*Review* **Pediatric Myeloid Sarcoma, More than Just a Chloroma: A Review of Clinical Presentations, Significance, and Biology**

**Kristin E. Zorn 1,2, Ashley M. Cunningham <sup>3</sup> , Alison E. Meyer <sup>2</sup> , Karen Sue Carlson 2,4 and Sridhar Rao 1,2,5,\***


**Simple Summary:** Childhood acute myeloid leukemia (AML) remains a cancer with poor overall outcomes. Myeloid sarcomas (MS) are extramedullary masses of leukemia cells that can develop in patients with AML. In children, MS occurs more frequently than described in adults. Their clinical significance in both pediatric and adult patients with AML is unclear. In this review, we aim to summarize the current knowledge of MS in children and its underlying biology in the hopes of sparking future studies and ultimately improving treatment options for children with AML.

**Abstract:** Myeloid sarcomas (MS), commonly referred to as chloromas, are extramedullary tumors of acute myeloid leukemia (AML) with varying incidence and influence on outcomes. Pediatric MS has both a higher incidence and unique clinical presentation, cytogenetic profile, and set of risk factors compared to adult patients. Optimal treatment remains undefined, yet allogeneic hematopoietic stem cell transplantation (allo-HSCT) and epigenetic reprogramming in children are potential therapies. Importantly, the biology of MS development is poorly understood; however, cell-cell interactions, epigenetic dysregulation, cytokine signaling, and angiogenesis all appear to play key roles. This review describes pediatric-specific MS literature and the current state of knowledge about the biological determinants that drive MS development. While the significance of MS remains controversial, the pediatric experience provides an opportunity to investigate mechanisms of disease development to improve patient outcomes. This brings the hope of better understanding MS as a distinct disease entity deserving directed therapeutic approaches.

**Keywords:** myeloid sarcoma; chloroma; acute myeloid leukemia; pediatric

### **1. Introduction**

Myeloid sarcomas (MS) are extramedullary tumors of myeloid blasts forming masses disrupting normal tissue architecture in patients with acute myeloid leukemia (AML) [1–3]. They are also known as myeloblastomas, granulocytic sarcomas, chloroleukemia, and chloromas given the historically green appearance of the tumors resulting from myeloperoxidase exposure to air. Importantly, there is no clearly accepted definition of what qualifies as MS. Most agree that discrete tumor masses of myeloid blasts are MS; however, whether gingival infiltration and masses within lymph nodes, the liver, or spleen should also be considered MS is debated given their propensity for generalized infiltration. Central nervous system (CNS) leukemia is also a challenge with categorization including both cerebral spinal fluid (CSF)-positive disease and CNS infiltrates/masses on imaging. This has made clear, consistent reporting of clinical presentation and outcome data difficult given the lack

**Citation:** Zorn, K.E.; Cunningham, A.M.; Meyer, A.E.; Carlson, K.S.; Rao, S. Pediatric Myeloid Sarcoma, More than Just a Chloroma: A Review of Clinical Presentations, Significance, and Biology. *Cancers* **2023**, *15*, 1443. https://doi.org/10.3390/ cancers15051443

Academic Editors: Saurabh Agarwal and Jianhua Yang

Received: 22 December 2022 Revised: 14 February 2023 Accepted: 21 February 2023 Published: 24 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of consensus within the literature. Given this limitation, the following terms will be used for this review: extramedullary disease and MS. Extramedullary disease will more broadly refer to leukemic disease outside the bone marrow/peripheral blood, while MS will be specific to myeloid blast tumors. These terms are not used interchangeably and are used as defined to more accurately portray the referenced literature, with extramedullary disease as an umbrella term that also includes MS.

MS most frequently presents with a mass in subcutaneous/soft tissue, bone, and skin (also known as leukemia cutis). Case reports include masses and infiltrative involvement in nearly every conceivable tissue including the GI tract, reproductive organs, CNS, heart, lungs, kidneys, and breast [4]. Interestingly, MS, while most often seen concurrently with intramedullary AML, can occur in isolation in the absence of bone marrow disease. MS can also occur in the setting of a preceding hematologic disease such as myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPN). Finally, MS can develop as a relapse following a hematopoietic malignancy, including after allogeneic hematopoietic stem cell transplantation (allo-HSCT) [1,2].

Although AML is seen primarily in older adults with a median age at diagnosis of 68 years, AML accounts for 10 to 15% of acute leukemias in children [5,6]. Pediatric AML differs from AML in adults in terms of clinical course, outcomes, and genomic landscape [7–9]. MS in pediatrics represent an inadequately understood aspect of AML. MS presentations offer another distinction between pediatric and adult AML with opportunities for improvement in diagnosis, management, and further investigation into the biological mechanisms of development and treatment resistance.

This review will discuss the clinical presentations and reported outcomes of pediatric patients with MS including post-allo-HSCT, imaging approaches to diagnosis, and finally, the biology of MS will be addressed. While this review focuses on pediatric MS, important comparisons with adults will also be discussed.

#### **2. Pediatric Clinical Presentation, Incidence, and Outcomes**

Although generally considered a rare presentation, MS and extramedullary AML are common in children with AML. Numerous cooperative groups and large institution studies have reported both characteristics and outcomes associated with extramedullary disease (Table 1). The inconsistent terminology surrounding MS and extramedullary disease prevents direct comparison across these studies. Despite this limitation, these studies provide helpful data about the clinical features and associations seen as well as insight into outcomes.


**Table 1.** Summary of survival outcomes of pediatric patients with AML and extramedullary disease. Described terminology as per original report. Abbreviations: YO, years old; EMD, extramedullary disease; CSF, cerebral spinal fluid; EML, extramedullary leukemia; EMI,


#### *2.1. Incidence* The incidence of MS varies widely, particularly when comparing adults and children.

*2.1. Incidence* 

The incidence of MS varies widely, particularly when comparing adults and children. This is predominantly related to the lack of consistency in MS evaluation and reporting. There is no standard recommendation for patients with AML to undergo screening evaluation for MS and the true incidence is likely higher than that reported given the potential for asymptomatic occult tumors. Pediatric studies describe an incidence of MS ranging from 5.7% to as high as 40% with the expansive definition of extramedullary disease, although most commonly it is between 10 and 25% [10,13]. The distribution of common anatomic sites in children is illustrated in Figure 1. A lower incidence of MS is generally reported in adults (4–9%) with newly diagnosed AML, although this is likely an underestimate considering a recent prospective study [24]. It is unclear why such differences exist between children and adults, but this may be related to differences in diagnostic evaluations performed or inherent differences in the leukemias that children develop compared to adults in terms of mutational spectrum [7–9]. This is predominantly related to the lack of consistency in MS evaluation and reporting. There is no standard recommendation for patients with AML to undergo screening evaluation for MS and the true incidence is likely higher than that reported given the potential for asymptomatic occult tumors. Pediatric studies describe an incidence of MS ranging from 5.7% to as high as 40% with the expansive definition of extramedullary disease, although most commonly it is between 10 and 25% [10,13]. The distribution of common anatomic sites in children is illustrated in Figure 1. A lower incidence of MS is generally reported in adults (4–9%) with newly diagnosed AML, although this is likely an underestimate considering a recent prospective study [24]. It is unclear why such differences exist between children and adults, but this may be related to differences in diagnostic evaluations performed or inherent differences in the leukemias that children develop compared to adults in terms of mutational spectrum [7–9].

*Cancers* **2023**, *15*, x FOR PEER REVIEW 4 of 18

**Figure 1.** Common locations of initial presentation of MS in children with percentage of MS site involvement. Abbreviations: CNS, central nervous system; MS, myeloid sarcoma [11–18,21–23,25]. Figure created with BioRender.com. **Figure 1.** Common locations of initial presentation of MS in children with percentage of MS site involvement. Abbreviations: CNS, central nervous system; MS, myeloid sarcoma [11–18,21–23,25]. Figure created with BioRender.com.

#### *2.2. Clinical Associations 2.2. Clinical Associations*

comes.

*2.3. Outcomes* 

In children, MS is typically associated with a younger age at diagnosis, particularly in infants, and more frequently in males, with 55–75% of patients with MS being male [19,21,22]. Higher WBC counts and hepatosplenomegaly are often seen in the presence of MS, although less consistently. Additionally, the FAB M4 (acute myelomonocytic) and M5 (acute monocytic) subtypes are most commonly associated with MS. Cytogenetically, the most frequently described associations are inv(16), t(8;21), and chromosome 11 abnormalities, namely 11q23, with both deletions and rearrangements [12,15,17–19,26]. By contrast, in adults, a recent study of 1,583 patients identified increased odds of extramedullary disease in patients with *PTPN11*, *NPM1,* and *FLT3-ITD* mutations with no association with inv(16) or t(8;21) and decreased odds of extramedullary disease with *IDH2* and *CEBPA* mutations [27]. Pediatric AML has distinct mutational profiles in comparison to adult AML, and similarly, the AML genetic profile associated with pediatric MS appears distinct from that of adults. This suggests different biologic drivers of pediatric AML and MS In children, MS is typically associated with a younger age at diagnosis, particularly in infants, and more frequently in males, with 55–75% of patients with MS being male [19,21,22]. Higher WBC counts and hepatosplenomegaly are often seen in the presence of MS, although less consistently. Additionally, the FAB M4 (acute myelomonocytic) and M5 (acute monocytic) subtypes are most commonly associated with MS. Cytogenetically, the most frequently described associations are inv(16), t(8;21), and chromosome 11 abnormalities, namely 11q23, with both deletions and rearrangements [12,15,17–19,26]. By contrast, in adults, a recent study of 1,583 patients identified increased odds of extramedullary disease in patients with *PTPN11*, *NPM1,* and *FLT3-ITD* mutations with no association with inv(16) or t(8;21) and decreased odds of extramedullary disease with *IDH2* and *CEBPA* mutations [27]. Pediatric AML has distinct mutational profiles in comparison to adult AML, and similarly, the AML genetic profile associated with pediatric MS appears distinct from that of adults. This suggests different biologic drivers of pediatric AML and MS or extramedullary disease development, and potential significance for treatment and outcomes.

or extramedullary disease development, and potential significance for treatment and out-

#### *2.3. Outcomes*

There is no consensus on the influence of MS on prognosis in children with AML. Table 1 summarizes many of the larger studies, with notably conflicting results. Although typically considered a presentation of advanced disease, event free survival (EFS) and relapse free survival (RFS) do not consistently demonstrate worse outcomes for MS.

Reports from Children's Oncology Group (COG) demonstrate an improved EFS in subsets of MS patients with non-skin MS, later defined as orbital MS and CNS MS, with otherwise similar outcomes to non-MS patients in those with other sites of MS disease including skin [12,15]. A single center report from India similarly describes improved EFS and overall survival (OS) in patients with MS excluding CSF-only disease [18].

By contrast, other studies describe no significant association between MS or more general extramedullary disease and EFS [11,16,17]. A Turkish single center study found significant effects on outcomes for patients with MS only when less intensive treatment was given [13]. The Japanese childhood AML cooperative study group also only found inferior EFS with extramedullary disease in the setting of WBC count > 100 <sup>×</sup> <sup>10</sup>9/L [14]. By contrast, other reports describe significantly worse EFS and OS for children with AML and MS compared to those without MS [10,19,25]. Collectively, this indicates that whether MS is a critical driver of outcomes remains an important unanswered question within the field.

Favorable cytogenetics, including core binding factor mutations, are common in patients with MS. In evaluation of children with low risk AML (e.g., inv(16), t(8;21), *NPM1* mutated without FLT3-ITD mutation, and *CEBPA* mutation), reports demonstrated worse RFS and EFS in patients with MS present compared to those without MS present [20–22]. This suggests that even in otherwise favorable AML, the presence of MS could be relevant for both prognosis and potentially risk stratification.

Although controversy remains in broadly assigning prognostic impact to the presence or absence of MS in pediatric leukemia, the significance should not be simply ignored. Particularly in patients with t(8;21), inv(16), or chromosome 11/*KMT2A* abnormalities, evaluating for the presence of MS may be significant in considering therapeutic approaches. Additional studies are needed to prospectively identify patients with MS, as defined by clear criteria, to determine the impact on prognosis, the potential need for altering risk stratification, and defining remission status. This will be important to identify which patients may benefit from specific or intensified therapy regimens to improve outcomes.

#### **3. Significance of Extramedullary Disease and Myeloid Sarcomas Post-Allogeneic Hematopoietic Stem Cell Transplant**

Relapse of AML remains the predominant cause of treatment failure and death with allo-HSCT as the only curative option for many patients. The role of allo-HSCT in the setting of MS is a moving target in children. However, new data are emerging to address this important point because anecdotal evidence suggests isolated MS relapse, in the absence of bone marrow relapse, is a common occurrence post-allo-HSCT.

The Japan Society for Hematopoietic Cell Transplantation (JSHCT) used their national database to identify pediatric AML patients that underwent allo-HSCT and found that the presence of extramedullary disease (both CNS disease and MS) had no impact on OS or leukemia-free survival (LFS) after transplant. However, the patients with extramedullary disease prior to transplant were more likely to have extramedullary relapses after transplant, with 41% of relapses being extramedullary. In comparison, those without prior extramedullary disease had only 6% extramedullary relapse, although the overall rates of recurrence were the same between the two groups [28]. Relapse with isolated MS was not separated out as a group and was therefore difficult to directly assess.

The Turkish Pediatric Bone Marrow Transplantation Registry recently reported on their experience with isolated extramedullary relapse (iEMR) in children following allo-HSCT, although they included both acute lymphoblastic leukemia (ALL) and AML. They found different risk factors for medullary relapse post-allo-HSCT versus iEMR. Transplant

in CR2 or later or active disease at time of transplant and matched sibling donor transplants were independently associated with increased risk of medullary relapse as well as iEMR. The presence of chronic graft versus host disease (cGVHD) was conversely associated with decreased risk of medullary relapse with no impact on the risk of iEMR. iEMR rates were, however, independently higher in those with prior extramedullary disease [29]. A higher rate of second iEMR was also seen following a first iEMR at 58.8% versus after a first medullary relapse at 13% [29]. Local radiotherapy of extramedullary disease sites prior to transplantation and the presence of cGVHD had no impact on post-allo-HSCT iEMR, while cGVHD was protective in preventing medullary relapse [29]. This suggests that although a graft versus leukemia effect is helpful in preventing medullary relapse, this immune-mediated mechanism is not effective against MS masses and extramedullary sites of leukemia. A single site report from the University of Michigan found that children were also three times more likely than adults to experience an extramedullary relapse with an associated higher pretransplant extramedullary disease incidence [30]. The significance of extramedullary relapse, particularly in these settings, provides insight into mechanisms of disease resistance specific to the MS phenotype following allo-HSCT. Despite these concerns, allo-HSCT remains the best disease management for patients with high-risk AML.

Extramedullary disease prior to transplant is consistently associated with increased risk of extramedullary relapse after HSCT in both children and adults [30,31]. The presence of prior extramedullary disease (including CNS disease and MS) in adults with AML was not found to be an independent risk factor for post-allo-HSCT relapse, DFS, or OS in both a large CIBMTR analysis and a Canadian report [32,33]. This confirms the anecdotal clinical concern that allo-HSCT is more effective for medullary versus extramedullary disease.

Adults with iEMR post-allo-HSCT are more likely to have had prior extramedullary disease and GVHD present compared to those with medullary relapse [30,31,34,35]. Additionally, extramedullary relapse has a higher incidence following allo-HSCT than intensive chemotherapy alone [36]. These iEMRs may represent sanctuary sites in which immunebased therapies may be less effective and may result from a different mechanism of pathogenesis compared to medullary relapse. There is currently no treatment consensus for iEMR post-allo-HSCT, with a range of treatment approaches taken including local radiotherapy and systemic chemotherapy [37–40]. The significance of iEMR on outcomes compared to medullary relapse is more controversial, with conflicting studies limited by inclusion of both ALL and AML patients with known differences in the efficacy of graft versus leukemia effect between the two diseases [31,34]. One retrospective adult study, however, did report that allo-HSCT was an effective treatment for patients with MS compared to chemotherapy alone, although lack of complete MS remission prior to transplant had independently worse OS and PFS [41].

In summary, the presence of known or occult MS prior to allo-HSCT may be clinically important for a subset of patients, although additional studies are needed to define which groups may benefit. Furthermore, identifying the increased risk for iEMR following allo-HSCT can inform evaluations and management of patients during their post-transplant course. This emphasizes the importance of identifying and following MS in patients with AML prior to allo-HSCT and remaining vigilant to the possibility of iEMR.

#### **4. Imaging Evaluation of Myeloid Sarcomas**

The use of imaging to identify occult MS as well as re-evaluation of disease presence has remained inconsistent, both in frequency and modality, particularly in children. Ultrasounds of MS lesions typically show homogenously hypoechoic lesions with hypervascularity [42]. Computed tomography (CT) scans identify MS as isodense lesions compared to muscle with moderate enhancement with IV contrast media. Enhancement is more commonly homogenous (65%) versus inhomogenous (35%) [43]. MRI scans demonstrate predominantly T2 hyperintense (82%) or isointense (18%) lesions compared to muscle and T1 isointense (61%) or hypointense (39%) lesions with homogenous contrast enhancement

and a mean apparent diffusion coefficient (ADC) on diffusion weighted imaging (DWI) of 0.57 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mm2/s [43].

Fluorodeoxyglucose (FDG)-positron emission tomography (PET) scans are increasingly being used for diagnosing extramedullary disease, with MS lesions displaying moderate uptake of FDG [44]. A retrospective study including pediatric patients showed a sensitivity of 93% and a specificity of 71.4% limited by difficulty differentiating extramedullary leukemia disease from infectious/inflammatory entities [45]. The recent PETAML trial however prospectively evaluated adult patients with AML prior to therapy initiation with total body <sup>18</sup>FDG PET/CT scans to determine prevalence of extramedullary disease. This showed a prevalence of 22% with a sensitivity of 77% and specificity of 97% [24]. Interestingly, leukemia cutis and CNS meningeal involvement were not necessarily <sup>18</sup>FDG-PET-avid [24,45]. In addition, there were four patients who remained with residual <sup>18</sup>FDG-PET-positive lesions despite complete marrow remission. Three of those four subsequently relapsed, suggesting there may be a specific role for <sup>18</sup>FDG-PET imaging for remission evaluation of patients with AML [24]. Consideration should be given to prospectively evaluating patients with AML to identify MS lesions requiring focused follow-up and possible treatment modifications and may be of value in designing de novo AML clinical trials, particularly in pediatrics with a high incidence of MS. *Cancers* **2023**, *15*, x FOR PEER REVIEW 7 of 18 T1 isointense (61%) or hypointense (39%) lesions with homogenous contrast enhancement and a mean apparent diffusion coefficient (ADC) on diffusion weighted imaging (DWI) of 0.57 × 10−3 mm2/s [43]. Fluorodeoxyglucose (FDG)-positron emission tomography (PET) scans are increasingly being used for diagnosing extramedullary disease, with MS lesions displaying moderate uptake of FDG [44]. A retrospective study including pediatric patients showed a sensitivity of 93% and a specificity of 71.4% limited by difficulty differentiating extramedullary leukemia disease from infectious/inflammatory entities [45]. The recent PETAML trial however prospectively evaluated adult patients with AML prior to therapy initiation with total body 18FDG PET/CT scans to determine prevalence of extramedullary disease. This showed a prevalence of 22% with a sensitivity of 77% and specificity of 97% [24]. Interestingly, leukemia cutis and CNS meningeal involvement were not necessarily 18FDG-PET-avid [24,45]. In addition, there were four patients who remained with residual 18FDG-PET-positive lesions despite complete marrow remission. Three of those four subsequently relapsed, suggesting there may be a specific role for 18FDG-PET imaging for remission evaluation of patients with AML [24]. Consideration should be given to pro-

#### **5. Pathology of Myeloid Sarcomas** spectively evaluating patients with AML to identify MS lesions requiring focused followup and possible treatment modifications and may be of value in designing de novo AML

MS are infiltrative tumor masses of myeloid blasts that efface or disrupt the normal architecture of the involved organ. The leukemic blasts found in MS have heterogeneous morphology; however, monocytic differentiation is common where the blasts will show either myelomonocytic or monoblastic morphology [46]. An example of the histology of MS is shown in Figure 2. Immunophenotypic profiling by flow cytometry or immunohistochemical stains is often necessary for a definitive diagnosis, as many of these tumor masses may resemble carcinoma. Evaluation of markers of immaturity, including CD34 and CD117, are helpful in addition to other markers which are variably expressed on myeloid blasts including CD13, CD33, CD68 (KP1), CD45, and myeloperoxidase (MPO). In the setting of monocytic differentiation, expression of monocytic markers such as CD68, CD163, CD14, and/or non-specific esterase (NSE) can be seen. clinical trials, particularly in pediatrics with a high incidence of MS. **5. Pathology of Myeloid Sarcomas**  MS are infiltrative tumor masses of myeloid blasts that efface or disrupt the normal architecture of the involved organ. The leukemic blasts found in MS have heterogeneous morphology; however, monocytic differentiation is common where the blasts will show either myelomonocytic or monoblastic morphology [46]. An example of the histology of MS is shown in Figure 2. Immunophenotypic profiling by flow cytometry or immunohistochemical stains is often necessary for a definitive diagnosis, as many of these tumor masses may resemble carcinoma. Evaluation of markers of immaturity, including CD34 and CD117, are helpful in addition to other markers which are variably expressed on myeloid blasts including CD13, CD33, CD68 (KP1), CD45, and myeloperoxidase (MPO). In the setting of monocytic differentiation, expression of monocytic markers such as CD68, CD163, CD14, and/or non-specific esterase (NSE) can be seen.

**Figure 2.** Histology of myeloid sarcoma involving the psoas muscle. (**A**, 500×). The tumor cells are blasts with irregular/convoluted nuclei, finely dispersed chromatin, and small distinct nucleoli. The inset (400×) shows the tumor cells infiltrating between skeletal muscle fibers. The tumor cells are immunoreactive for CD33 (**B**, 100×), CD163 (**C**, 100×), and CD45 (**D**, 100×).

Several translational studies have begun to investigate the genetic landscape of MS lesions in isolation as well as in comparison to their paired intramedullary leukemia counterparts. One recent study evaluated 7 adult trios of AML, MS, and normal tissue using capture-based next generation sequencing (NGS) of 479 cancer genes. Genes recurrently altered in these patients included *KMT2A*, *FLT3*, *NRAS*, *CEBPA*, *TP53*, *WT1*, and *NPM1*, with 84% of variants found in the AML also present in the MS [47]. Three of the seven patients had additional variants detected in the MS compared to the AML including additional *FLT3*, *SETD2*, and *NF1* mutations in the MS, while two had additional variants of *U2AF1* and *RAD21* in the AML but not the MS [47]. In the relapsed MS samples, there were increased single nucleotide variants (SNV) in the MS [47]. Another study evaluated 6 isolated MS tumors (without concurrent AML) and performed a 21 gene targeted panel of AML and MDS associated genes. They found recurrent variants in the genes for *FLT3* (50%), *NPM1* (33%), and *KIT* (67%) and additional variants in *WT1*, *SF3B1*, *EZH2*, *ASXL1*, and *TET2* in one MS each [48]. The genomic reports of patient-derived MS are all limited by targeted NGS sequencing without exploration of novel gene variants that may be specific to MS pathogenesis. Additionally, RNA transcriptome analysis of MS is lacking in the literature and provides an opportunity for investigation of transcriptome-based changes that may contribute to MS development outside of genetic mutations. Such studies may also facilitate the identification of cryptic translocations, which are common in pediatric AML [7]. Although, typically, the genomic profile of MS is in concordance with the AML and marrow, this is not always true. Particularly in cases of isolated MS, NGS and molecular evaluation may inform targeted treatment options and should be included in diagnostic evaluation of patients. *Cancers* **2023**, *15*, x FOR PEER REVIEW 8 of 18 **Figure 2.** Histology of myeloid sarcoma involving the psoas muscle. (**A**, 500×). The tumor cells are blasts with irregular/convoluted nuclei, finely dispersed chromatin, and small distinct nucleoli. The inset (400×) shows the tumor cells infiltrating between skeletal muscle fibers. The tumor cells are immunoreactive for CD33 (**B**, 100×), CD163 (**C**, 100×), and CD45 (**D**, 100×). Several translational studies have begun to investigate the genetic landscape of MS lesions in isolation as well as in comparison to their paired intramedullary leukemia counterparts. One recent study evaluated 7 adult trios of AML, MS, and normal tissue using capture-based next generation sequencing (NGS) of 479 cancer genes. Genes recurrently altered in these patients included *KMT2A*, *FLT3*, *NRAS*, *CEBPA*, *TP53*, *WT1,* and *NPM1*, with 84% of variants found in the AML also present in the MS [47]. Three of the seven patients had additional variants detected in the MS compared to the AML including additional *FLT3*, *SETD2*, and *NF1* mutations in the MS, while two had additional variants of *U2AF1* and *RAD21* in the AML but not the MS [47]. In the relapsed MS samples, there were increased single nucleotide variants (SNV) in the MS [47]. Another study evaluated 6 isolated MS tumors (without concurrent AML) and performed a 21 gene targeted panel of AML and MDS associated genes. They found recurrent variants in the genes for *FLT3* (50%), *NPM1* (33%), and *KIT* (67%) and additional variants in *WT1*, *SF3B1*, *EZH2*, *ASXL1,* and *TET2* in one MS each [48]. The genomic reports of patient-derived MS are all limited by targeted NGS sequencing without exploration of novel gene variants that may be specific to MS pathogenesis. Additionally, RNA transcriptome analysis of MS is lacking in the literature and provides an opportunity for investigation of transcriptome-based changes that may contribute to MS development outside of genetic mutations. Such studies may also facilitate the identification of cryptic translocations, which are common in pediatric AML [7]. Although, typically, the genomic profile of MS is in concordance with

#### **6. Biological Understandings of Pathogenesis** and molecular evaluation may inform targeted treatment options and should be included

The biology underlying development of MS remains poorly defined with no clear molecular determinants. Biological features such as cytogenetic changes, molecular abnormalities, and cell surface marker expression are not consistent across studies. Much of the work on MS development surrounds the invasiveness of AML as studied using in vitro transwell assays and infiltration in the spleen and liver. The simple infiltration of hematopoietic organs, however, appears distinct from MS development in non-hematopoietic sites with no clear biological explanation. The development of MS appears to require leukemia mobilization/release from the marrow environment, tissue invasion, and further changes leading to a tumor/mass phenotype, as illustrated in Figure 3. These steps will be further discussed below. in diagnostic evaluation of patients. **6. Biological Understandings of Pathogenesis**  The biology underlying development of MS remains poorly defined with no clear molecular determinants. Biological features such as cytogenetic changes, molecular abnormalities, and cell surface marker expression are not consistent across studies. Much of the work on MS development surrounds the invasiveness of AML as studied using in vitro transwell assays and infiltration in the spleen and liver. The simple infiltration of hematopoietic organs, however, appears distinct from MS development in non-hematopoietic sites with no clear biological explanation. The development of MS appears to require leukemia mobilization/release from the marrow environment, tissue invasion, and further changes leading to a tumor/mass phenotype, as illustrated in Figure 3. These steps will be further discussed below.

the AML and marrow, this is not always true. Particularly in cases of isolated MS, NGS

**Figure 3.** Depiction of MS development in AML with proposed mediators. Initial release/migration of leukemic blasts from the bone marrow into the peripheral blood circulation, followed by extravasation and invasion into distant tissue spaces (e.g., subcutaneous tissue). This results in organization of those extravasated cells into a mass with subsequent tissue architectural distortion and gross observation and clinical symptoms. Abbreviations: CAMs, cell adhesion molecules; MMPs, matrix metalloproteinases; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Figure created with BioRender.com.

#### *6.1. CXCR4*

CXCR4 (CXC chemokine receptor 4, CD184) is the receptor for the chemokine matrix cell derivative-1 (SDF-1/CXCL12) and is expressed by most tissues as well as hematopoietic stem cells and leukemic blasts wherein it facilitates the retention of hematopoietic stem cells in the bone marrow niche [49,50]. The CXCR4/SDF-1 axis may contribute to chemoresistance through downstream signaling cascade dysregulation within leukemia cells [49,51]. Higher *CXCR4* expression has been seen in AML patients with extramedullary infiltration at diagnosis and extramedullary infiltration in childhood ALL [49,52]. The proposed mechanism of extramedullary involvement in acute leukemias is altered bone marrow homing and increased peripheral blood dissemination via a chemotactic gradient of SDF-1 with increased CXCR4 expression on the leukemia cells [53]. CXCR4/SDF-1 can promote the retention of AML cells within the skin of children with AML; however, CXCR4 expression by peripheral blood blasts was no different in patients with or without skin involvement [54]. Furthermore, a lack of association between SDF-1 polymorphisms and MS implies that small variants do not contribute to extramedullary disease development, although these have been previously of interest and described [55]. More common in adults, *NPM1*-mutated AML is associated with extramedullary disease and is associated with downregulation of *CXCL12* and *CXCR4* gene pathways [50].

While CXCR4 expression and signaling may be a contributing factor to extramedullary disease, its impact appears limited to initial release and migration of leukemia cells from the marrow and is not specific to MS development. Further work is needed to better characterize this mechanism and whether or how CXCR4 is contributing to discrete MS formation.

#### *6.2. CD56*

CD56 (also known as neural cell adhesion molecule-1 or NCAM1) is normally expressed by natural killer (NK) cells and other immune cell subtypes and is housed on chromosome 11q23.1. It is frequently described as part of the immunophenotype of AML with MS [1,56,57]. Expression patterns of CD56 are not consistently described, however, and in a population of adult t(8;21) AML patients, there was no association between CD56 expression and presence of extramedullary disease [58]. AML in adults with CD56 positivity is more commonly associated with worse 5-year EFS and OS; however, a report in low-risk patients shows no association with outcome [21,25,59]. Additionally, post-allo-HSCT CD56 positivity is not associated with extramedullary relapse [30]. Despite the frequent CD56+ immunophenotype, there is no described mechanism or in vitro data to suggest the significance of this finding. Additional experimental studies are required to determine if CD56 is simply a biomarker of MS or is required for MS development.

#### *6.3. Integrins and Cell Adhesion Molecules (CAMs)*

An AML-extracellular matrix interaction is likely critical to the development of MS. This is illustrated in transcriptome analysis of adult patient-derived AMLs demonstrating enrichment of cell surface gene sets in those AMLs with concomitant MS [60]. This includes integrin-α7 *(ITGA7*), which showed a higher expression in AML with associated MS in addition to high expression in MS samples [60]. Laminin 211 is a specific ligand of integrinα7 that signals through the ERK signaling cascade [60]. While there is much described about the role of integrins and selectins in migration and homing of hematopoietic stem cells, there remains no clear mechanism by which these molecules facilitate MS formation [61,62]. Further study is needed to better evaluate the role of cell adhesion molecules in MS development and whether targeting these cell interactions may provide therapeutic benefit for patients with MS.

#### *6.4. Vascular Endothelial Growth Factor (VEGF) and Receptor (VEGFR)*

Angiogenesis plays a notable role in acute leukemia with increased microvascular density in AML and adult MS [63]. VEGFR2, the major mediator of the mitogenic, angiogenic, and permeability effects of VEGF, may contribute to the development of MS [64]. VEGF

signaling via the PI3K/Akt pathway in the setting of *hERG1* expression was necessary for an in vitro migratory phenotype in AML cells [65]. In adults, the small molecule VEGFR2 tyrosine kinase inhibitor apatinib (also known as TN968D1) demonstrated enhanced antileukemic effects in ex vivo cytotoxicity studies from patient-derived AML samples with associated extramedullary disease [66]. Angiogenesis is well-described in the pathogenesis of other malignancies and it is reasonable to think that a unique perturbation may play a role in the migration or tumor formation of MS.

#### *6.5. Matrix Metalloproteinases (MMP)*

In vitro studies have described the role of MMP secretion (MMP-2 and MMP-9) by leukemia cells contributing to invasion capacity, most notably of the blood-brain barrier, with upstream regulation by mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3-K)/AKT pathways [67,68]. Additionally, TIMP-2 (tissue inhibitor of metalloproteinase 2) upregulation has been seen with increased leukemia cell line (i.e., SHI-1) invasion both in vitro and in vivo with more extensive and severe extramedullary infiltration through both MMP-2-dependent and independent activities [69,70]. Other in vitro studies propose a role for the β2 integrin-proMMP-9 complex in the extramedullary phenotype of AML [71]. Type IV collagenase secretion enhanced by TNFα and TGFβ from a patient-derived MS cell line increased in vitro cell invasion with collagenase secretion demonstrated in the MS AML cell line but not other leukemia cell lines [72]. While tissue invasion by leukemia cells is likely required for MS development, not all of the critical players have been identified.

#### *6.6. Epigenetic Dysregulation*

Epigenetic dysregulation has been reported in the context of extramedullary disease and infiltration in AML. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), which deposits Histone 3 Lysine 27 trimethylation (H3K27me3). High *EZH2* expression is correlated with higher peripheral blood blast percentages as well as extramedullary infiltration in patients with AML with numerous well-established biological roles. In vitro studies suggest that migration of AML cells appears to be regulated by EZH2/p-ERK/p-cmyc/MMP-2 and E-cadherin signaling pathways [73]. *EZH2* is a frequently mutated gene in AML; however, *EZH2* has a variety of biologic influences and a unique role in MS formation remains undefined [7,9].

Altered DNA methylation is another described mechanism in the development of extramedullary disease with key enzymes frequently mutated in AML. DNA methyltransferase 3A (*DNMT3A*) mutations contribute to altered DNA methylation, subsequently resulting in increased expression of a subset of genes with specific roles in myeloproliferation and extramedullary hematopoiesis [74]. *DNMT3A* mutation appears to contribute to extramedullary CNS infiltration mediated by overexpression of *TWIST1*, a key epithelial mesenchymal transition transcription factor, which is not otherwise well described in AML [75]. Furthermore, *TET2* is a member of the ten-eleven translocation (TET) gene family and is a key enzyme for DNA demethylation and a critical regulator for hematopoietic stem cell homeostasis. Models using *TET2*-deficient mice demonstrated not only high incidence of MS development but also transplant ability of the MS cells as well as an in vivo response to azacitidine treatment [76]. Decreased *TET2* expression was also seen in patient-derived MS samples with further suggestion of methylation changes impacting MS development [77].

AML has many examples of mutations in epigenetic pathways that are enriched in AML more than many other disease entities and may not be directly related to their involvement in MS [9]. The role of epigenetic dysregulation in leukemia migration and invasion with described MS phenotypes is intriguing yet requires further study. Additional research may uncover future targetable pathways for MS treatment, and as noted below, case reports have demonstrated the safety and efficacy of hypomethylating agent use for patients with MS.

#### *6.7. Other Biological Associations*

Mesothelin (MSLN) is a cell surface protein hypothesized to be involved in cell adhesion and is overexpressed in a subset of AML patients. MSLN overexpression was strongly associated with KMT2A-R, t(8;21), and inv(16) as well as the presence of extramedullary disease in children and young adults with AML. Methylation profiling further demonstrated an inverse association between MSLN promoter methylation and MSLN expression, suggesting another impact of epigenetic dysregulation [78].

Versican (VCAN) overexpression in the setting of *NPM1*-mutated AML is associated with an invasive phenotype and higher expression levels in patients with skin infiltration [79]. Lysyl oxidase (LOX), which has roles in pediatric acute megakaryoblastic leukemia and in the creation of a growth permissive fibrotic microenvironment, was associated with increased extramedullary disease in adults with AML and high plasma LOX activity [80]. *WT1* overexpression has also been described in MS cases as well as in extramedullary relapsed disease [81,82]. *ERG* transcription factor overexpression, similar to that of Ewing sarcoma, has been seen in patient-derived MS samples [83]. Multiple studies describe other associations observed in AML and extramedullary disease, including increased expression of amyloid precursor protein (APP) in *AML1/ETO* leukemia cells perhaps mediating the p-ERK/c-Myc/MMP-2 pathway, expression of miR-29c&b2, circular RNA expression patterns, and expression of CD25 and CD117 [84–90]. Polo-like kinase 1 (PLK1), which is involved in cell cycle control, was effectively inhibited in vivo using a patient-derived leukemia in mice with improvement in extramedullary disease [91]. PD-1 and PD-L1 have been investigated given the described efficacy of checkpoint inhibitors; however, there was no difference in expression of *PD-1*/*PD-L1* in MS tested, and they may instead have more impact in the surrounding tumor microenvironment [92,93]. Using mouse models, others observe a maturation plasticity of leukemia cells, with potential implications for chemotherapy resistance as a mechanism for extramedullary relapse [94]. A *PIM2/MYC* co-expressed mouse model demonstrated consistent and lethal in vivo MS development with *MYC* expression likely contributing to the phenotype [95]. Mouse models have also demonstrated cooperation between *MLL/AF10* and activating *KRAS* mutations, with increased cell adhesion properties contributing to in vivo MS formation via *Adgra3* and *Hoxa11* [96,97].

While many different mechanisms have been suggested in the development of MS, there remains no clear understanding of the pathogenesis. As such, it is hard to definitively identify the potential molecular determinants causing MS formation is some AML patients but not others. While the pathogenesis remains to be fully elucidated, prior studies suggest that there are likely multiple steps leading to MS development, including release from the bone marrow (which may be represented by higher WBC counts associated with MS), tissue invasion, and discrete mass formation, with the latter being the most consequential with regards to leukemia and the least described. Investigating how these different steps may cooperate and ultimately how the leukemia cells aggregate and sustain an aggregated phenotype requires dedicated study. Furthermore, the immune evasive or immunosuppressive microenvironment of MS illustrated in the post-allo-HSCT setting highlights that there is much more to learn about the pathogenesis of MS and its uniqueness with respect to its systemic/intramedullary AML counterpart.

#### **7. Treatment Considerations**

There is no consensus on the best treatment approach for management of MS, particularly isolated extramedullary disease. In pediatrics, systemic chemotherapy has been favored with consideration of allo-HSCT independent of the presence of MS.

The general approach to management in pediatric patients has evolved over the last three decades. In prior large cooperative group study treatment protocols (e.g., CCG 2961), children with MS would receive radiation therapy to the affected sites following initial induction chemotherapy, given MS responsiveness to irradiation. Although part of protocol therapy, many patients were not irradiated and outcomes demonstrated no difference in 5-year EFS, similar to smaller cohorts [12,98]. In adults, radiation therapy is more commonly used to treat isolated relapses, but the effects are not typically sustained and both localized and medullary relapse following radiotherapy are common [30,99]. Although prior COG studies included radiotherapy for treatment of MS sarcomas, this is no longer standard of care in the US; however, it continues to be recommended for MS in Berlin–Frankfurt–Munster (BFM) studies [17,100].

Given the epigenetic basis of AML development, inclusion of novel epigenetic approaches in treatment are increasing in utility for AML [9,101,102]. The hypomethylating agents azacitidine and decitabine have demonstrated efficacy in management of extramedullary disease in AML, including in pediatric patients [103]. Case reports in pediatrics have demonstrated complete response to monotherapy with azacitidine in MDS patients who received allo-HSCT as consolidation [103]. In the setting of post-allo-HSCT relapses of MS, multiple case reports in adults demonstrate efficacy of azacitidine or decitabine including complete response [104,105]. Hypomethylating agents in adults with AML and extramedullary disease showed improvement after one–two cycles and complete or near complete resolution of MS following four–five cycles [106–109]. Venetoclax has also shown activity against MS [109–111]. The utility of venetoclax and hypomethylating agents suggests a role for epigenetic reprogramming as a means for MS treatment, although DNA methylation-based mutations including *DNMT3A*, *IDH1*, *IDH2,* and *TET2* are far less common in children than in adults and translation of utility is more challenging [9,112]. While no consensus exists regarding optimal treatment of MS, particularly in pediatrics, radiation therapy is unlikely to contribute to durable remission of disease and expanding chemotherapeutic options to include hypomethylating agents and venetoclax in both initial chemotherapy regimens or as maintenance therapy following allo-HSCT should be considered and deserves further investigation.

#### **8. Conclusions: Knowledge Gaps and Areas for Improvement**

Children with AML and MS are distinct from adults. Given these differences, it is necessary to further study MS in the context of the driver lesions specific to children. MS remains a known clinical presentation with unclear impact on prognosis, risk stratification, and potential consequences in the setting of allo-HSCT. The advancement of imaging techniques and data for MS provides the opportunity for more directed and prospective evaluation in children. Collectively, this highlights the need for further large-scale cooperative group studies with clear criteria for the identification of MS in children.

While there is no specific treatment approach for children with MS, the use of intensive systemic chemotherapy remains at the forefront. However, additional studies are required to determine if epigenetic or immuno-oncology therapies may be beneficial. The role of allo-HSCT continues to be important as a curative option for many patients with high-risk AML; although, considering its potential lack of efficacy in the setting of extramedullary disease, the role of an immunosuppressive microenvironment in MS requires additional study. Further investigation into the potentially immunosuppressive MS microenvironment will be crucial to improving efficacy of allo-HSCT and managing isolated extramedullary relapses post-HSCT.

Although many different biological associations exist, there is an ongoing lack of clarity as to how leukemic blasts can not just invade tissues but form discrete tumors. Experiments delineating the potential epigenetic and transcriptomic differences between medullary AML disease and MS are required to identify the underlying molecular mechanisms that promote MS development. Understanding how leukemic blasts transform into and sustain an MS phenotype is critical to identifying specific targetable mechanisms. Understanding and combatting chemotherapy resistance and immune escape will ultimately improve survival in patients with AML and MS.

**Author Contributions:** Conceptualization, K.E.Z. and S.R.; writing—original draft preparation, K.E.Z., A.M.C. and S.R.; writing—review and editing, K.E.Z., A.M.C., A.E.M., K.S.C. and S.R.; visualization, K.E.Z. and A.M.C.; supervision, S.R.; project administration, S.R.; funding acquisition, S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** S.R. is funded by CA204231 and HL149620.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **The Battlefield of Chemotherapy in Pediatric Cancers**

**Letao Bo <sup>1</sup> , Youyou Wang <sup>1</sup> , Yidong Li <sup>1</sup> , John N. D. Wurpel <sup>1</sup> , Zoufang Huang 2,\* and Zhe-Sheng Chen 1,3,\***


**Simple Summary:** The survival rate for pediatric cancers has improved significantly over the last decades. Conventional chemotherapies play a vital role in pediatric cancer treatment, especially in low- and middle-income countries, and the roster of chemo drugs for use in children has expanded. However, patients suffer from chemotherapy as a result of its countless side effects. Furthermore, multidrug resistance (MDR) continues to be an insurmountable obstacle that limits survival for a considerable number of patients. In this review, we discuss severe side effects in pediatric chemotherapies such as doxorubicin-induced cardiotoxicity (DIC) and vincristine-induced peripheral neuropathy (VIPN). Here, MDR mechanisms in chemotherapy are elucidated with the aim of improving survival, while also reducing the intensity and toxicity of chemotherapy. Furthermore, we focus on various drug transporters in common types of pediatric tumors, which could provide different therapeutic strategies.

**Abstract:** The survival rate for pediatric cancers has remarkably improved in recent years. Conventional chemotherapy plays a crucial role in treating pediatric cancers, especially in low- and middle-income countries where access to advanced treatments may be limited. The Food and Drug Administration (FDA) approved chemotherapy drugs that can be used in children have expanded, but patients still face numerous side effects from the treatment. In addition, multidrug resistance (MDR) continues to pose a major challenge in improving the survival rates for a significant number of patients. This review focuses on the severe side effects of pediatric chemotherapy, including doxorubicin-induced cardiotoxicity (DIC) and vincristine-induced peripheral neuropathy (VIPN). We also delve into the mechanisms of MDR in chemotherapy to the improve survival and reduce the toxicity of treatment. Additionally, the review focuses on various drug transporters found in common types of pediatric tumors, which could offer different therapeutic options.

**Keywords:** chemotherapy; pediatric cancers; VIPN; DIC; MDR; drug transporters

### **1. Introduction**

Pediatric cancer is relatively rare and has a high survival rate compared with adult cancer. Pediatric cancer includes 12 major types and over 100 subtypes [1]. In the United States, pediatric cancer therapies have made remarkable advances over the last 70 years, and the overall survival (OS) rate for pediatric cancer patients has increased to 80% [2,3]. However, cancer is still the top cause of death by disease in children. In 2023, diagnosed pediatric cancer patients in the USA will include ~9910 children (birth to age 14) and ~5280 adolescents (aged 15–19 years). In addition, approximately 1040 children and 550 adolescents in the USA will die from cancer in 2023 [4]. An improvement in these amounts can be achieved by an increased understanding of the molecular basis of cancer through international collaborative efforts.

**Citation:** Bo, L.; Wang, Y.; Li, Y.; Wurpel, J.N.D.; Huang, Z.; Chen, Z.-S. The Battlefield of Chemotherapy in Pediatric Cancers. *Cancers* **2023**, *15*, 1963. https://doi.org/10.3390/ cancers15071963

Academic Editor: David Wong

Received: 10 February 2023 Revised: 12 March 2023 Accepted: 21 March 2023 Published: 24 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Despite these successes, the incidences and mortalities between countries are greatly different. In 2017, the 5-year survival rate of childhood cancer was 80% in high-income countries. However, data suggest that far fewer children survive in most low- and middleincome countries—the 5-year survival rate of children is ~40%, while more than 90% of children at risk of potential pediatric cancer live in these countries [5,6]. Because of major advances in modern science over the past several decades, the improvement in pediatric cancer in high-income countries has not translated to most low- and middle-income countries. There are still gaps among these countries regarding recognition, diagnosis, and treatment [7].

Despite financial hardship or treatment results, conventional chemotherapy is still a standard option for childhood cancer treatments in low- and middle-income countries, where molecular-targeted drugs are broadly accessible [8–10]. Severe side effects and resistance mechanisms against cancer therapies remain the primary reasons limiting chemotherapy outcomes [11]. Here, we consider the advancement of chemotherapy in pediatric cancer, including drugs and specific considerations. Beyond the previous focus on adult cancer alone, in the current review, we discuss severe side effects such as neuropathy and myopathy, the effects of the MDR phenomenon, and the role of various drug transporters in common types of pediatric tumors that could lead to more efficient pediatric chemotherapy.

#### **2. Current Chemotherapy in Pediatric Cancer**

There are currently various cancer treatment approaches for cancer. The main four lines of treatment are surgical removal, immunotherapy, radiotherapy, and chemotherapy [12]. Surgical removal has the longest history in cancer treatment. It is an essential treatment option and often is often performed together with other cancer treatments, depending on the type of cancer [13]. Immunotherapy is generally considered to have been applied in 1890 by Dr. Coley, who injected a mixture of live and inactivated *Streptococcus* and *Serratia* to achieve responses [14]. It modulates the immune system to act better against cancer, and it includes immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies, and treatment vaccines. Radiotherapy precisely delivers radiation to kill cancer cells and shrink tumors, and was started in 1896 by Emile Grubbé, who treated a breast cancer patient using X-rays [15]. Chemotherapy was first introduced after the Second World War, and works by interfering with cell proliferation. Other cancer treatment approaches include stem cell transplantation, targeted therapy, photodynamic therapy, and hormone therapy.

The efficient treatment of pediatric cancer started after 1945. Sidney Farber treated a 3-year-old patient with acute lymphoblastic leukemia (ALL) with aminopterin [16]. As a conventional cancer therapy, chemotherapy has played an important role in pediatric cancer over the past several decades. In pediatric diagnosis, ALL is the most common type of cancer. The OS of ALL increased from 57% in the 1970s to 96% in recent years [17]. This enormous progress is partly attributed to advances in chemotherapy [3]. Currently, chemotherapy has reached its limit, as severe toxicities have been observed in pediatric patients during treatment. Modifications to chemotherapy are made in order to reduce multiple toxicities. For instance, studies have provided evidence that chronomodulated chemotherapy, based on the body's intrinsic circadian clock, might minimize toxicity while maintaining an anticancer activity [18]. High-dose chemotherapy has been explored for treating childhood malignant glioma so as to pass the blood−brain barrier (BBB), reduce cell chemoresistance, and achieve a more comprehensive response [19].

Chemo drugs are grouped by function, chemical structure, and interaction with other medications. Types of chemo drugs include alkylating agents, antimetabolites, antibiotics, topoisomerase inhibitors, mitotic inhibitors, and corticosteroids [20,21]. In 1997, under the Food and Drug Administration Modernization Act2 (FDAMA), a pediatric exclusivity provision was enacted by the U.S. Congress. Few chemo drugs were explicitly approved for treating pediatric cancer. The FDAMA later reauthorized as the Best Pharmaceuticals Act for Children (BPCA), which encourages pediatric drug studies in companies by providing a financial incentive. A total of 16 chemo drugs have been approved by the FDA with

pediatric indications in the U.S. Before the FDAMA, only nine chemo drugs were approved for pediatric indications, see Table 1. After the FDAMA (1997–2022), seven chemo drugs were approved to treat childhood cancer (see Table 2).

There are less drugs with pediatric indications worldwide compared with adult indications [22]. For some chemo drugs, the initial approval was only for adult indications. On 20 May 2022, Azacitidine was newly approved for childhood cancers, while it first approved on 19 May 2004 for treating all subtypes of myelodysplastic syndrome. Moreover, FDA approved these chemo drugs (Clofarabine, Nelarabine, Erwinia, and Mercaptopurine) for both adult and pediatric indications at the same time (Table 2). Although efforts have been made in pediatric chemotherapy, only a few chemo drugs have gained pediatric indications—there is a gap between pediatric and adult approval.

The therapy to treat pediatric cancers requires specific prerequisites and considerations, as patients are still growing and developing. Some adverse effects of cancer treatments are more severe for children than adults, as developing organs are more susceptible. Infectionrelated serious adverse events are more common in ALL patients receiving chemotherapy, which enhances the risk of death [23]. Therefore, it is necessary to identify drug resistance mechanisms to reduce chemotherapy intensity and toxicity. The counterstrategies against drug resistance are discussed in the following section.

**Table 1.** Chemo drugs approved prior to FDAMA, for which labeling includes pediatric indications [24].


Data provided by National Cancer Institute (https://www.cancer.gov/), accessed on 30 January 2023, updated: 20 December 2022, and U.S. FDA (https://www.fda.gov/). The drugs and drug combinations are not listed here.

**Table 2.** Chemo drugs approved post FDAMA with pediatric specific indications (1997–2022) [24].


Data provided by National Cancer Institute (https://www.cancer.gov/), updated: 20 December 2022, and U.S. FDA (https://www.fda.gov/). JMML: juvenile myelomonocytic leukemia. The drugs and drug combinations are not listed here.

Asparaginase Erwinia Chrysanthemi

#### **3. Severe Neuropathy and Myopathy Side Effects in Chemotherapy** In cancer treatment, chemotherapeutic agents are considered a double edge sword,

**3. Severe Neuropathy and Myopathy Side Effects in Chemotherapy**

*Cancers* **2023**, *15*, 1963 4 of 18

**(Drugs approved post FDAMA with pediatric specific indications (1997–2022))** Azacitidine (Vidaza) 19 May 2004 20 May 2022 JMML

Everolimus 1 November 2010 29 August 2012 Giant Cell

(Erwinaze) same 18 November 2011 ALL Clofarabine (Clolar) same 28 December 2004 ALL Pegaspargase (Oncaspar) same 24 July 2006 ALL

Nelarabine (Arranon) same 28 October 2005 Non-Hodgkin

Calaspargase Pegol-mknl (Asparlas) same 20 December 2018 ALL

**Drugs Original Approval Pediatric**

drug combinations are not listed here.

In cancer treatment, chemotherapeutic agents are considered a double edge sword, as it kills cancer cells and other healthy, fast-growing cells, without differentiation. As a matter of fact, patients suffer from various non-negligible side effects such as hair loss, fatigue, nausea, vomiting, and diarrhea. Currently, doxorubicin (DOX) and vincristine (VCR) are two commonly used chemotherapeutic agents in pediatric cancer treatment [25,26]. However, the potential harm of neuropathy and myopathy has become a considerate risk factor that limits the effect of DOX and VCR [27,28]. The pathway mechanism of neuropathy and myopathy induced by DOX and VCR in illustrated in Figure 1. Thus, it is necessary to discuss the opposing side and corresponding solutions for these two chemo drugs, mainly when applied to pediatric cancer patients. as it kills cancer cells and other healthy, fast-growing cells, without differentiation. As a matter of fact, patients suffer from various non-negligible side effects such as hair loss, fatigue, nausea, vomiting, and diarrhea. Currently, doxorubicin (DOX) and vincristine (VCR) are two commonly used chemotherapeutic agents in pediatric cancer treatment [25,26]. However, the potential harm of neuropathy and myopathy has become a considerate risk factor that limits the effect of DOX and VCR [27,28]. The pathway mechanism of neuropathy and myopathy induced by DOX and VCR in illustrated in Figure 1. Thus, it is necessary to discuss the opposing side and corresponding solutions for these two chemo drugs, mainly when applied to pediatric cancer patients.

Data provided by National Cancer Institute (https://www.cancer.gov/), updated: 20 December 2022, and U.S. FDA (https://www.fda.gov/). JMML: juvenile myelomonocytic leukemia. The drugs and

**Table 2.** Chemo drugs approved post FDAMA with pediatric specific indications (1997–2022) [24].

**Approval**

**Indications for Pediatric Cancer**

Astrocytoma

Lymphoma

**Figure 1.** Neuropathy and myopathy induced by two commonly used chemotherapeutic agents (DOX and VCR) in pediatric cancer treatment. **Figure 1.** Neuropathy and myopathy induced by two commonly used chemotherapeutic agents (DOX and VCR) in pediatric cancer treatment.

DOX, an anthracycline drug to treat solid tumors in children, such as Wilm's tumor, was approved by the FDA in 1974 to become the first clinical liposomal encapsulated anticancer drug [29]. DOX acts as an intercalation that inhibits topoisomerase II and further obstructs DNA replication [30]. The recommended dosage (2 mg/mL) is the same in both pediatric and adult patients. Symptoms of the side effects may include abnormality in electrocardiography, rhythm disturbances, and even left ventricular hypertrophy that leads to a reduction in ejection fraction [31].

DIC is a severe dose-dependent risk factor in cancer treatment [32]. It has been reported that SNP (rs2229774) in retinoic acid receptor-γ (RARG) has a significant impact on the increased occurrence of DIC. Further investigation found that a RARG agonist CD1530 showed a cardioprotective effect in an in vivo mouse model of DIC [33]. Other drug transporter genomic variants in the *SLC28A3* locus were also identified. Based on that, a single dosage of 3 µmol/L desipramine per day before the administration of DOX is recommended to prevent DIC [34]. Moreover, circular RNA (circRNA)-based therapy is a novel approach for treating DIC [35]. The overexpression of insulin receptor encoded circRNA (Circ-INSR) successfully prevented and reversed DIC in an in vivo mouse model [36]. Han. et al. reported another promising therapeutic target for DIC, namely tumor-suppressive human circular RNA CircITCH [37]. Other recent potential strategies that possibly reverse DIC

include *atg7*-based autophagy activation [38], meteorin-like (METRNL) protein [39], sirtuin 1 (SIRT1) [40], berberine [41], ADAR2 [42], elabela (ELA) [43], phenylalanine-butyramide (FBA) [44], gasdermin D [45], melatonin [46], levosimendan [47], paeonol [48], SNX17 [49], irisin [50], isorhapontigenin [51], liensinine [52], etc.

VCR, known initially as Leurocristine, is the first-line chemotherapeutic medication often administered in the combination chemotherapeutic treatment of pediatric hematologic malignancies and solid tumors [53]. VCR acts as mitotic inhibitors by binding to the βtubulin subunit of αβ-tubulin heterodimers, thus functionally destabilizing microtubule fibers, which ultimately leads to the termination of cancer cell division [54]. Previous studies have shown a cumulative effect for its neurotoxicity and overdosage may cause very serious or fatal outcomes [55,56]. Until 6 June 2022, it has been approved by FDA in the treatment of pediatric ALL, neuroblastoma, non-Hodgkin lymphoma, rhabdomyosarcoma, Wilm's tumor, and other childhood kidney cancers. USP recommended dose of VCR injection for pediatric patients is 1.5–2 mg/m<sup>2</sup> compared to adults is 1.4 mg/m<sup>2</sup> . For neonate and infant patients weighing 10 kg or less, the first dose should be 0.05 mg/kg, administered once a week. The latest research showed in the treatment of ALL and Wilm's tumors, compared with older children, neonates or infants have similar clearance in vincristine. Thus, doses less than 0.05 mg/kg should not be applied in neonate and infant patients due to inappropriate suboptimal VCR exposures [57].

The common side effect encountered with VCR use is VIPN [58]. Clinical patterns of VIPN can be classified as sensory neuropathy (e.g., numbness and paresthesia), motor neuropathy (e.g., extremity weakness and walking difficulties), and autonomic neuropathy (e.g., constipation and urinary retention) [59]. VIPN can be assessed by the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), the pediatricmodified Total Neuropathy Scale (ped-m TNS), and the Total Neuropathy Score-Pediatric version (TNS-PV) [28,60]. There are currently no effective strategies for reducing vincristineinduced neurotoxicity. Also, whether VIPN exists chronically in the survivors remains ambiguous [61]. Some studies showed that a significant proportion of patients receiving VCR would undergo a certain extent VIPN [62,63]. In 2017, Tay et al. reported that ~16% of pediatric ALL survivors suffer from VIPN [64]. Nevertheless, in 2020, an investigation among 150 pediatric patients with ALL and Wilm's tumors showed that significant side effects of the vincristine regimen are mostly neurotoxic, which is at a mild to moderate level [65]. In recent years, more and more studies have revealed the deep connection between genetic polymorphisms and VIPN [66–68]. The *CEP72* genetic variant is an optimistic VIPN signature marker since the *CEP72* gene encodes centrosome proteins that participate in the development of microtubules. Patients that carry *CEP72* TT genotype take potentially higher risk and severity of VIPN than CC or CT genotype patients [69]. Besides, the enzyme CYP3A5 contributes to hepatic clearance of VCR, which means CYP3A5 genetic polymorphisms, and its allelic variants are assumed to be associated with VCR neurotoxicity in different human populations. CYP3A5 genotyping analysis results showed over 70% of African Americans had been found to have one or more CYP3A5\*1 alleles (such as CYP3A5 expresser), which is about five times higher compared to Caucasians [70]. Moreover, VCR is transported by some members of ATP-binding cassette (ABC) transporter superfamily such as ABCB1, ABCC1, ABCC2, and ABCB4 [71]. Lopez-Lopez et al. reported that ABCC1 is the critical mediator acts transporting VCR into the blood, while ABCB1 and ABCC2 are indispensable in the biliary excretion of VCR. The genotypes rs3740066 GG and rs12826 GG of ABCC2 were identified with significant associations with increased VIPN, which suggested that ABCC2 polymorphism could be used as a potential biomarker for VIPN in screening and diagnosis of pediatric ALL [72].

Recently, numerous updates have been related to the strategy to overcome VIPN. Zhou et al. reported levo-corydalmine ameliorates VIPN in mice by inhibiting Cx43 expression and NFκB-dependent CXCL1/CXCR2 signalling pathway [73,74]. Also, both VIPN and tumor growth were alleviated by the inhibition of histone deacetylase 6 (HDAC6) in mice. Other prospective targets that possibly prevent VIPN include mitoquinone [75],

puerarin [76], nerve growth factor (NGF) monoclonal antibody DS002 [77], minocycline [78], bergapten [79], etc.

#### **4. MDR: The Challenge in Pediatric Cancer Chemotherapy**

Cancer cells can develop resistance to one chemotherapeutic drug, as well as other chemotherapeutic drugs that may have different chemical properties and mechanisms of action, which is called multi-drug resistance (MDR) [80]. MDR can occur in both adult and pediatric cancer chemotherapy. Although the mechanisms of MDR have been studied for a few decades, MDR is a very limiting factor to the success of cancer chemotherapies. MDR exists not only in chemotherapy and radiation therapy, but also in newly developed therapies such as targeted therapy and immunotherapy [81,82]. Drug resistance can be divided into intrinsic resistance or extrinsic resistance based on the cause of its occurrence. Cancer cells may have inherent drug resistance before receiving chemotherapy. However, various adaptive responses of cancer cells during the treatment cause extrinsic or acquired drug resistance of cancer cells. Several MDR mechanisms have been discovered, yet they are not yet fully understood. As shown in Figure 2, the factors of MDR include increased drug inactivation, reduction of influx and increased efflux of drugs, decreased activation of prodrugs, epigenetic dysregulation, changes in cell surface markers, tumor microenvironment (TME), epithelial−mesenchymal transition (EMT), altered miRNA, disruption of responses to DNA damage, inhibition of apoptotic pathways, tumor heterogeneity, and cancer stem cells [80,83,84]. *Cancers* **2023**, *15*, 1963 7 of 18

**Figure 2.** Mechanisms of MDR in cancer treatment. MDR occurs as a result of various causes, which ultimately lead to tumor recurrence. **Figure 2.** Mechanisms of MDR in cancer treatment. MDR occurs as a result of various causes, which ultimately lead to tumor recurrence.

#### **5. MDR-Related Drug Transporters and Their Roles in Pediatric Cancers 5. MDR-Related Drug Transporters and Their Roles in Pediatric Cancers**

Drug transporters are membrane proteins involved in the absorption, distribution, and excretion of drugs. Two transporter superfamilies have been identified in humans: the solute carrier (SLC) superfamily and the ABC superfamily [85]. ABC transporters are generally involved with the efflux of drugs, and SLC transporters have been chiefly described as influx transporters [86]. In addition to transporting therapeutic drugs across Drug transporters are membrane proteins involved in the absorption, distribution, and excretion of drugs. Two transporter superfamilies have been identified in humans: the solute carrier (SLC) superfamily and the ABC superfamily [85]. ABC transporters are generally involved with the efflux of drugs, and SLC transporters have been chiefly described as influx transporters [86]. In addition to transporting therapeutic drugs across membranes, these transporters also mediate the transport of endogenous compounds.

proteins involved in the influx and efflux of a wide array of substrates, such as ions, metabolites, and drugs [87]. SLC transports substrates based on the electrochemical potential difference between the biological membrane or the ion gradient originally generated by the primary active transporters. Genetic variants of SLC transporters and clinical outcomes of methotrexate (MTX) have been studied in pediatric patients with ALL [88]. Although SLC transporter families are essential in human health, related studies focusing on

pediatric cancer therapy are rare. We limited our discussion to ABC transporters.

SLC family transporters are a family that includes more than 300 membrane-bound

The ABC transporter superfamily, one of the most prominent transporter families, is

responsible for MDR by mediating drug efflux, which subsequently leads to a low intracellular concentration of antineoplastic agents in cancer cells and deteriorates therapeutic outcome [89]. In addition, more than efflux pumps, other critical roles in cancer development of this transporter superfamily have been revealed step by step [90–94]. So far, 7 subfamilies (ABC-A to ABC-G) and at least 48 additional subfamily members have been found and characterized depending on their structural differences and similarities [95,96]. Among these subfamily members, three members have been demonstrated that are closely related to MDR in chemotherapy, including P-glycoprotein (P-gp/ABCB1/MDR1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2)

confer MDR to cancer cells.

*5.1. SLC Family Transporters*

*5.2. ABC Transporters*

[97,98].

membranes, these transporters also mediate the transport of endogenous compounds.

There is considerable interest in transporters from both families, as they are known to confer MDR to cancer cells.

#### *5.1. SLC Family Transporters*

SLC family transporters are a family that includes more than 300 membrane-bound proteins involved in the influx and efflux of a wide array of substrates, such as ions, metabolites, and drugs [87]. SLC transports substrates based on the electrochemical potential difference between the biological membrane or the ion gradient originally generated by the primary active transporters. Genetic variants of SLC transporters and clinical outcomes of methotrexate (MTX) have been studied in pediatric patients with ALL [88]. Although SLC transporter families are essential in human health, related studies focusing on pediatric cancer therapy are rare. We limited our discussion to ABC transporters.

#### *5.2. ABC Transporters*

The ABC transporter superfamily, one of the most prominent transporter families, is responsible for MDR by mediating drug efflux, which subsequently leads to a low intracellular concentration of antineoplastic agents in cancer cells and deteriorates therapeutic outcome [89]. In addition, more than efflux pumps, other critical roles in cancer development of this transporter superfamily have been revealed step by step [90–94]. So far, 7 subfamilies (ABC-A to ABC-G) and at least 48 additional subfamily members have been found and characterized depending on their structural differences and similarities [95,96]. Among these subfamily members, three members have been demonstrated that are closely related to MDR in chemotherapy, including P-glycoprotein (P-gp/ABCB1/MDR1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) [97,98].

#### 5.2.1. MDR1

The ABCB subfamily involves four full transporters (ABCB1/4/5/11) and 7 half transporters (ABCB2/3/6/7/8/9/10) that can transport a vast variety of molecules, including peptides, drugs, and ions [99]. Half transporters include two polypeptides, each having a transmembrane binding domain (TMD) and (nucleotide-binding domains) NBD to form a homo- or hetero-dimer. Full transporters are characterized as all four domains reside on a single polypeptide [97].

P-gp (also named ABCB1 or MDR1) is the first discovered and well-studied ABCB subfamily transporter that mediates MDR in cancer cells [100]. It is most expressed in the blood−brain barrier, liver, placenta, gallbladder, and endocrine tissues. In 2020, Nosol et al. revealed the protein structure of P-gp and found that inhibitors are bound in pairs and interact with structural features to block the function of P-gp [101]. As P-gp is the substrate of a broad range of antineoplastic agents, overexpressed P-gp has been demonstrated to lead to the development of drug resistance, including chemotherapeutic agents Vinca alkaloids (vinblastine and vincristine), Taxanes (paclitaxel and docetaxel), Anthracyclines (doxorubicin, daunorubicin, and epirubicin), and imatinib mesylate [102,103]. Meanwhile, a high-level expression of P-gp that causes cancer drug resistance has also been found in some tyrosine kinase inhibitors (Imatinib [104], GSK1070916 [105], and WYE354 [106]).

Altered expression levels of MDR1 have been detected in various tumors, such as neuroblastoma, rhabdomyosarcoma, and Wilm's tumor. Neuroblastoma is the most common pediatric solid tumor, which accounts for 7–8% of childhood malignancies and 15% of all pediatric cancer deaths [107]. A study showed that the mRNA expression of MDR1 was increased in neuroblastoma patients with previous chemotherapy [108]. In addition, Qiu et al. [109] found that MDR1 hypermethylation expression can be associated with the pathogenesis and progression of neuroblastoma. MDR1 expression was observed to increase after chemotherapy in rhabdomyosarcoma and Wilm's tumor [110]. These findings may be helpful to understand the role of MDR1 in pediatric malignancies regarding drug resistance and allow researchers to come up with strategies for therapeutic intervention.

#### 5.2.2. MRPs

Multidrug resistance proteins (MRPs) include 9 transporters from 13 members in the ABCC subfamily due to their ability to mediate cancer MDR [111].

Although each of the MRPs have slight differences in structures and amino acid compositions, the mechanism of transport driven by ATP hydrolysis is much the same. Unlike P-gp, which extrudes mostly xenobiotics, MRPs account for the extruding of both endo- and xenobiotics, thus showing its crucial role in regulating MDR processes in cancer development [112]. The structure of MRP1 has shown a novel substrate recruitment mechanism in that substrates are recruited straight from the cytoplasm, whereas P-gp attaches substrates from the inner leaflet of the lipid bilayer [113]. MRPs are distributed in the human body in various tissues, including the blood−brain barrier, brain, lung, kidney, liver, etc. [111].

The expression of MRPs has been investigated in several pediatric malignant tumors as they are a vital factor causing cytotoxic drug resistance and chemotherapy failure. Abnormal MDR expressions have been observed in pediatric malignancies, such as ALL, neuroblastoma, rhabdomyosarcoma, Wilm's tumor, and retinoblastoma [114]. After chemotherapy, MRP1 expression has been observed to be upregulated in neuroblastoma, hepatoblastoma, and rhabdomyosarcoma patients [110]. Increased expression of MRP2-6 and decreased expression of MRP1 and MRP10 have been observed in ALL patients, which are associated with high doses of three chemotherapies [115]. Henderson et al. found that the inhibition of MPR1 is associated with reduced neuroblastoma development in transgenic mice [116]. These studies indicate their role in the chemotherapeutic drug efflux of MPRs and cancer prognosis.

#### 5.2.3. BCRP

The human BCRP has 665 amino acid residues with a molecular weight of 72 kDa. It is a half-transporter that is prominently expressed in various tissues, including, but not limited to, the brain, placenta, testis, liver, breast, and BBB [117]. A high overexpression of BCRP can be observed in different drug resistance cancer types, including in solid tumors and hematopoietic malignancies [118,119]. Although the clinical significance of BCRPmediated drug resistance remains unclear, many studies have shown strong evidence to support that modulating the expression of BCRP could enhance drug sensitivity in chemotherapy [120,121].

BCRP was first identified in 1998, followed by expression studies to explore its potential role in chemoresistance [122]. The expression of the *BCRP* gene in childhood ALL has been observed at a low expression level [123]. However, compared with a diagnosis when the co-expression of BCRP and MDR1 was observed, a higher RNA level of BCRP was expressed at the relapsed/refractory state in acute myeloid leukemia (AML) [124]. Correlations between BCRP and MRPs have also been reported. The combined high expression of BCRP and MRP4 is correlated with reduced antileukemia drug methotrexate accumulation. Similarly, evaluated expression of the *BCRP* gene was found in primary neuroblastoma mitoxantrone-resistant cells [125]. These results underscore the potential value of BCRP as a predictor of chemoresistance drug efflux.

Essentially, MDR can arise because of altered targeted proteins or cell signaling pathways. Changes in cellular or non-cellular processes is also a significant factor of MDR. However, most encountered MDRs are related to drug efflux, which is mainly caused by ABC transporters. Once MDR transporters are overexpressed, the efflux of a chemo drug can increase. For example, the overexpression of P-gp, MRP1, and BCRP decreases the chemosensitivity of cancer cells by limiting exposure to anticancer drugs [126]. The overexpression of P-gp in cancer cells is associated with increased drug resistance to DOX and paclitaxel [127,128]. After anticancer agent treatment, the overexpression of P-gp has been found in acute myeloid leukemia [129]. Decreased chemosensitivity and a high expression of P-gp and BCRP were noticed in MDR patients with chronic lymphocytic

leukemia, metastatic breast cancer, and multiple myeloma [130]. BCRP is regulated by proteins such as TGF-β1 and VEGFR-2 [131,132].

#### **6. Tackling Strategies Regarding Drug Transporters**

MDR is one of the barrier mechanisms against chemotherapy and thus is considered a key factor leading to the failure of chemotherapeutics. The overexpression of MDR1 that can enhance the efflux of cytotoxic agents is one of the targets to improve the effect of chemotherapeutics. One of the strategies is to reverse resistance mechanisms [133]. Several P MDR1 inhibitors have been investigated and shown successful MDR reversal on a drugresistant prostate cancer cell line, without exhibiting a toxic potential [134]. Lei et al. [135] observed intracellular accumulation of paclitaxel and decreased drug efflux activity in the MDR1-knockout colorectal cancer cell line.

Furthermore, applying a nano-drug delivery system (NDDS) is a practical approach to enhance chemotherapy validity due to the targeted co-delivery, reduced sides affected, and long-time blood circulation achieved [136]. Curcumin has an antitumor activity and reverses the tumor MDR effect by regulating the MDR1 protein [137]. Degradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles coloaded with curcumin and DOX could directly target cells or xenografted tumors and inhibit the growth of DOX-resistant esophageal carcinoma with a high biosafety [138]. NDDS-containing compounds have been developed that can inhibit MDR1, including tariquidar (XR9576), tetrandrine, verapamil, and cyclosporin A [139].

Therapy regimens can be different for treating children and adults as they have different drug-resistance profiles. The triple combination of fludarabine, ara-C, and G-CSF has been used in the treatment of childhood AML and caused an additive cell kill [140]. Tipifarnib can target the malignancies, such as leukemia, by activating RAS proteins (HRAS, KRAS, and NRAS) [141].

#### **7. Special Considerations of Resistance in Pediatric Cancer Treatment**

Pediatric malignancies have significant differences in their treatment compared with adult tumors, and thus require special considerations. Pan-cancer analyses have shown that pediatric cancers have comparatively lower mutation frequencies compared with adult cancers [142]. Epigenetic dysregulation, however, seems to be a particular factor in many types of pediatric cancers [143–145]. These genetic and non-genetic changes suggest therapeutic implications regarding chemotherapy.

Children with ALL have better prognoses and outcomes than adult patients with ALL. The 5-year OS rate is 87% for children aged 0–15 years, as opposed to 44% for adults aged 20–29 years [146]. A plethora of factors are responsible for the different outcomes, including socio-economic factors, resistance, disease heterogeneity, host responses, therapeutic treatment, etc. [147]. Resistance is one of the main factors leading to variables among ALL patients of different ages. For example, the activation of P-gp has a higher expression in adults [148]. In addition, the accumulated mutations in the *p53* gene and lower methotrexate polyglutamate may also contribute to the differences in the responses of drug resistance mechanisms [148,149]. Genetic lesions of polycomb repressor complex 2 (PRC2) have been reported in pediatric T-cell ALL (T-ALL), which promotes mutations of the IL7R/JAK/STAT pathway. Poor prednisone response and persistent MRD have been connected to adult T-ALL patients with loss-of-function alterations of PRC2 [150]. Genescà et al. reported that a complex karyotype (≥3 cytogenetic alterations) in adult T-ALL was associated with a minimal residual disease (MRD) level, but no correlation regarding the prednisone response [151]. Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) is a phosphatase suppressing a gene in T-cell ALL. Deletions of PTPN2 in pediatric patients were associated with a higher glucocorticoid response and improved survival in children, yet these trends were not found in adults [149]. Other studies have suggested that a single subclone with additional mutations confers resistance to therapy, although half of the leukemia patients had multiple subclonal mutations [152]. Because children are not

simply small adults, the difference in outcomes across age categories should be considered in diagnosis and therapy.

#### **8. Clinical Trials and Recommendations for Risk Surveillance**

With typically more aggressive protocols being used in children than in adults, chemotherapy is the standard treatment in cancers such as ALL, AML, and Hodgkin lymphoma [153]. The number of clinical trials related to chemotherapy-induced side effects is on the rise.

Notably, VIPN is being studied in two clinical trials. A Phase 4 clinical trial (NCT02923388) is testing Vitamin B12 and vitamin B6 in ALL patients treated with VCR.

The results from a study of 102 patients showed that vitamin B6 and B12 significantly reduced the incidence, relative risk, and severity of VIPN. The amount needed to treat was encouragingly low, and vitamin B6 and B12 were recommended as promising neuroprotective agents against VIPN [154]. A clinical trial (NCT02796365) is currently underway to evaluate the effectiveness of exercise rehabilitation as a preventive measure to DIC. The studies are summarized in Table 3.

**Table 3.** Clinical trials of DIC and VIPN.


Data provided by Clinical Trials (https://clinicaltrials.gov). \* DIC-doxorubicin-induced cardiotoxicity; # Vincristine Induced Peripheral Neuropathy.

Significantly, four clinical trials are investigating or studying DIC. A Phase 2 clinical trial (NCT04166253) is testing vitamin D in breast cancer patients treated with DOX. A clinical trial (NCT02796365) is currently underway to evaluate the effectiveness of exercise rehabilitation as a preventive measure for DIC. The studies are summarized in Table 3.

PanCare is Pan-European Network for care of pediatric cancer survivors. The risk for significant and potentially life-threatening late effects can be identified by certain long-term follow-up projects such as PanCareSurPass and PanCareFollowUp. Based on the reports from 10 countries, ototoxicity following platinum-based chemotherapy has been evaluated regarding the quality of evidence. The ototoxicity surveillance recommendations for pediatric cancer survivors' future studies should focus on the evaluation of otoprotectants and the identification of optimal threshold doses to prevent ototoxicity [155]. In addition, candidate genetic markers are useful for identifying childhood cancer patients at risk of severe late effects, such as *SLC22A2,* which detects those at risk of platinum-induced hearing loss [156]. Moreover, adequate knowledge of cancer history, subsequent treatment exposure, and potential risks of late effects are needed to enhance survivors' health and self-management skills. Accessible and reliable information is essential to increase awareness about late effects, which is necessary for providing personal recommendations for surveillance and prevention [157].

### **9. Conclusions**

Together with the significant advancements in target therapy and immunotherapy, chemotherapy remains the primary treatment option for childhood cancer patients in most low- and middle-income countries. Recognizing the absolute validity of chemo drugs is crucial as they have shown to be highly effective for obtaining long-term survival in childhood cancers. Until now, in total, 16 chemo drugs have been approved by the FDA for pediatric chemotherapy. Unlike adult cancer patients, children diagnosed with cancer are less tolerant of chemotherapy. The numerous adverse effects and occurrence of MDR have become a significant obstacle in pediatric chemotherapy. It is crucial to alleviate severe side effects such as VIPN and DIC. Meanwhile, a comprehensive understanding of MDR and its reversal mechanism is essential. To improve follow-up care quality, the electronic document summarizes the clinical history of childhood/adolescent cancer survivors, including treatments received, and provides personalized follow-up and screening recommendations. By overcoming the challenges of traditional chemotherapy, we will hopefully be able to improve both the survival rate and overall quality of life of pediatric cancer patients.

**Author Contributions:** L.B. and Y.W. wrote the original draft of the manuscript; Y.L. was responsible for preparing figures and tables; J.N.D.W. and Z.H. reviewed and edited the manuscript; Z.-S.C. supervised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** Science and Technology Plan of Jiangxi Provincial Health Commission (no: 202210918); the Guiding Science and Technology Programme of Ganzhou city (no: GZ2022ZSF231).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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