studies in the systematic review. For example, if the study group included patients with AL and myelodysplastic syndromes (MDS), the study was included in the current systematic review. Other exclusion criteria were insufficient data on hepcidin (e.g., lack of numerical values of hepcidin levels) and studies published in non-English languages. Clinical trials, reviews, case reports, editorials, comments, position articles, guidelines, chapters of books, conference proceedings, and nonhuman studies were also excluded.

Data Extraction
Relevant data from the included studies were extracted by two reviewers (M.Ł. and J.S.). From each eligible study, data were captured on the following: general study information (first author, year of publication, study design, and study location), participant characteristics (sample size, age, sex, diagnosis, and therapeutic modalities), details relating to the assessment of hepcidin (type of biospecimen, measurement time with corresponding detection method, and hepcidin levels), and, lastly, each study's main findings. All data were extracted from the published studies; we did not contact the corresponding authors to collect further information.

Quality Assessment
We assessed the methodological quality of the included studies by using the Newcastle-Ottawa (NOS) scale for case-control and cohort studies [27], adapted for cross-sectional studies [28]. The NOS score is recommended for assessing the quality of nonrandomized studies [29]. As originally developed, NOS consists of eight items in three domains: selection (total score 4), comparability (total score 2), and exposure for case-control studies or outcome for cohort studies (total score 3). The highest total score is nine points. The NOS, as adapted for cross-sectional studies, consists of seven items in three domains: selection (total score 5), comparability (total score 2), and outcome (total score 3), with the highest total score being ten points. A total score of 3 or less was considered low quality, 4 to 6 was considered moderate quality, and 7 to 9 (10 for cross-sectional studies) was deemed high quality [30]. Any discrepancies in the quality assessment were discussed and resolved by the two reviewers (A.S. and M.Ł.).

Literature Search
Our systematic search identified 3607 unique citations. Of these, 3439 (95%) were found in Scopus, 106 (3%) were found in Web of Science, and 62 (2%) were found in PubMed. No additional studies were identified through our hand search of references from published studies, relevant reviews, and previous meta-analyses.
After adjusting for duplicates, the searches provided a total of 2949 citations, of which 2802 were excluded based on abstract and title. One hundred forty-seven articles underwent full-text review, and one hundred thirty-four were excluded. The remaining thirteen met all inclusion criteria [20][21][22]24,[31][32][33][34][35][36][37][38][39]. There was no disagreement about any of the studies selected for final inclusion in the systematic review. Most studies were excluded because they were irrelevant to the current study subject (n = 45). A full list of excluded studies and reasons for exclusion is available in Supplementary Table S3. Due to the study design's high heterogeneity, we did not use formal meta-analysis techniques. The flow of study selection is reported in Figure 1. of the studies selected for final inclusion in the systematic review. Most studies were excluded because they were irrelevant to the current study subject (n = 45). A full list of excluded studies and reasons for exclusion is available in Supplementary Table S3. Due to the study design's high heterogeneity, we did not use formal meta-analysis techniques. The flow of study selection is reported in Figure 1.

Hepcidin Assays
All included studies specified the time at which biological samples were obtained. Eight studies have documented pre-transplant hepcidin levels [22,24,[31][32][33][34][35]38], six posttransplant hepcidin levels [21,31,32,34,35,38], four before treatment [20,21,36,37], two during treatment [36,39], three at remission [20,36,37], and one after treatment [21]. Five studies had three time points of assessment [20,34,35,38,39]. Data on the collection, preparation, or storage of biological samples were described in five studies [21,22,24,33,37]. For the testing of biological materials, twelve of the studies evaluated hepcidin levels in the blood (serum or plasma) [20][21][22]24,31,32,[34][35][36][37][38][39] and the other one in blood and urine [33]. Different hepcidin assays were applied in the selected studies. Nine studies used enzyme-linked immunosorbent assay (ELISA) kits from different suppliers [20,21,32,[34][35][36][37][38][39]. However, the most commonly employed kit in the four studies was DRG Instruments GmbH (Marburg, Germany) [34,35,37,38]. The mass spectrometry (MS) methods were also used to measure hepcidin levels in four studies [22,24,31,33]. Most of the selected studies did not provide sufficient data regarding the methods applied for hepcidin measurement. Only one study reported assay range, detection limit, and intra-and inter-assay coefficient of variation (CV) for hepcidin assay [21]. Two studies reported a normal range for serum hepcidin, according to manufacturers [32,38]. Blinding of laboratory personnel to the clinical characteristics and patients' outcomes was reported in one study [21]. For the standardization of the results and straightforward comparisons between studies in this systematic review, the hepcidin levels are presented in nanograms per milliliter (ng/mL) for each study included. Table 3 compares blood hepcidin levels with a commonly used marker illustrating iron metabolism, i.e., serum ferritin levels. As expected, ferritin levels were very high in the patients in the included studies, which is also related to the number of packed red blood cells (PRBCs) transfused (Table 3).    Tables 1 and 2; however, the re-presentation of the hepcidin levels facilitates its comparison with those of ferritin. † The units of the ferritin levels are standardized and presented as micrograms per deciliter (µg/dL). The reference values were based on data from the "WHO guideline on use of ferritin concentrations to assess iron status in individuals and populations" (https://www.who.int/ publications/i/item/9789240000124 (accessed on 4 July 2022)). ‡ We wrote 'pre-transplant' on purpose, not pre-HCT because the patient group included those who underwent HCT and those who underwent liver and kidney transplants. # The unit in which the authors reported the ferritin levels was left. However, it seems that a mistake was made here (e.g., when calculating the levels for all patients (n = 112), we receive extremely high values, i.e., 1730.0 µmol/L = 76985000.0 µg/dL). Abbreviations: AL = acute leukemia; ALL = acute lymphoblastic leukemia; eLPI = enhanced labile plasma iron; HCT = hematopoietic cell transplantation; ND = not determined; PRBC = packed red blood cells; SC = stem cell.

Hepcidin Levels in Childhood Leukemia
Three case-control studies [21,36,37] and one cross-sectional study [39] evaluated hepcidin levels in childhood leukemia. The studies included ranged in size from 40 [36] to 67 patients [21] and from 17 [37] to 20 controls [36], for a total number of 213 patients and 55 controls. Most of the included patients were diagnosed with acute lymphoblastic leukemia (ALL). The main message from case-control studies is that hepcidin levels are significantly higher in children with AL than in controls, regardless of the stage of the disease [21,36,37]. Hepcidin levels vary between phases of the disease [21,36,37,39], and they appear to decrease for cases of childhood leukemia in remission when compared to the levels at the time of diagnosis [36,37]; however, a significant difference was found only in one study [36]. Two studies demonstrated that hepcidin levels are lower during maintenance therapy [36,39]. Particular attention should also be paid to the wide range of serum hepcidin levels in both AL and controls (Table 1), e.g., 58.45 ng/mL [37] to 387.6 ng/mL [36] at the diagnosis of the disease. In the controls, the differences are even more noticeable (ten times lower levels in one study [37] compared to another [36]). This is a supplemental factor, due to which we did not perform a meta-analysis.

Hepcidin Levels in Adult Leukemia
Four case-control studies [20,31,32,34], four cohort studies [22,24,35,38], and one crosssectional study [33] were used to evaluate hepcidin levels in adulthood leukemia. The studies included ranged in size from 31 [31] to 166 patients [24] and from 17 [31] to 50 controls [34], for a total number of 568 patients and 99 controls. In the case of studies in adult patients, we found significant heterogeneity in the populations; however, AML seems to be the most frequently diagnosed.
The main message from case-control studies performed in adults with AL is similar to the conclusion involving childhood studies. Hepcidin levels are significantly higher in adults with AL than in the controls, regardless of the stage of the disease or of patients' iron storage [20,31,32,34]. A single study found a decrease in serum hepcidin levels in patients in remission compared to pre-treatment levels [20]. As the remaining studies [22,24,[31][32][33]35,38] largely concerned patients undergoing HCT, we described their results in the next subsection of our systematic review. As in the pediatric population, studies in adult patients and adult controls showed a wide spread of the mean or median of hepcidin levels (Tables 1 and 2).

Hepcidin Levels in HCT Patients
HCT was performed in adult patients in eight studies (n = 498) [22,24,[31][32][33][34][35]38]. In two studies, a significant increase in serum hepcidin levels was observed one week after HCT compared to pretransplant levels, with normalization of the levels one month after transplantation [31,34]. One study found no differences in the serum levels of hepcidin 10 days before HCT compared to the third month afterward [32]. In turn, the lowest level of serum hepcidin was found before the start of conditioning rather than before stem cell (SC) infusion or on engraftment [35]. High serum hepcidin levels before transplantation are also associated with a higher risk of bacterial infections [22], invasive fungal disease [34], and lower overall survival [24] after HCT. The remaining studies found a positive relationship between pre-transplant serum hepcidin levels and other markers of iron metabolism [33,38].
Only one study investigated hepcidin levels in children undergoing HCT (n = 21): it found that one month after transplantation, serum hepcidin levels were significantly higher compared to the levels at diagnosis or after the end of intensive chemotherapeutic treatment [21].

Discussion
Iron overload is a common secondary complication in patients treated for AL or undergoing HCT and is caused by frequent packed red blood cell concentrate (PRBC) transfusions. Each milliliter of transfused PRBC contains 0.8 mg of iron [40]; thus, repeated transfusions ponderously contribute to iron accumulation. No other diagnoses in oncology bearing this complication in such an aggrandized grade. Iron overload is the long-term sequelae of blood component therapy, and intensive treatment damages cells, causing clinically relevant homeostasis imbalance. The pathophysiological processes following one another include: PRBC repeated transfusions; iron delivery; ferritin production and storage; iron overload; imbalance in iron regulation; cellular, tissue, and organ toxicity; and finally, organ failure.
There are four significant findings of our study. First, hepcidin increases during intensive chemotherapy of AL, then partially decreases during maintenance therapy or after its completion. Second, in patients undergoing allogeneic HCT, hepcidin increases during the pre-engraftment period and might partially decrease after engraftment. Third, these profiles of serum hepcidin levels seem to be similar in children and adults. Fourth, hepcidin levels correlate with ferritin levels and iron overload status in patients treated for leukemia or undergoing allo-HCT.
From a pathophysiological point of view, hepcidin production in hepatocytes is stimulated by various factors, including iron and inflammatory status, expressed substantially by the upregulation of interleukin-6 (IL-6). The serum levels of hepcidin correlate with the serum levels of ferritin, and both proteins are upregulated by systemic iron overload. In this context, an increase in serum hepcidin levels reflects a regulation mechanism secondary to iron overload. Therefore, it is unsurprising that the ongoing disease process and applied treatment induce inflammation, leading to increased hepcidin synthesis. As such, our observation of high serum hepcidin levels is widely reported in children and adults. New data emerging from our analysis shows that, regardless of the phase of the disease (and thus also of treatment), hepcidin levels remain high in patients compared to controls.
Increased serum hepcidin levels are also the result of the compensation of iron overload. Nevertheless, mechanisms of homeostasis, and return of effective myelopoiesis, including erythropoiesis, cannot cause full utilization of iron excess. In this context, hepcidin in patients treated for AL or undergoing HCT is an ineffective marker of iron overload and metabolism.
No studies directly compare hepcidin levels in children and adults with AL. The profile of hepcidin levels during and after intensive anti-leukemic chemotherapy seems similar and age-independent. However, it is possible that mechanisms of homeostasis and organ abilities to compensate for organ toxicities are better in children than in adults; thus, some improvement, expressed as a decrease in iron overload, ferritin or hepcidin levels, can be expected in the pediatric population [41][42][43].
Hepcidin levels peaked after the conditioning and pre-engraftment phase in transplanted patients, then decreased during engraftment [44]; however, not in each study [35]. This process clinically correlates with the need for frequent blood transfusions before engraftment, which is not the case afterward due to more efficient myelopoiesis and partial iron utilization.
HCT is a perfect model showing the profile of changes in serum hepcidin levels related to iron overload, followed by effective erythropoiesis, presented in three phases. The first phase, on patients' referral to HCT, reflects the status of patients heavily transfused with PRBC, often in chronic inflammatory status caused by chemotherapy-induced mucositis and possible organ toxicity. Thus, pre-transplant serum hepcidin levels are usually high, at least doubled in most studies, compared to leukemic patients on diagnosis, both in children and adults [21,31,34]. In the second phase, during the conditioning and preengraftment phase, the serum hepcidin levels usually increase [34,35], as the intensity of PRBC transfusion is even higher than in non-transplant patients. High hepcidin levels cause a delay in platelet engraftment after HCT [24]. Finally, the third phase starts from the day of engraftment, followed by effective erythropoiesis, resulting in the utilization of iron and decreased ferritin and hepcidin levels. Obviously, it is not possible to utilize all excesses of iron; hence, the status of iron overload persists, causing cellular, tissue, and organ damage impairing their function and leading to worse overall survival [14,24]. In the case of non-transplant leukemic patients, this becomes a two-phasic model. The first phase, during intensive chemotherapy, is characterized by frequent PRBC transfusions and increasing iron overload, followed by increased ferritin and hepcidin levels [21,36]. In the second phase, during maintenance therapy or after cessation of treatment, when effective erythropoiesis is present, the content of iron and levels of ferritin and hepcidin is lowered [36].
The strength of the study is the first systematic review on hepcidin in AL/HCT patients with a new insight into the assessment of serum hepcidin levels in pediatric and adult patients, showing variability in serum profile before, during, and after AL/HCT treatment.
This study has several limitations due to heterogeneity of the studies, relatively low number of patients included in basic studies, lack of studies comparing other novel parameters of iron metabolism, lack of studies comparing children and adults, and lack of long-term analyses of survival outcomes.

Conclusions
To the best of our knowledge, our study is the first to summarize the observational studies on hepcidin in AL and HCT. Thanks to the rigorous methodology used, we were able to trace the available literature conscientiously and draw the following conclusions: (1) in both children and adults with AL and qualified for HCT, hepcidin levels are high regardless of the phase of the disease or iron resources; (2) AL therapy, and HCT in particular, may affect hepcidin levels, but the data, especially for children, are fragmented; (3) pre-HCT hepcidin levels may help predict post-HCT outcomes; (4) there is a need to standardize the determination of hepcidin levels in the clinical setting. We find a very large discrepancy in the reported mean and median hepcidin levels, both in healthy subjects and in AL. This significantly hinders the interpretation and comparison of the results.
In conclusion, we can show that the profile of hepcidin levels in patients treated for AL/HCT is presumably similar in children and adults. Hepcidin levels increase relatively quickly with RBC transfusions during intensive chemotherapy for AL or between the start of conditioning and engraftment after HCT. However, in both settings, it tends to decrease: during maintenance therapy for AL or in the post-engraftment phase of HCT. Nevertheless, the homeostasis mechanisms are not efficient enough, and increased hepcidin levels might be a risk factor for overall survival.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers14194936/s1, Table S1: Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist; Table S2: PubMed search strategy; Table S3: A complete list of excluded studies along with reasons for exclusion; Table S4: The Newcastle-Ottawa Scale (NOS) for case-control studies; Table S5: The Newcastle-Ottawa Scale (NOS) for cohort studies; Table S6: The Newcastle-Ottawa Scale (NOS) for cross-sectional studies.
Author Contributions: A.S. was responsible for the study design and supervision, developed and performed the search strategy, performed full-text literature screening and assessed the quality, extracted the data, analyzed the data and drafted the manuscript, and validated the final version of the manuscript. M.Ł. performed the literature search, performed full-text literature screening and assessed the quality, extracted the data, analyzed the data, drafted tables, and revised the manuscript. J.S. was responsible for the study design and supervision, drafted the manuscript, and validated the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.