Preface

Genome and exome sequencing together with targeted deep-sequencing studies have defined several gene mutations in almost every myelodysplastic syndrome (MDS) patient. These mutations can impact disease phenotype, prognosis and disease progression. However, there are many non-pathogenic mutations, and most mutations are not specific for MDS. Applying this information clinically is in progress even if the clinical impact of some mutations remains controversial. Several mutations will likely be incorporated into future prognostic scoring systems. Great progress has also been reached in the studies of non-coding RNAs in MDS. It is possible that they will be used in MDS diagnostics and prognosis. Big advancements in the studies of immune mechanisms in MDS have been achieved and translated into clinical studies of immunotherapies in MDS. Innate and adaptive immune pathways are excessively active in the niche of hematopoietic cells in MDS. Common etiological mechanisms of MDS and autoimmune diseases is possible because these diseases are often associated with MDS. Unfortunately, no new drugs have been approved for MDS since approval by the Food and Drug Administration of azacitidine in 2004, lenalidomide in 2005, and decitabine in 2006. The European Medicines Agency approved azacitidine in 2008 and lenalidomide in 2013. This progress in many aspects of MDS warrants this new book about this heterogeneous group of clonal neoplasms arising from hematopoietic stem cells, and characterized by inefficient hematopoiesis, peripheral cytopenias, frequent karyotypic abnormalities, risk of clonal evolution, and transformation to acute myeloid leukemia, three years after the last book about MDS published by IntechOpen.

This book provides a review of several fields of MDS not only for research workers and clinicians, but also for medical students with an interest in MDS. The first introductory chapter deals with recent progress in this area. The second chapter provides a review of diagnosis and classification of MDS. Chapter 3 discusses the association of MDS and autoimmune disorders. Chapter 4 introduces immune dysregulation in MDS. The fifth chapter analyzes an update on pathophysiology and management of MDS. The last chapter discusses non-coding RNAs in MDS.

> **Ota Fuchs, PhD** Institute of Hematology and Blood Transfusion, Department of Genomics, Prague, Czech Republic

**1**

**Chapter 1**

*Ota Fuchs*

**gene mutations**

trisomy 8 or trisomy 21 [7].

complex) [4, 8–10].

often controversial.

Introductory Chapter: Progress in

Myelodysplastic Syndrome Area

**1. Advances in our knowledge of cytogenetic abnormalities and**

Myelodysplastic syndromes (MDS) constitute a group of age-associated heterogeneous clonal hematopoietic disorders characterized by ineffective hematopoiesis with peripheral cytopenias, dysplasia, and an increased risk of progression to acute myeloid leukemia (AML) [1–6]. About 50% of cases of MDS are characterized by the presence of cytogenetic abnormalities. Losses of chromosomal material as del(5q), del(20q), monosomy 7 or del(7q), and del(Y) are most common cytogenetic abnormalities and are more frequent than gains of chromosomal material as

MDS are caused by abnormalities in many genes. The great progress in analysis of these mutations and in elucidation of relationships between gene mutations and clinical phenotypes of these disorders was achieved. Somatic mutations were found in more than 90%. Next-generation sequencing (NGS) detected about 10 different mutations in almost every patient with MDS. The majority of these mutations are nonpathogenic passenger mutations. However, one or more driver mutations in most patients with MDS are associated with the pathogenesis of MDS. Gene mutations affect proteins involved in various important cell processes as RNA-splicing, DNA methylation, histone and chromatin modifications, signal transduction, transcription (transcription factors), tumor suppressor (*TP53*), *RAS* pathway, and separation of sister chromatids during cell division (cohesion

RNA-splicing and DNA methylation mutations occur early and are known as founding mutations. Other mutations are called subclonal mutations. No MDSspecific mutations exist. Strongly represented mutations in genes coding for proteins involved in DNA methylation, such as *TET2*, *DNMT3A*, and *ASXL1*, are common also in older individuals with normal blood count (clonal hematopoiesis of indeterminate potential/CHIP/) [11, 12]. Until now, mutations in *TP53*, *EZH2*, *RUNX1*, and *SF3B1* predict independently overall survival (OS) of MDS patients. The first three mutations are associated with shorter OS but the last mutation is connected with better survival in refractory anemia with ring sideroblast (MDS-RS) and with thrombocytosis (RARS-T) [13, 14]. *SF3B1* mutations are present in about 80% of MDS-RS and correlates with its development. *SF3B1* mutations could alter the expression of the gene for ABCB7 transporter and abnormally regulate iron homeostasis in mitochondria mediating the phenotype of acquired MDS-RS [15]. Effects of other mutations are not clear up to now and results are

We lack clinical methods to stop clonal development from relatively benign state of CHIP to malignancy. Especially, *TP53*-mutant clones induce progress to therapyrelated MDS/AML. Therapy-related myeloid neoplasms have mutations in *TP53* and

### **Chapter 1**

## Introductory Chapter: Progress in Myelodysplastic Syndrome Area

*Ota Fuchs*

### **1. Advances in our knowledge of cytogenetic abnormalities and gene mutations**

Myelodysplastic syndromes (MDS) constitute a group of age-associated heterogeneous clonal hematopoietic disorders characterized by ineffective hematopoiesis with peripheral cytopenias, dysplasia, and an increased risk of progression to acute myeloid leukemia (AML) [1–6]. About 50% of cases of MDS are characterized by the presence of cytogenetic abnormalities. Losses of chromosomal material as del(5q), del(20q), monosomy 7 or del(7q), and del(Y) are most common cytogenetic abnormalities and are more frequent than gains of chromosomal material as trisomy 8 or trisomy 21 [7].

MDS are caused by abnormalities in many genes. The great progress in analysis of these mutations and in elucidation of relationships between gene mutations and clinical phenotypes of these disorders was achieved. Somatic mutations were found in more than 90%. Next-generation sequencing (NGS) detected about 10 different mutations in almost every patient with MDS. The majority of these mutations are nonpathogenic passenger mutations. However, one or more driver mutations in most patients with MDS are associated with the pathogenesis of MDS. Gene mutations affect proteins involved in various important cell processes as RNA-splicing, DNA methylation, histone and chromatin modifications, signal transduction, transcription (transcription factors), tumor suppressor (*TP53*), *RAS* pathway, and separation of sister chromatids during cell division (cohesion complex) [4, 8–10].

RNA-splicing and DNA methylation mutations occur early and are known as founding mutations. Other mutations are called subclonal mutations. No MDSspecific mutations exist. Strongly represented mutations in genes coding for proteins involved in DNA methylation, such as *TET2*, *DNMT3A*, and *ASXL1*, are common also in older individuals with normal blood count (clonal hematopoiesis of indeterminate potential/CHIP/) [11, 12]. Until now, mutations in *TP53*, *EZH2*, *RUNX1*, and *SF3B1* predict independently overall survival (OS) of MDS patients. The first three mutations are associated with shorter OS but the last mutation is connected with better survival in refractory anemia with ring sideroblast (MDS-RS) and with thrombocytosis (RARS-T) [13, 14]. *SF3B1* mutations are present in about 80% of MDS-RS and correlates with its development. *SF3B1* mutations could alter the expression of the gene for ABCB7 transporter and abnormally regulate iron homeostasis in mitochondria mediating the phenotype of acquired MDS-RS [15]. Effects of other mutations are not clear up to now and results are often controversial.

We lack clinical methods to stop clonal development from relatively benign state of CHIP to malignancy. Especially, *TP53*-mutant clones induce progress to therapyrelated MDS/AML. Therapy-related myeloid neoplasms have mutations in *TP53* and epigenetic modifying genes, instead of mutations in tyrosine kinase and spliceosome genes [16]. The possible treatments are now the use of hypomethylating agents or in future anti-inflammatory therapy and clonally selective immunotherapies.

MDS are associated with genomic instability and extensive DNA damage caused by deficient repair mechanisms. Aberrations in DNA damage response/repair genes other than *TP53* and some genes involved in DNA damage checkpoints are rare. Differential expression of homologous recombination DNA repair-associated genes during MDS progression was detected and could be confirmed as new biomarkers related to pathogenesis and poor prognosis in MDS [17, 18].

### **2. Advance in our understanding of del(5q) myelodysplastic syndrome pathogenesis and its treatment with lenalidomide**

The greatest progress was achieved in the study of molecular pathogenesis of del(5q) MDS disease phenotype and its treatment by immunomodulatory or cereblon-binding drug lenalidomide [2, 19–35]. Ebert et al. described that impaired ribosome biosynthesis due to *RPS14* (ribosomal protein 14 of the small ribosome subunit) gene haploinsufficiency leads to the E3 ubiquitin ligase HDM2 (human homolog to mouse double minute 2, major negative regulator of p53) inactivation by free ribosomal proteins, particularly RPL11 [36]. HDM2 degradation drives p53-mediated apoptosis of erythroid cells carrying the del(5q) aberration. This p53-mediated apoptosis of erythroid cells is a key effector of hypoplastic anemia in MDS patients with del(5q) [36]. *RPS14* haploinsufficiency causes a block in erythroid differentiation mediated by calprotectin (the heterodimeric S100 calciumbinding proteins S100A8 and S100A9) [37]. Proinflammatory proteins, S100A9 and tumor necrosis factor-α, suppress the effect of erythropoietin in MDS [38]. Some patients originally considered as MDS patients without del(5q) can have a phenotype of atypical 5q− syndrome and can be sensitive to lenalidomide therapy because they have diminutive somatic deletions in the 5q region. These deletions were not identified by fluorescence in situ hybridization or cytogenetic testing but by single nucleotide polymorphism array genotyping [39]. Low RPS14 expression in 50–70% MDS patients without del(5q) confers higher apoptosis rate of nucleated erythrocytes and predicts prolonged survival [40, 41].

What is the mechanism of lenalidomide in del(5q) MDS based on what has been achieved and elucidated to date? Lenalidomide stabilizes E3 ubiquitin ligase HDM2, thereby accelerating p53 degradation [42, 43]. Lenalidomide inhibits phosphatases PP2a and Cdc25c (coregulators of cell cycle which genes are very commonly deleted in del(5q) MDS) with consequent G2 arrest of del(5q) MDS progenitors and their apoptosis. PP2a and Cdc25c inhibition by lenalidomide suppress HDM2 autoubiquitination and subsequent degradation. Thus, lenalidomide has been shown to not only reverse apoptosis within the erythroid compartment, but also directly induce apoptosis of the myeloid clone in del(5q) MDS [44, 45]. Lenalidomide upregulates expression of other two haploinsufficient genes located on chromosome 5q, genes for microRNAs (miR-145 and miR-146a) [46]. These miRs are involved in Toll-like receptor pathway, IL-6 induction, and regulation of megakaryopoiesis [20].

Ito et al. discovered that thalidomide (founding member of immunomodulatory drugs/IMiDs/) binds cereblon (CRBN) in the terminal C-region (parts of exons 10 and 11 of the *CRBN* gene code this IMiD binding region) [47]. Several researchers confirmed CRBN as target of lenalidomide in multiple myeloma (MM), lymphoma, chronic lymphocytic leukemia, and del(5q) MDS [48]. CRBN is the ubiquitously expressed 51 kDa protein with a putative role in cerebral development, especially memory and learning [49, 50].

**3**

*Introductory Chapter: Progress in Myelodysplastic Syndrome Area*

subgroup known to be especially sensitive to lenalidomide [51].

mutations on lenalidomide treatment in del(5q) MDS [57, 58].

of GATA2 transcriptional complex [59].

**treatment and new possible therapies**

Even if the treatment of del(5q) MDS patients with lenalidomide is very efficient, 50% of treated patients relapse after 2–3 years. Martinez-Hoyer et al. found that low platelet count and occurrence of additional mutations, mainly *TP53* mutations induce lenalidomide resistance [59–61]. They used whole genome sequencing and observed in several resistant patients mutations in *RUNX1* gene or decreased amount of *RUNX1* transcript without aberration in *TP53* [59]. Results were verified in model system of two human del(5q) lines, MDS-L and KG-1a. *RUNX1* knock-out or RUNX1 shRNA increased proliferation and reduced apoptosis in lenalidomidetreated cells with decreased *RUNX1* transcript. Therefore, effect of lenalidomide in del(5q) requires functional RUNX1. Similar results were obtained with *TP53* knock-out cells. Both *RUNX1* and *TP53* transcripts cooperate and alter the activity

**3. Studies on lenalidomide use also in lower risk non-del(5q) MDS**

While CSNK1A1 is CRL4CRBN target in del(5q) MDS, CRL4CRBN targets in lower risk non-del(5q) remain to be determined. The mechanism of action of lenalidomide is still unclear in non-del(5q) MDS cells. Recent evidence shows that lenalidomide directly improves erythropoietin receptor (EPOR) signaling by EPOR upregulation mediated by a posttranscriptional mechanism [62]. Lenalidomide

Our group found that del(5q) MDS patients (the so-called 5q minus syndrome) have higher levels of full-length CRBN mRNA than other patients with lower risk MDS, linking higher levels of a known lenalidomide target CRBN and del(5q) MDS

CRBN is a member and substrate receptor of the cullin 4 RING E3 ubiquitin ligase complex (CRL4). CRBN recruits substrate proteins to the CRL4 complex for ubiquitination and the subsequent degradation in proteasomes. IMiDs binds to CRBN in CRL4 complex and block normal endogenous substrates (CRBN and the homeobox transcription factor MEIS2 in multiple myeloma/MM/) from binding to CRL4 for ubiquitination and degradation [52]. After IMID binding to CRBN, CRL4 complex is recruiting transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) for ubiquitination and degradation in MM cells [53]. Degradation of these transcription factors explains lenalidomide's growth inhibition of MM cells and increased interleukin-2 (IL-2) release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide's activity in MDS with del(5q). Fink et al. identified a novel target casein kinase1A1 (CSNK1A1) by quantitative proteomics in the myeloid cell line KG-1 [54]. CSNK1A1 is encoded in the del(5q) commonly deleted region and the gene is haploinsufficient. Lenalidomide treatment leads to increased ubiquitination of the remaining CSNK1A1 and decreased protein abundance. CSNK1A1 negatively regulates β-catenin which drives stem cell self-renewal, and *CSNK1A1* haploinsufficiency causes the initial clonal expansion in patients with the del(5q) MDS and contributes to the pathogenesis of del(5q) MDS. The further inhibition of CSNK1A1 in del(5q) MDS is associated with del(5q) failure and p53 activation. The inhibition of CSNK1A1 reduced RPS6 phosphorylation, induced p53 expression and growth inhibition, and triggered myeloid differentiation program. TP53-null leukemia did not respond to CSNK1A1 inhibition, strongly supporting the importance of the p53 expression for the yield of CSNK1A1 inhibition. *CSNK1A1* mutations have been recently found in 5–18% of MDS patients with del(5q) [55]. These mutations are associated similarly to the effect of *TP53* mutations with rise to a poor prognosis in del(5q) MDS [56]. Other studies did not find impact of *CSNK1A1*

*DOI: http://dx.doi.org/10.5772/intechopen.84594*

#### *Introductory Chapter: Progress in Myelodysplastic Syndrome Area DOI: http://dx.doi.org/10.5772/intechopen.84594*

*Recent Developments in Myelodysplastic Syndromes*

related to pathogenesis and poor prognosis in MDS [17, 18].

**pathogenesis and its treatment with lenalidomide**

cytes and predicts prolonged survival [40, 41].

epigenetic modifying genes, instead of mutations in tyrosine kinase and spliceosome genes [16]. The possible treatments are now the use of hypomethylating agents or in future anti-inflammatory therapy and clonally selective immunotherapies.

MDS are associated with genomic instability and extensive DNA damage caused by deficient repair mechanisms. Aberrations in DNA damage response/repair genes other than *TP53* and some genes involved in DNA damage checkpoints are rare. Differential expression of homologous recombination DNA repair-associated genes during MDS progression was detected and could be confirmed as new biomarkers

**2. Advance in our understanding of del(5q) myelodysplastic syndrome**

The greatest progress was achieved in the study of molecular pathogenesis of del(5q) MDS disease phenotype and its treatment by immunomodulatory or cereblon-binding drug lenalidomide [2, 19–35]. Ebert et al. described that impaired ribosome biosynthesis due to *RPS14* (ribosomal protein 14 of the small ribosome subunit) gene haploinsufficiency leads to the E3 ubiquitin ligase HDM2 (human homolog to mouse double minute 2, major negative regulator of p53) inactivation by free ribosomal proteins, particularly RPL11 [36]. HDM2 degradation drives p53-mediated apoptosis of erythroid cells carrying the del(5q) aberration. This p53-mediated apoptosis of erythroid cells is a key effector of hypoplastic anemia in MDS patients with del(5q) [36]. *RPS14* haploinsufficiency causes a block in erythroid differentiation mediated by calprotectin (the heterodimeric S100 calciumbinding proteins S100A8 and S100A9) [37]. Proinflammatory proteins, S100A9 and tumor necrosis factor-α, suppress the effect of erythropoietin in MDS [38]. Some patients originally considered as MDS patients without del(5q) can have a phenotype of atypical 5q− syndrome and can be sensitive to lenalidomide therapy because they have diminutive somatic deletions in the 5q region. These deletions were not identified by fluorescence in situ hybridization or cytogenetic testing but by single nucleotide polymorphism array genotyping [39]. Low RPS14 expression in 50–70% MDS patients without del(5q) confers higher apoptosis rate of nucleated erythro-

What is the mechanism of lenalidomide in del(5q) MDS based on what has been achieved and elucidated to date? Lenalidomide stabilizes E3 ubiquitin ligase HDM2, thereby accelerating p53 degradation [42, 43]. Lenalidomide inhibits phosphatases PP2a and Cdc25c (coregulators of cell cycle which genes are very commonly deleted in del(5q) MDS) with consequent G2 arrest of del(5q) MDS progenitors and their apoptosis. PP2a and Cdc25c inhibition by lenalidomide suppress HDM2 autoubiquitination and subsequent degradation. Thus, lenalidomide has been shown to not only reverse apoptosis within the erythroid compartment, but also directly induce apoptosis of the myeloid clone in del(5q) MDS [44, 45]. Lenalidomide upregulates expression of other two haploinsufficient genes located on chromosome 5q, genes for microRNAs (miR-145 and miR-146a) [46]. These miRs are involved in Toll-like receptor pathway, IL-6 induction, and regulation of megakaryopoiesis [20].

Ito et al. discovered that thalidomide (founding member of immunomodulatory drugs/IMiDs/) binds cereblon (CRBN) in the terminal C-region (parts of exons 10 and 11 of the *CRBN* gene code this IMiD binding region) [47]. Several researchers confirmed CRBN as target of lenalidomide in multiple myeloma (MM), lymphoma, chronic lymphocytic leukemia, and del(5q) MDS [48]. CRBN is the ubiquitously expressed 51 kDa protein with a putative role in cerebral development, especially

**2**

memory and learning [49, 50].

Our group found that del(5q) MDS patients (the so-called 5q minus syndrome) have higher levels of full-length CRBN mRNA than other patients with lower risk MDS, linking higher levels of a known lenalidomide target CRBN and del(5q) MDS subgroup known to be especially sensitive to lenalidomide [51].

CRBN is a member and substrate receptor of the cullin 4 RING E3 ubiquitin ligase complex (CRL4). CRBN recruits substrate proteins to the CRL4 complex for ubiquitination and the subsequent degradation in proteasomes. IMiDs binds to CRBN in CRL4 complex and block normal endogenous substrates (CRBN and the homeobox transcription factor MEIS2 in multiple myeloma/MM/) from binding to CRL4 for ubiquitination and degradation [52]. After IMID binding to CRBN, CRL4 complex is recruiting transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) for ubiquitination and degradation in MM cells [53]. Degradation of these transcription factors explains lenalidomide's growth inhibition of MM cells and increased interleukin-2 (IL-2) release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide's activity in MDS with del(5q). Fink et al. identified a novel target casein kinase1A1 (CSNK1A1) by quantitative proteomics in the myeloid cell line KG-1 [54]. CSNK1A1 is encoded in the del(5q) commonly deleted region and the gene is haploinsufficient. Lenalidomide treatment leads to increased ubiquitination of the remaining CSNK1A1 and decreased protein abundance. CSNK1A1 negatively regulates β-catenin which drives stem cell self-renewal, and *CSNK1A1* haploinsufficiency causes the initial clonal expansion in patients with the del(5q) MDS and contributes to the pathogenesis of del(5q) MDS. The further inhibition of CSNK1A1 in del(5q) MDS is associated with del(5q) failure and p53 activation. The inhibition of CSNK1A1 reduced RPS6 phosphorylation, induced p53 expression and growth inhibition, and triggered myeloid differentiation program. TP53-null leukemia did not respond to CSNK1A1 inhibition, strongly supporting the importance of the p53 expression for the yield of CSNK1A1 inhibition. *CSNK1A1* mutations have been recently found in 5–18% of MDS patients with del(5q) [55]. These mutations are associated similarly to the effect of *TP53* mutations with rise to a poor prognosis in del(5q) MDS [56]. Other studies did not find impact of *CSNK1A1* mutations on lenalidomide treatment in del(5q) MDS [57, 58].

Even if the treatment of del(5q) MDS patients with lenalidomide is very efficient, 50% of treated patients relapse after 2–3 years. Martinez-Hoyer et al. found that low platelet count and occurrence of additional mutations, mainly *TP53* mutations induce lenalidomide resistance [59–61]. They used whole genome sequencing and observed in several resistant patients mutations in *RUNX1* gene or decreased amount of *RUNX1* transcript without aberration in *TP53* [59]. Results were verified in model system of two human del(5q) lines, MDS-L and KG-1a. *RUNX1* knock-out or RUNX1 shRNA increased proliferation and reduced apoptosis in lenalidomidetreated cells with decreased *RUNX1* transcript. Therefore, effect of lenalidomide in del(5q) requires functional RUNX1. Similar results were obtained with *TP53* knock-out cells. Both *RUNX1* and *TP53* transcripts cooperate and alter the activity of GATA2 transcriptional complex [59].

### **3. Studies on lenalidomide use also in lower risk non-del(5q) MDS treatment and new possible therapies**

While CSNK1A1 is CRL4CRBN target in del(5q) MDS, CRL4CRBN targets in lower risk non-del(5q) remain to be determined. The mechanism of action of lenalidomide is still unclear in non-del(5q) MDS cells. Recent evidence shows that lenalidomide directly improves erythropoietin receptor (EPOR) signaling by EPOR upregulation mediated by a posttranscriptional mechanism [62]. Lenalidomide

stabilizes the EPOR protein by inhibition of the E3 ubiquitin ligase RNF41 (ring finger protein 41, also known as neuregulin receptor degradation protein-1/Nrdp1/ and fetal liver ring finger/FLRF/) responsible for EPOR polyubiquitination and next degradation [62] and induces lipid raft assembly to enhance EPOR signaling in MDS erythroid progenitors [63, 64].

After failure of ESAs, lenalidomide yields red blood cell transfusion independence in 20–30% of lower risk non-del(5q) MDS. Indeed, several observations suggest an additive effect of ESA and lenalidomide in this situation [65, 66] and also in del(5q) MDS patients [67]. Synthetic corticosteroids (dexamethasone and prednisone) are also able to potentiate the effect of lenalidomide or combination of lenalidomide and erythropoietin [67–69].

Basiorka et al. and Sallman et al. reported activation of the NLRP3 inflammasome in MDS [70, 71]. NRLP3 drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins. Activated NLRP3 complexes direct then activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX) and increasing levels of reactive oxygen species (ROS). ROS initiate cation influx, cell swelling, and β-catenin activation. Knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs cause a common redox-sensitive inflammasome circuit. They are new candidates for therapeutic intervention.

Not only apoptosis and pyroptosis are involved in increased cell death in MDS. Recently, possible further mechanism of cell death, necroptosis, in MDS has been described [72, 73]. Necroptosis is like pyroptosis associated with membrane permeabilization and the release of damage-associated molecular patterns (DAMPs) such as alarmins. Alarmins bind Toll-like receptor 4 (TLR4) and activate the transcription factor NF-κB and inflammation [74].

The effects of lenalidomide in non-del(5q) are thought to be secondary to modulation of the immune system. Hyperactivated T cells inhibit hematopoiesis. Immunosuppressive therapies with antithymocyte globulin alone and in combination with prednisone or cyclosporine show response rates between 25 and 40% [75, 76].

The studies discussed in this and other chapters of this book will help to translate our knowledge of genetic aberrations and of pathophysiological mechanisms in MDS into clinical use in diagnosis, prognosis, and therapy. Novel agents developed on the basis of this knowledge (luspatercept, rigosertib, immune checkpoint inhibitors, venetoclax, and others) are in clinical trials and will help in relapsed/ refractory MDS.

The work of our group in this area was supported by the research project for conceptual development of research organization (00023736; Institute of Hematology and Blood Transfusion, Prague) from the Ministry of Health of the Czech Republic.

**5**

**Author details**

Ota Fuchs

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
*Introductory Chapter: Progress in Myelodysplastic Syndrome Area*

*DOI: http://dx.doi.org/10.5772/intechopen.84594*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Institute of Hematology and Blood Transfusion, Prague, Czech Republic

*Introductory Chapter: Progress in Myelodysplastic Syndrome Area DOI: http://dx.doi.org/10.5772/intechopen.84594*

*Recent Developments in Myelodysplastic Syndromes*

MDS erythroid progenitors [63, 64].

lenalidomide and erythropoietin [67–69].

therapeutic intervention.

refractory MDS.

Czech Republic.

stabilizes the EPOR protein by inhibition of the E3 ubiquitin ligase RNF41 (ring finger protein 41, also known as neuregulin receptor degradation protein-1/Nrdp1/ and fetal liver ring finger/FLRF/) responsible for EPOR polyubiquitination and next degradation [62] and induces lipid raft assembly to enhance EPOR signaling in

After failure of ESAs, lenalidomide yields red blood cell transfusion independence in 20–30% of lower risk non-del(5q) MDS. Indeed, several observations suggest an additive effect of ESA and lenalidomide in this situation [65, 66] and also in del(5q) MDS patients [67]. Synthetic corticosteroids (dexamethasone and prednisone) are also able to potentiate the effect of lenalidomide or combination of

Basiorka et al. and Sallman et al. reported activation of the NLRP3 inflammasome in MDS [70, 71]. NRLP3 drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins. Activated NLRP3 complexes direct then activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX) and increasing levels of reactive oxygen species (ROS). ROS initiate cation influx, cell swelling, and β-catenin activation. Knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs cause a common redox-sensitive inflammasome circuit. They are new candidates for

Not only apoptosis and pyroptosis are involved in increased cell death in MDS. Recently, possible further mechanism of cell death, necroptosis, in MDS has been described [72, 73]. Necroptosis is like pyroptosis associated with membrane permeabilization and the release of damage-associated molecular patterns (DAMPs) such as alarmins. Alarmins bind Toll-like receptor 4 (TLR4) and activate

The effects of lenalidomide in non-del(5q) are thought to be secondary to modulation of the immune system. Hyperactivated T cells inhibit hematopoiesis. Immunosuppressive therapies with antithymocyte globulin alone and in combination with prednisone or cyclosporine show response rates between 25 and 40% [75, 76]. The studies discussed in this and other chapters of this book will help to translate our knowledge of genetic aberrations and of pathophysiological mechanisms in MDS into clinical use in diagnosis, prognosis, and therapy. Novel agents developed on the basis of this knowledge (luspatercept, rigosertib, immune checkpoint inhibitors, venetoclax, and others) are in clinical trials and will help in relapsed/

The work of our group in this area was supported by the research project for conceptual development of research organization (00023736; Institute of Hematology and Blood Transfusion, Prague) from the Ministry of Health of the

the transcription factor NF-κB and inflammation [74].

**4**

## **Author details**

Ota Fuchs Institute of Hematology and Blood Transfusion, Prague, Czech Republic

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

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[3] Shastri A, Will B, Steidl U, Verma A. Stem and progenitor cell alterations in myelodysplastic syndromes. Blood. 2017;**129**(12):1586-1594. DOI: 10.1182/ blood-2016-10-696062

[4] Nazha A, Sekeres MA. Precision medicine in myelodysplastic syndromes and leukemias: Lessons from sequential mutations. Annual Review of Medicine. 2017;**68**:127-137. DOI: 10.1146/ annurev-med-062915-095637

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[51] Jonasova A, Bokorova R, Polak J, Vostry M, Kostecka A, Hajkova H, et al. High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide. European Journal of Haematology. 2015;**95**(1): 27-34. DOI: 10.1111/ejh.12457

[52] Fischer ES, Böhm K, Lydeard JR, Yang H, Stadler MB, Cavadini S, et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature. 2014;**512**(7512): 49-53. DOI: 10.1038/nature13527

[53] Krönke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;**343**(6168):301-305. DOI: 10.1126/ science. 1244851

[54] Krönke J, Fink EC, Hollenbach PW, MacBeth KJ, Hurst SN, Udeshi ND, et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature. 2015;**523**(7559):183-188. DOI: 10.1038/nature14610

[55] Schneider RK, Ademà V, Heckl D, Järås M, Mallo M, Lord AM, et al. Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS. Cancer Cell. 2014;**26**(4):509-520. DOI: 10.1016/j.ccr. 2014.08.001

[56] Smith AE, Kulasekararaj AG, Jiang J, Mian S, Mohamedali A, Gaken J, et al. CSNK1A1 mutations and isolated del(5q) abnormality in myelodysplastic syndrome: A retrospective mutational analysis. The Lancet Haematology. 2015;**2**(5):e212-e221. DOI: 10.1016/ S2352-3026(15)00050-2

[57] Heuser M, Meggendorfer M, Cruz MM, Fabisch J, Klesse S, Köhler L, et al. Frequency and prognostic impact of casein kinase 1A1 mutations in MDS patients with deletion of chromosome 5q. Leukemia. 2015;**29**(9):1942-1945. DOI: 10.1038/leu.2015.49

[58] Negoro E, Radiovoyevitch T, Polprasert C, Adema V, Hosono N, Makishima H, et al. Molecular predictors of response in patients with myeloid neoplasms treated with lenalidomide. Leukemia. 2016;**30**(12):2405-2409. DOI: 10.1038/ leu.2016.228

[59] Martinez-Høyer S, Mo A, Deng D, Jiang J, Docking R, Li J, et al. Resistance to lenalidomide in del(5q) MDS is mediated by inhibition of drug-induced megakaryocytic differentiation. Blood. 2018;**132**:176. DOI: 10.1182/ blood-2018-176

[60] List A, Ebert BL, Fenaux P. A decade of progress in myelodysplastic syndrome with chromosome 5q deletion. Leukemia. 2018;**32**(7):1493- 1499. DOI: 10.1038/s41375-018-0029-9

[61] Belickova M, Vesela J, Jonasova A, Pejsova B, Votavova H, Merkerova MD, et al. TP53 mutation variant allele frequency is a potential predictor for clinical outcome of patients with lower-risk myelodysplastic syndromes. Oncotarget. 2016;**7**(24):36266-36279. DOI: 10.18632/oncotarget.9200

[62] Basiorka AA, McGraw KL, De Ceuninck L, Griner LN, Zhang L, Clark JA, et al. Lenalidomide stabilizes the erythropoietin receptor by inhibiting

the E3 ubiquitin ligase RNF41. Cancer Research. 2016;**76**(12):3531-3540. DOI: 10.1158/0008-5472.CAN-15-1756

[63] McGraw KL, Fuhler GM, Johnson JO, Clark JA, Caceres GC, Sokol L, et al. Erythropoietin receptor signaling is membrane raft dependent. PLoS One. 2012;**7**(4):e34477. DOI: 10.1371/journal. pone.0034477

[64] McGraw KL, Basiorka AA, Johnson JO, Clark J, Caceres G, Padron E, et al. Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors. PLoS One. 2014;**9**(12):e114249. DOI: 10.1371/ journal.pone.0114249

[65] Komrokji RS, Lancet JE, Swern AS, Chen N, Paleveda J, Lush R, et al. Combined treatment with lenalidomide and epoetin alfa in lower-risk patients with myelodysplastic syndrome. Blood. 2012;**120**(17):3419-3424. DOI: 10.1182/ blood-2012-03-415661

[66] Toma A, Kosmider O, Chevret S, Delaunay J, Stamatoullas A, Rose C, et al. Lenalidomide with or without erythropoietin in transfusiondependent erythropoiesis-stimulating agent-refractory lower-risk MDS without 5q deletion. Leukemia. 2016;**30**(4):897-905. DOI: 10.1038/ leu.2015.296

[67] Jonasova A, Neuwirtova R, Polackova H, Siskova M, Stopka T, Cmunt E, et al. Lenalidomide treatment in lower risk myelodysplastic syndromes—The experience of a Czech hematology center. (Positive effect of erythropoietin ± prednisone addition to lenalidomide in refractory or relapsed patients). Leukemia Research. 2018;**69**:12-17. DOI: 10.1016/j. leukres.2018.03.015

[68] Narla A, Dutt S, McAuley JR, Al-Shahrour F, Hurst S, McConkey M, et al. Dexamethasone and

**11**

*Introductory Chapter: Progress in Myelodysplastic Syndrome Area*

Immunosuppressive therapy: Exploring an underutilized treatment option for myelodysplastic syndrome. Clinical Lymphoma, Myeloma & Leukemia. 2016;**16**(Suppl):S44-S48. DOI: 10.1016/j.

[76] Stahl M, Zeidan AM. Lenalidomide use in myelodysplastic syndromes: Insights into the biologic mechanisms and clinical applications. Cancer. 2017;**123**(10):1703-1713. DOI: 10.1002/

clml.2016.02.017

cncr.30585

*DOI: http://dx.doi.org/10.5772/intechopen.84594*

lenalidomide have distinct functional effects on erythropoiesis. Blood. 2011;**118**(8):2296-2304. DOI: 10.1182/

[69] Komrokji RS, Al Ali NH, Padron E, Cogle C, Tinsley S, Sallman D, et al. Lenalidomide and prednisone in low and intermediate-1 IPSS risk, nondel(5q) MDS patients: A phase II clinical trial. Clinical Lymphoma, Myeloma & Leukemia. 2019. DOI: 10.1016/j.

[70] Basiorka AA, McGraw KL, Eksioglu EA, Chen X, Johnson J, Zhang L, et al. The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype. Blood.

2016;**128**(25):2960-2975. DOI: 10.1182/

[71] Sallman DA, Cluzeau T, Basiorka AA, List A. Unraveling the pathogenesis of MDS: The NLRP3 inflammasome and pyroptosis drive the MDS phenotype. Frontiers in Oncology. 2016;**6**:151. DOI:

[72] Croker BA, Kelliher MA. BIDding on necroptosis in MDS. Blood. 2019;**133**(2):103-104. DOI: 10.1182/

[73] Wagner PN, Shi Q , Salisbury-Ruf CT, Zou J, Savona MR, Fedoriw Y, et al. Increased Ripk1-mediated bone marrow necroptosis leads to myelodysplasia and bone marrow failure in mice. Blood. 2019;**133**(2):107-120. DOI: 10.1182/

blood-2010-11-318543

clml.2018.12.014

blood-2016-07-730556

10.3389/fonc.2016.00151

blood-2018-11-886242

blood-2018-05-847335

[74] Ping Z, Chen S, Hermans SJF, Kenswil KJG, Feyen J, van Dijk C, et al. Activation of NF-κB driven inflammatory programs in mesenchymal elements attenuates

hematopoiesis in low-risk

myelodysplastic syndromes. Leukemia. 2018. DOI: 10.1038/s41375-018-0267-x

[75] Haider M, Al Ali N, Padron E, Epling-Burnette P, Lancet J, List A, et al. *Introductory Chapter: Progress in Myelodysplastic Syndrome Area DOI: http://dx.doi.org/10.5772/intechopen.84594*

lenalidomide have distinct functional effects on erythropoiesis. Blood. 2011;**118**(8):2296-2304. DOI: 10.1182/ blood-2010-11-318543

*Recent Developments in Myelodysplastic Syndromes*

the E3 ubiquitin ligase RNF41. Cancer Research. 2016;**76**(12):3531-3540. DOI: 10.1158/0008-5472.CAN-15-1756

[63] McGraw KL, Fuhler GM, Johnson JO, Clark JA, Caceres GC, Sokol L, et al. Erythropoietin receptor signaling is membrane raft dependent. PLoS One. 2012;**7**(4):e34477. DOI: 10.1371/journal.

[64] McGraw KL, Basiorka AA, Johnson JO, Clark J, Caceres G, Padron E, et al. Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors. PLoS One. 2014;**9**(12):e114249. DOI: 10.1371/

[65] Komrokji RS, Lancet JE, Swern AS, Chen N, Paleveda J, Lush R, et al. Combined treatment with lenalidomide and epoetin alfa in lower-risk patients with myelodysplastic syndrome. Blood. 2012;**120**(17):3419-3424. DOI: 10.1182/

[66] Toma A, Kosmider O, Chevret S, Delaunay J, Stamatoullas A, Rose C, et al. Lenalidomide with or without erythropoietin in transfusion-

dependent erythropoiesis-stimulating agent-refractory lower-risk MDS without 5q deletion. Leukemia. 2016;**30**(4):897-905. DOI: 10.1038/

treatment in lower risk myelodysplastic syndromes—The experience of a Czech hematology center. (Positive effect of erythropoietin ± prednisone addition to lenalidomide in refractory or relapsed patients). Leukemia Research. 2018;**69**:12-17. DOI: 10.1016/j.

[67] Jonasova A, Neuwirtova R, Polackova H, Siskova M, Stopka T, Cmunt E, et al. Lenalidomide

[68] Narla A, Dutt S, McAuley JR, Al-Shahrour F, Hurst S, McConkey M, et al. Dexamethasone and

pone.0034477

journal.pone.0114249

blood-2012-03-415661

leu.2015.296

leukres.2018.03.015

[56] Smith AE, Kulasekararaj AG, Jiang J, Mian S, Mohamedali A, Gaken J, et al. CSNK1A1 mutations and isolated del(5q) abnormality in myelodysplastic syndrome: A retrospective mutational analysis. The Lancet Haematology. 2015;**2**(5):e212-e221. DOI: 10.1016/

[57] Heuser M, Meggendorfer M, Cruz MM, Fabisch J, Klesse S, Köhler L, et al. Frequency and prognostic impact of casein kinase 1A1 mutations in MDS patients with deletion of chromosome 5q. Leukemia. 2015;**29**(9):1942-1945.

S2352-3026(15)00050-2

DOI: 10.1038/leu.2015.49

leu.2016.228

blood-2018-176

[58] Negoro E, Radiovoyevitch T, Polprasert C, Adema V, Hosono N, Makishima H, et al. Molecular predictors of response in patients with myeloid neoplasms treated with lenalidomide. Leukemia.

2016;**30**(12):2405-2409. DOI: 10.1038/

[59] Martinez-Høyer S, Mo A, Deng D, Jiang J, Docking R, Li J, et al. Resistance to lenalidomide in del(5q) MDS is mediated by inhibition of drug-induced

megakaryocytic differentiation. Blood. 2018;**132**:176. DOI: 10.1182/

[60] List A, Ebert BL, Fenaux P. A decade of progress in myelodysplastic syndrome with chromosome 5q deletion. Leukemia. 2018;**32**(7):1493- 1499. DOI: 10.1038/s41375-018-0029-9

[61] Belickova M, Vesela J, Jonasova A, Pejsova B, Votavova H, Merkerova MD, et al. TP53 mutation variant allele frequency is a potential predictor for clinical outcome of patients with lower-risk myelodysplastic syndromes. Oncotarget. 2016;**7**(24):36266-36279. DOI: 10.18632/oncotarget.9200

[62] Basiorka AA, McGraw KL, De Ceuninck L, Griner LN, Zhang L, Clark JA, et al. Lenalidomide stabilizes the erythropoietin receptor by inhibiting

**10**

[69] Komrokji RS, Al Ali NH, Padron E, Cogle C, Tinsley S, Sallman D, et al. Lenalidomide and prednisone in low and intermediate-1 IPSS risk, nondel(5q) MDS patients: A phase II clinical trial. Clinical Lymphoma, Myeloma & Leukemia. 2019. DOI: 10.1016/j. clml.2018.12.014

[70] Basiorka AA, McGraw KL, Eksioglu EA, Chen X, Johnson J, Zhang L, et al. The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype. Blood. 2016;**128**(25):2960-2975. DOI: 10.1182/ blood-2016-07-730556

[71] Sallman DA, Cluzeau T, Basiorka AA, List A. Unraveling the pathogenesis of MDS: The NLRP3 inflammasome and pyroptosis drive the MDS phenotype. Frontiers in Oncology. 2016;**6**:151. DOI: 10.3389/fonc.2016.00151

[72] Croker BA, Kelliher MA. BIDding on necroptosis in MDS. Blood. 2019;**133**(2):103-104. DOI: 10.1182/ blood-2018-11-886242

[73] Wagner PN, Shi Q , Salisbury-Ruf CT, Zou J, Savona MR, Fedoriw Y, et al. Increased Ripk1-mediated bone marrow necroptosis leads to myelodysplasia and bone marrow failure in mice. Blood. 2019;**133**(2):107-120. DOI: 10.1182/ blood-2018-05-847335

[74] Ping Z, Chen S, Hermans SJF, Kenswil KJG, Feyen J, van Dijk C, et al. Activation of NF-κB driven inflammatory programs in mesenchymal elements attenuates hematopoiesis in low-risk myelodysplastic syndromes. Leukemia. 2018. DOI: 10.1038/s41375-018-0267-x

[75] Haider M, Al Ali N, Padron E, Epling-Burnette P, Lancet J, List A, et al. Immunosuppressive therapy: Exploring an underutilized treatment option for myelodysplastic syndrome. Clinical Lymphoma, Myeloma & Leukemia. 2016;**16**(Suppl):S44-S48. DOI: 10.1016/j. clml.2016.02.017

[76] Stahl M, Zeidan AM. Lenalidomide use in myelodysplastic syndromes: Insights into the biologic mechanisms and clinical applications. Cancer. 2017;**123**(10):1703-1713. DOI: 10.1002/ cncr.30585

**13**

**Chapter 2**

**Abstract**

**1. Introduction**

*and Safa Shukry*

Diagnosis and Classification of

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by morphological dysplastic changes in one or more of the major hematopoietic cell lines. MDS can present with varying degrees of single or multiple cytopenias including neutropenia, anemia and thrombocytopenia. Presentation of MDS can range from asymptomatic to life threatening. MDS diagnosis and classification present important challenges, particularly in the distinction from benign conditions. French-American-British (FAB) classification proposed a classification based on easily obtainable laboratory information and was recommended in early and as modified by guidelines of new classification of World Health Organization (WHO). The strategy of diagnostic laboratory in MDS depends on morphological changes and is based on existence of dysplastic changes in the peripheral blood and bone marrow including peripheral blood smear, bone marrow aspirate smear and bone marrow trephine biopsy. The correct morphological interpretation and the use of cytogenetics, immunophenotyping, immunohistochemistry and molecular

Myelodysplastic Syndrome

*Gamal Abdul Hamid, Abdul Wahab Al-Nehmi*

analysis will give valuable information on diagnosis and prognosis.

**Keywords:** myelodysplasia, cytopenia, diagnostic criteria, classification

Myelodysplastic syndromes (MDS) are clonal stem cell disorders with a relatively heterogeneous spectrum, characterized by morphological dysplasia in hematopoietic cells and by bone marrow failure and varying degrees of peripheral blood cytopenias. MDS have been recognized for more than 70 years and named

The risks of MDS include infection, anemia, bleeding and transformation to acute myeloblastic leukemia (AML) in approximately 30% of cases. MDS incidence increased from less than 5/100,000 for patients less than 60 years to 36.2 per 100,000 in patients more than 80 year old and more common among men.

In the last 20 years, different MDS classification and prognostic scoring systems have been proposed [1]. French-American-British (FAB) classification was recommended in early and as modified by the World Health Organization (WHO). The WHO classification system uses percentages of blasts in bone marrow, ring sideroblasts and dysplastic changes to differentiate MDS subtypes. The International Prognostic Scoring System (IPSS) is based on a multivariate to evaluate the prognosis. The updated and recent scoring system combine with WHO classification

refractory anemia, oligoblastic leukemia and smoldering acute leukemia.

## **Chapter 2**


**Appendix A**

**Table A1.** Land use and land cover change from 1980 to 2015.

**Table A2.** Land use change transfer matrix between 1980 and 2015.

Note: The bold letters indicate that there is no change in the land use change over the time period.


*Review* **NSAID Use and Effects on Pediatric Bone Healing: A Review of Current Literature**

**Stephanie Choo and Julia A. V. Nuelle \***

Department of Orthopaedic Surgery, Missouri Orthopaedic Institute, University of Missouri, Columbia, MO 65212, USA<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** This systematic review evaluates and synthesizes the available peer-reviewed evidence regarding the impact of non-steroidal anti-inflammatory drugs (NSAIDs) on fracture healing in skeletally immature patients. Evidence supports the use of NSAIDs in this patient population for adequate pain control without increasing the risk of nonunion, particularly in long bone fractures and pseudoarthrosis after spine fusion. However, further clinical studies are needed to fill remaining gaps in knowledge, specifically with respect to the spectrum of available NSAIDs, dosage, and duration of use, in order to make broad evidence-based recommendations regarding the optimal use of NSAIDs during bone healing in skeletally immature patients.

**Keywords:** NSAIDs; ibuprofen; fracture healing; pediatrics; evidence-based recommendations

### **1. Introduction**

Nonsteroidal anti-inflammatory drugs (NSAIDs) have consistently been some of the most widely used medications for decades, based on their anti-inflammatory and analgesic mechanisms of action, in conjunction with their safety profiles and non-addictive characteristics [1]. The opioid epidemic has generated a strong impetus for the use of multimodal pain regimens, aimed at effectively managing pain with less reliance on opioids and the associated risks of misuse, abuse, addiction, morbidity, and mortality [2–4]. Decreased reliance on opioids also mitigates the known side effects of this class of medication, including nausea, vomiting, dizziness, and constipation. As such, NSAIDs provide an attractive choice as a mainstay of multimodal pain management protocols. There is hesitancy, however, to prescribe NSAIDs within the period following a fracture, arising from concern around delayed healing or non-union, given the potential blockade of the required inflammatory phase of bone healing.

Fracture nonunions are painful, can negatively impact the patient's quality of life, and are costly to the healthcare system [5]. In general, the incidence of fracture nonunion has been reported to range from 5% to 10% in the skeletally mature patient population, with even higher rates associated with at-risk patients and fractures of the scaphoid (15.5%), tibia and fibula (14%), and femur (13.9%) [6,7]. Pseudoarthrosis in the pediatric population is substantially less, at a rate of <1% [8]. Several animal models and retrospective cohort studies have demonstrated the potential for NSAIDs to increase the risk of non-union in the skeletally mature population [6,9,10]. However, recent prospective studies have shown that NSAIDs do not increase the risk of nonunion, while improving pain control with a potential opioid-sparing effect [11–13]. In addition to these contrasting results in skeletally mature population studies, evidence-based recommendations regarding the use of NSAIDs for pain management after fractures in the pediatric, skeletally immature population have similarly not been conclusive to date.

Fracture risk in pediatric patients is high, however, the incidence for nonunion is relatively low [8]. Still, while NSAIDs are highly desirable for effective pain management with a reduction in opioid use in this patient population, there has been an apparent

**Citation:** Choo, S.; Nuelle, J.A.V. NSAID Use and Effects on Pediatric Bone Healing: A Review of Current Literature. *Children* **2021**, *8*, 821. https://doi.org/10.3390/children 8090821

Academic Editor: Christiaan J. A. van Bergen

Received: 7 July 2021 Accepted: 12 September 2021 Published: 18 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

hesitancy in prescribing NSAIDs to pediatric patients with fractures due to physicians' concerns for non-unions based on some of the published data for adults. The concerns, based on anecdotal extrapolation to the pediatric population, does not have a sound physiologic basis in light of distinct differences in bone healing between patients that are skeletally immature and skeletally mature [14], and may unnecessarily inhibit best practice. Therefore, the purpose of this study was to (1) systematically review available evidence regarding skeletally immature fracture healing patterns based on exposure to NSAIDs to synthesize recommendations on NSAID use for pain control post-injury, and (2) determine critical gaps in knowledge towards establishing evidence-based care for analgesia after fractures in the pediatric population.

#### **2. Materials and Methods**

Using PRISMA guidelines, two electronic databases—PUBMED and Cochrane—were systematically searched using combinations of the keywords "NSAIDS", "ibuprofen", "nonsteroidal", "anti-inflammatory drugs", "naproxen", "ketorolac", "bone healing", "fracture healing", "bone fractures", "bone union", "non union", "children", "pediatric", "child", and "skeletally immature" as terms, title words, and abstract words. Reference lists from identified articles were also reviewed for inclusion. Studies in the English language and published between the years 2000–2021 were considered for inclusion. Studies were included when they assessed NSAID effects on bone healing in individuals who are skeletally immature and/or evaluated pediatric outcomes after NSAIDs use in association with fracture management. The exclusion criteria, included fracture or bone healing following orthopaedic surgical intervention not included as a study outcome, as well as studies of patients who are skeletally mature. The title and abstract of each qualifying article were screened and full-text manuscripts were retrieved in cases of uncertainty for inclusion. The following data from included studies were then extracted: Date of publication, number of patients, number of males and females, mean age, the incidence of nonunion or pseudoarthrosis, length of follow-up, type and amount of medication used, and the study type/level of evidence. An independent reviewer determined the final eligibility of studies included for systematic review. The synthesis of included articles followed the PICO (Population, Intervention, Comparison, Outcome) reporting methodology. The Cochrane Risk of Bias Tool was also utilized to assess and report bias risk.

#### **3. Results**

reported in Table 2.

**Study Year Author Number of Pa-**

2018 Zura et al. [8] 237,033 fractures

2008 Sucato et al. [13] 319 total

2003 Vitale et al. [15] 208 total

2010 Kay et al. [16] 221 total

2011 Kay et al. [17] 327 total

2016 DePeter et al. [18] 808 total

2009 Drendel et al. [19] 336 total

2020 Nuelle et al. [20] 95 total

The database search produced a total of 37 studies for review. A reference lists review produced an additional 4 relevant studies. Based on a full review of these 41 articles, 8 met inclusion criteria for systematic review (Figure 1). *Children* **2021**, *8*, x FOR PEER REVIEW 3 of 11

**Table 1.** Demographics.

**tients Male/Female**

**Figure 1.** PRISMA 2020 flow diagram for new systematic reviews which included searches of databases and registers only. From these 10 studies, 6 were retrospective cohort studies, 2 were prospective ran-**Figure 1.** PRISMA 2020 flow diagram for new systematic reviews which included searches of databases and registers only.

 Synthesis of Examined Studies Evaluating Effect of NSAIDs on Bone Healing in Skeletally Immature Patients.

(169 exposed) 126/210 (8.2 years/ 7.4

(158 exposed) 50/269 14.2 years 39 months

(60 exposed) 59/149 13.4 years 67 months

(299 exposed) 181/136 (8.4 years/9.2 years) (38 months/44

(49 exposed) 58/37 7.6 years 6 months

domized control trials, and 2 were meta-analysis. The meta-analysis studies were excluded from this analysis as the 4 retrospective pediatric studies, cited in this literature, are also included in this current review. Demographic data for each study are included in

From these 10 studies, 6 were retrospective cohort studies, 2 were prospective randomized control trials, and 2 were meta-analysis. The meta-analysis studies were excluded from this analysis as the 4 retrospective pediatric studies, cited in this literature, are also included in this current review. Demographic data for each study are included in Table 1. Qualitative synthesis, including risk of bias assessment for these 10 studies, were reported in Table 2.

#### **Table 1.** Demographics.

**Table 2.** Synthesis of Examined Studies Evaluating Effect of NSAIDs on Bone Healing in Skeletally Immature Patients.

**Table 2.** *Cont.*

**Table 2.** *Cont.*

LOE = Level of Evidence; RCT = Randomized Controlled Trial.

#### *3.1. Retrospective Cohort Studies*

Vitale et al. [15] conducted a retrospective study to determine whether an association exists between the use of ketorolac and postoperative complications in children undergoing spinal fusion surgery for scoliosis. Ketorolac was shown to be an effective alternative to opioid medications for analgesia [21]. This study investigated potentially related complications, including bony fusion failure and the requirement for revision surgery due to pseudoarthrosis, as well as infected hardware, hardware failure, and bleeding risk represented by need for transfusion.  Sixty patients (29%) received ketorolac postoperatively and the remaining 148 (71%) did not. The group of patients that were given ketorolac in the post-operative period was composed primarily of complicated patients (typically a greater degree of curvature) as determined by the operative surgeon. They reported an average dose of ketorolac to be 0.5 mg/kg intravenous (IV), every 6 h for 2–3 days, starting 24 to 48 h postoperatively. Patients were followed through clinical and radiographic assessments for an average of 62 months (ketorolac group) and 69 months (no ketorolac group). Analyses from this study produced no statistically significant differences between ketorolac versus no ketorolac for fusion rate, pseudarthrosis, or other potentially related complications. The only independent variable that was significantly associated with the need for revision surgery, due to pseudoarthrosis, hardware failure, or hardware infection was the degree of curvature of the spine prior to intervention.

The study by Sucato et al. [13] referenced a study by Glassman and colleagues, which demonstrated the significant inhibitory effects of ketorolac on spinal fusion when used as an adjunct for postoperative pain in adult patients [22], and served as the impetus for the former authors' similar study in a pediatric population. This retrospective study compared patients who had postoperative ketorolac and those who did not, following posterior spinal fusion and instrumentation for adolescent idiopathic scoliosis (AIS). Overall, 161 patients did not receive ketorolac postoperatively and 158 did. Pseudoarthrosis was defined by surgical exploration and confirmation in the patients who presented with ongoing symptoms

after initial fixation. This study noted the average number of ketorolac doses was 6.7 for a duration of 48 h after surgery. Patients in the ketorolac group augmented their medical pain management with an additional 5.8 doses of ibuprofen after surgery on average in comparison to the non-ketorolac group's average of 0.7 doses. Mean follow-up for both groups was 39 months after fixation and the overall incidence of pseudoarthrosis during this time was 2.5% (8 of 319 patients) with no statistically significant difference between the ketorolac and non-ketorolac groups (1.9% vs 3.1%). The data from this study led the investigators to conclude that pseudoarthrosis risks are similar for AIS patients, regardless of ketorolac use, a finding consistent with those of Vitale et al. [15].

Kay et al. [16] performed a retrospective review of 221 children who sustained fractures (supracondylar humerus (42%), lateral condyle (12%), forearm (10%), femur (18%), tibia (7%), or ankle (7%)) requiring operative fixation. Of the 221 patients, 169 were treated with ketorolac postoperatively and 52 were not. The decision to use ketorolac for treatment was based on the preference of the anesthesiologist or the pain management team. The ketorolac group received weight-based dosing every 6 h while in the hospital, and the non-ketorolac group was treated with ibuprofen. These patients, on average, stayed 2.5 days in the hospital, discharged with ibuprofen, and followed for an average of 6.2 ± 7.5 months after fixation. No complications related to ketorolac or ibuprofen administration were reported, and there were no cases of delayed union or malunion in this pediatric fracture cohort.

In their 2011 study, Kay et al. [17] performed a retrospective review of 327 children undergoing lower extremity osteotomy surgery, of which 299 patients received ketorolac perioperatively and 28 patients did not. Delayed union occurred in 5 of 682 osteotomies (0.7%), which included 0.6% in the ketorolac group and 1.8% in the non-ketorolac group (*p* = 0.893). No nonunions were documented. The data in this study led the investigators to conclude that perioperative ketorolac is acceptable for pain management in patients who are skeletally immature and undergoing lower extremity osteotomies without significant risks regarding bone healing.

Zura et al. [8] performed a retrospective cohort study of 237,033 fractures in pediatric patients to identify risk factors for nonunion. Data were acquired from Truven Health Analytics health claims in a single calendar year (1 January 2011—31 December, 2012). The study population maintained a low nonunion rate until age 11 where a strong inflection point occurred such that a significant increase in nonunion rate was noted for each fracture location analyzed (*p* < 0.001), with tibia, fibula, femoral neck, and scaphoid having >5% nonunion risk after age 11. Interestingly, this study reported that pediatric fracture patients that used NSAIDs with opioids (*p* < 0.01; OR = 2.52) or opioids alone (*p* < 0.01; OR = 2.47) had a nearly 2.5 times increased risk of non-union, while patients (age 0 to 18 years) who consumed NSAIDs alone did not show a statistically significant increase in risk. This study did not specify which NSAIDs were used, or the dosage and duration of use. This data led the authors to conclude that NSAID use is appropriate for pain management during bone healing in pediatric patients, but should not be used in conjunction with opioids, especially after age 11.

DePeter et al. [18] performed a retrospective study in children aged 6 months to 17 years, assessing patients with common fractures that have been reported to have a higher incidence of healing complications, including in the tibia, femur, humerus, scaphoid, and fifth metatarsal fractures. A total of 808 patients were included in this study, of which a total of 27 (3%) showed bone healing complications, 1% from nonunion, 0.4% delayed union, and 2% loss of reduction. Of the 338 patients (42%) exposed to ibuprofen, 10 patients (3%) went on to non-union, which was not significantly (*p* = 0.61) different from patients not consuming ibuprofen. These data led the authors to conclude that there was no significant association between ibuprofen use and the development of bone healing complications in this pediatric high-risk fracture population.

#### *3.2. Prospective Randomized Studies*

Nuelle et al. [20] performed a randomized, parallel, single-blinded clinical trial to assess the impact of NSAIDs on acute-phase bone healing in patients who are skeletally immature with a variety of long bone fractures. This study assessed 97 fractures in pediatric patients, randomized to control (*n* = 47) or NSAID (*n* = 50) cohorts. The control group was provided weight-based acetaminophen for pain control and oxycodone for breakthrough pain. If a patient was in the emergency department, weight-based fentanyl was available for administration. Patients in the NSAID group were prescribed weight-based ibuprofen for pain control, as well as oxycodone for breakthrough pain. NSAID patients in the emergency department were permitted to have ketorolac for pain control if needed. Clinical visits involving radiographs of the fractured extremity occurred at 1–2 weeks, 6 weeks, 10–12 weeks, and 6 months after injury. Fracture healing was determined by clinical examination, absence of tenderness to palpation at fracture site, and radiographs demonstrating callus formation bridging in 3 cortices. As a secondary outcome measure, pain control was assessed by the patient's caregiver recording the patient's visual analog scale pain score, as well as the amount and type of pain medication used, for a total of 3 weeks after the injury. This study demonstrated that, by 6 weeks following initial fracture treatment, 82% of the control group had united fractures and 92% of the NSAID group had healed fractures, with no significant difference (*p* = 0.22) between both groups. By the 10–12 week appointment, 98% of control and 100% of NSAID patients had healed fractures, and the 6-month follow-up demonstrated 100% healed fractures for both cohorts. Of note, pain scores were similar for both groups with acetaminophen used for 3.9 days on average versus ibuprofen use for 4.3 days on average. Oxycodone was used for breakthrough pain for a mean of 2.4 days in the control cohort and 1.9 days in the NSAID cohort. These data led the authors to conclude that ibuprofen can be used to effectively manage pain in pediatric patients during the acute post-treatment period following long bone fractures without impairing fracture healing.

Drendel et al. [19] performed a randomized, double-blind, clinical trial assessing ibuprofen versus acetaminophen with codeine in pediatric patients treated for upper extremity fractures. In contrast to Nuelle et al. [20], the primary outcome measure for this study was pain control, with fracture healing as a secondary outcome measure. Pain was assessed using the visual Bieri Faces Pain Scale and caregivers were also asked to keep a diary documenting the patient's tolerability of the medication. Non-unions were assessed by reviewing medical records for 1 year following treatment, with a follow-up phone call to the patient's caregiver for verification when an adverse event related to fracture healing was noted. The study enrolled 336 children, and 169 patients were randomized to the ibuprofen group and 167 to the acetaminophen with codeine group. Of the 244 patients that met the criteria and were analyzed, the proportion of "treatment failures" and the need for rescue IV pain medication was lower in the ibuprofen group than the acetaminophen with codeine group (20.3% vs 31.0%, respectively). However, this difference was not statistically significant. Pain scores were similar between the two groups. However, medication-related adverse effects reported during the first 3 days of treatment were significantly higher for the acetaminophen with codeine group at 50.9% versus the ibuprofen group at 29.5% (% difference 21.4, confidence interval (CI) 9.1 to 33.7). Chart review and telephone calls determined that four (1.6%) of the children sustained re-fracture within 1 year of the original fracture, and of those, three (75%) received acetaminophen with codeine. There were no reports of nonunion in either group. These data led the authors to conclude that ibuprofen was as effective as an acetaminophen with codeine in providing analgesia for children with upper extremity fractures. The drug was associated with less medication-related adverse effects and was not associated with re-fracture or nonunion in this patient population.

#### **4. Discussion**

This systematic review of available evidence regarding NSAID exposure, and bone healing in patients who are skeletally immature, produced six retrospective studies and two prospective clinical trials for inclusion. The qualitative synthesis of these studies suggested that the best current evidence supports the use of two NSAIDs, ibuprofen and ketorolac, as safe and effective medications for pain management. These medications did not show an increased risk of pseudoarthrosis after spinal fusions or nonunion after fractures of the upper and lower extremity, scaphoid, or metatarsals in the pediatric population.

Concerns regarding the use of NSAIDs in the acute phase of bone healing have been based on animal studies and clinical assessments of patients that are skeletally mature. To date, there have been no studies demonstrating that NSAID use in the acute phases of bone healing result in higher rates of nonunion.

Bone healing involves a complex cascade of events with the end-goal being the formation of bridging vascularized woven bone capable of functional remodeling over time [23]. Studies have indicated that prostaglandins, which are important in the regulation of osteoclastic bone resorption and osteoblastic bone formation, required for bone healing, are released at the time of fracture via cyclooxygenase enzyme activity (COX-2) [8]. As COX inhibitors, NSAIDs can arrest the cell cycle and increase cytotoxicity and apoptosis of osteoblasts, leading to ineffective mineralization of newly formed extracellular bone matrix [24]. In studies of skeletally mature animals [25–28], non-selective NSAIDs were shown to delay long bone healing without any detrimental effects on long-term outcomes. Whereas selective NSAIDs were associated with detrimental long-term effects on material and structural properties of healing bone. Research that focused on dose and time effects suggested avoiding NSAID treatment during the initial weeks of fracture healing, in order to avoid these negative effects on fracture callus [7]. However, the physiology of the skeletally immature system is distinct from the skeletally mature. Some contributors to this unique physiology, which may make skeletally immature patients less prone to pseudoarthrosis, include thicker periosteoum, greater subperiosteal hematoma, active physes, and enhanced metabolic activity. Capello et al. explored the effects of NSAID administration during the acute phase of fracture healing in skeletally immature animals, and found no significant difference in strength, stiffness, or histologic characteristics of fracture callus. These finds support the view that the bone healing physiology of the skeletally immature is not perturbed by NSAID administration at physiologic doses [29].

A large inceptive cohort study by Zura et al. demonstrated the combination of NSAIDs and opioids (multivariate odds ratio (OR), 1.84; 95% CI, 1.73–1.95) is a risk factor for nonunion. However, this study found no significant association of NSAIDs alone with nonunion (multivariate OR, 0.98, 95% CI, 0.89–1.07, *p* = 06), but found opioids alone to be a moderately strong positive risk factor for nonunion (multivariate OR, 1.43, 95% CI, 1.34–1.52, *p* ≤ 0.001) [6]. Zura et al. expanded on this original study to isolate pediatric patient risk factors for nonunion and found similar results, further supporting the finding that NSAIDs are not significant contributors to fracture nonunion, but NSAIDs combined with opioids, lead to significant risks [8].

This analysis is limited by the number of studies available, as well as heterogeneity of the bone healing types included in the reviewed studies, including fractures treated operatively compared with those treated by closed means, osteotomies, as well as spinal fusions. As with all reviews, this review is an analysis of the best available published data. Certainly, future studies on the potential multicenter participation and non-inferiority designs, examining each of these bone healing types, are warranted.

#### **5. Conclusions**

Clinical data investigating the effects of NSAIDS on bone healing in the pediatric population is sparse. The retrospective studies of pediatric patients who used NSAID following posterior spinal fusion did not demonstrate inhibition of bone healing [13,15]. Two studies by Kay et al. found no cases of nonunion in pediatric patients who received ketorolac around the time of operative fixation of common pediatric fractures or lower extremity osteotomy [16,17]. DePeter et al. similarly found no healing complications

arose for the pediatric population exposed to ibuprofen during fracture healing. The two prospective studies by Nuelle et al. [20] and Drendel et al. [19] support the use of ibuprofen for long bone fractures in the acute healing period for adequate pain control without compromising bone healing. In conclusion, based on the current available evidence, NSAID use in the acute phase of bone healing in skeletally immature patients is not associated with a higher rate of pseudoarthrosis.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to this study being a literature review.

**Informed Consent Statement:** Patient consent was waived due to this study being a literature review.

**Acknowledgments:** The authors would like to thank Adelina Colbert for her assistance with the review and submission of this manuscript.

**Conflicts of Interest:** J.A.V.N. receives research support from the Henry M. Jackson Foundation. She serves on the Editorial Board for Arthroscopy: The Journal of Arthroscopic and Related Surgery. These entities had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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