**8. Epigenetic landscape of JMML**

Aberrant DNA methylation was found to be an initiating event in JMML. This finding has instigated the research in the field of epigenetics and led to the landmark discovery of methylation classes in 2017. In addition, epigenetic modifications are implicated in JMML disease progression and together with established clinical and genetic markers fully recapitulate the clinical and biological heterogeneity of JMML. The genome-wide DNA methylation analysis of JMML patients identified three biologically distinct JMML subgroups [24]:


The results of all DNA methylome studies in JMML were exceptionally consistent with the fact that DNA hypermethylation is a recurrent feature of JMML cells that confers treatment resistance. The dysregulated genomic DNA methylation is a crucial component of RAS-driven malignant cell transformation proposing a possible functional links between RAS pathway mutational patterns and methylation classes. However, DNA hypermethylation was only weakly associated with the canonical genotypes or cytogenetic aberrations. Instead, it correlated strongly with the known clinical risk factors predictive of aggressive disease and poor outcome, especially older age and increased HbF level (**Table 3**). In fact, DNA methylation status is an independent prognostic factor and better predictor of clinical outcome than JMML genetic types. Frequent hypermethylation in 4 genes (*BMP4, CALCA, CDKN2B*, and *RARB*) were associated with a poor prognosis. Moreover, *RASA4* hypermethylation was related to poor prognosis and disease relapse after HSCT. DNA methylation could be used as a biomarker that can both identify patients who are predicted to fail HSCT as well as those who are most likely to experience spontaneous resolution and could be observed to avoid the acute and late side effects of HSCT [25].

#### **9. Treatment**

The heterogeneity of the disease is reflected by the varied clinical outcomes. Although the current standard of care for JMML is allogeneic HSCT, continued controversy exists about identifying those patients who need to be moved quickly to HSCT versus those rare patients who might be observed [26]. The majority of children with *CBL*-mutated JMML and some *NRAS*- mutated patients experience spontaneous disease regression. These children are clinically well with a low HbF. A careful watch-and-wait strategy may be indicated as the treatment of choice. On the other hand, JMML with somatic *PTPN11* mutations appear to represent cases with aggressive biology with a high risk of relapse even after HSCT.

**29**

*Juvenile Myelomonocytic Leukemia (JMML): A Mimicker of* KMT2A*-Rearranged Acute…*

Nevertheless, allogeneic HSCT remains the most effective means of stopping the uncontrolled production of monocytic cells in the majority of patients. If left untreated, survival for most children is less than one year. The predominant cause of JMML-related death is respiratory failure as a result of pulmonary infiltration by leukemic cells (blastic transformation is infrequent in JMML). In fact, HSCT early in the course of disease significantly improved the dismal prognosis of JMML patients. However, the 5-year event-free survival (EFS) rate after HSCT is only 44–53%. Disease recurrence is the most important cause of failure, occurring with a cumulative incidence of 35%. Of note, patients with JMML who transform into AML (defined as >20% blasts in bone marrow) generally have dismal outcomes

Pre-transplant chemotherapy for JMML had no benefit on EFS or overall survival. A variety of pre-HSCT treatments have been employed to control symptoms of JMML (such as high white blood cell count, pulmonary problems, and/or prominent organomegaly) as well as theoretically improve outcomes. However, none of these agents induced durable responses or reduced the relapse rate. The main aim of the pre-HSCT treatments remains to bridge HSCT. Given the current lack of convincing evidence for traditional myelosuppressive chemotherapy pre-HSCT, other treatment modalities has been sought. In particular, molecular classification of cancer based on DNA methylomes has revolutionized the diagnostic and prognostic parameters of JMML and aid our understanding of the mechanistic link between epigenetic dysregulation and resistance to treatment. A DNA methyltransferase–inhibiting azanucleoside is assumed to reverse the epigenetic dysregulation in malignant cells. This has led to a European protocol to employ DNA hypomethylating agents such as azacitidine for therapy in JMML [27]. The first case report of azacitidine for JMML before HSCT was published in 2009. The child achieved a complete clinical, hematologic, cytogenetic (monosomy 7) and molecular (*KRAS* index mutation) remission during eight monthly cycles of 5-day azacitidine. The reduction of *BMP4* promoter DNA methylation preceded the disappearance of leukemic cells, demonstrating the DNA-hypomethylating activity of azacitidine in JMML cells. Although azacytidine may induce complete clinical, cytogenetic and/or molecular remission before allogeneic HSCT, complete remission has not been sustained without transplant. On the other hand, the acceptable toxicity of low-dose azacitidine and its cytoreductive potential make it an attractive option as a bridging therapy before HSCT or as palliation after 1 or more transplants have failed [28].

JMML is a puzzling disease with blurring clinical presentation that can commonly mimic a wide variety of other diseases. Overall, the most consistent features of the JMML phenotype are young patient age, splenomegaly and increased synthesis of hemoglobin F (HbF). The molecular basis of JMML is closely linked to Rasopathies, a family of inherited cancer predisposition syndromes characterized by cardiac defects, defective growth, facial dysmorphism and variable cognitive deficits. Defining aberrant RAS signaling transduction pathway as the common denominator linking RASopathies to JMML and a transient JMML-like disease made JMML a fundamentally a disease of uncontrolled hyperactivation of RAS signaling. The identification of RAS pathway mutations in JMML has advanced the understanding of molecular mechanisms underlying the progression from cancer predisposition to neoplasia. Deciphering of the mutational spectrum had led to the classification of JMML into 5 genetic subtypes which have distinctive genotype–phenotype characteristics. However, the clinical outcome of JMML

*DOI: http://dx.doi.org/10.5772/intechopen.94537*

**10. Conclusion and future perspectives**

following HSCT.

*Juvenile Myelomonocytic Leukemia (JMML): A Mimicker of* KMT2A*-Rearranged Acute… DOI: http://dx.doi.org/10.5772/intechopen.94537*

Nevertheless, allogeneic HSCT remains the most effective means of stopping the uncontrolled production of monocytic cells in the majority of patients. If left untreated, survival for most children is less than one year. The predominant cause of JMML-related death is respiratory failure as a result of pulmonary infiltration by leukemic cells (blastic transformation is infrequent in JMML). In fact, HSCT early in the course of disease significantly improved the dismal prognosis of JMML patients. However, the 5-year event-free survival (EFS) rate after HSCT is only 44–53%. Disease recurrence is the most important cause of failure, occurring with a cumulative incidence of 35%. Of note, patients with JMML who transform into AML (defined as >20% blasts in bone marrow) generally have dismal outcomes following HSCT.

Pre-transplant chemotherapy for JMML had no benefit on EFS or overall survival. A variety of pre-HSCT treatments have been employed to control symptoms of JMML (such as high white blood cell count, pulmonary problems, and/or prominent organomegaly) as well as theoretically improve outcomes. However, none of these agents induced durable responses or reduced the relapse rate. The main aim of the pre-HSCT treatments remains to bridge HSCT. Given the current lack of convincing evidence for traditional myelosuppressive chemotherapy pre-HSCT, other treatment modalities has been sought. In particular, molecular classification of cancer based on DNA methylomes has revolutionized the diagnostic and prognostic parameters of JMML and aid our understanding of the mechanistic link between epigenetic dysregulation and resistance to treatment. A DNA methyltransferase–inhibiting azanucleoside is assumed to reverse the epigenetic dysregulation in malignant cells. This has led to a European protocol to employ DNA hypomethylating agents such as azacitidine for therapy in JMML [27]. The first case report of azacitidine for JMML before HSCT was published in 2009. The child achieved a complete clinical, hematologic, cytogenetic (monosomy 7) and molecular (*KRAS* index mutation) remission during eight monthly cycles of 5-day azacitidine. The reduction of *BMP4* promoter DNA methylation preceded the disappearance of leukemic cells, demonstrating the DNA-hypomethylating activity of azacitidine in JMML cells. Although azacytidine may induce complete clinical, cytogenetic and/or molecular remission before allogeneic HSCT, complete remission has not been sustained without transplant. On the other hand, the acceptable toxicity of low-dose azacitidine and its cytoreductive potential make it an attractive option as a bridging therapy before HSCT or as palliation after 1 or more transplants have failed [28].

#### **10. Conclusion and future perspectives**

JMML is a puzzling disease with blurring clinical presentation that can commonly mimic a wide variety of other diseases. Overall, the most consistent features of the JMML phenotype are young patient age, splenomegaly and increased synthesis of hemoglobin F (HbF). The molecular basis of JMML is closely linked to Rasopathies, a family of inherited cancer predisposition syndromes characterized by cardiac defects, defective growth, facial dysmorphism and variable cognitive deficits. Defining aberrant RAS signaling transduction pathway as the common denominator linking RASopathies to JMML and a transient JMML-like disease made JMML a fundamentally a disease of uncontrolled hyperactivation of RAS signaling. The identification of RAS pathway mutations in JMML has advanced the understanding of molecular mechanisms underlying the progression from cancer predisposition to neoplasia. Deciphering of the mutational spectrum had led to the classification of JMML into 5 genetic subtypes which have distinctive genotype–phenotype characteristics. However, the clinical outcome of JMML

*Acute Leukemias*

progression and poor clinical outcomes.

**8. Epigenetic landscape of JMML**

three biologically distinct JMML subgroups [24]:

*KRAS* mutations and monosomy 7.

of JMML [23]. Secondary mutations of *SETBP1* and *JAK3* were the most frequent mutations (around 15% of children) and were presumed to be involved in tumor

Aberrant DNA methylation was found to be an initiating event in JMML. This finding has instigated the research in the field of epigenetics and led to the landmark discovery of methylation classes in 2017. In addition, epigenetic modifications are implicated in JMML disease progression and together with established clinical and genetic markers fully recapitulate the clinical and biological heterogeneity of JMML. The genome-wide DNA methylation analysis of JMML patients identified

1.The high-methylation group (HM) was dominated by older children and cases

2.The intermediate methylation group (IM) showed enrichment for somatic

3.The low methylation group (LM) is enriched for somatic *NRAS* and *CBL* mutations, as well as for Noonan patients, and has a good prognosis.

The results of all DNA methylome studies in JMML were exceptionally consistent with the fact that DNA hypermethylation is a recurrent feature of JMML cells that confers treatment resistance. The dysregulated genomic DNA methylation is a crucial component of RAS-driven malignant cell transformation proposing a possible functional links between RAS pathway mutational patterns and methylation classes. However, DNA hypermethylation was only weakly associated with the canonical genotypes or cytogenetic aberrations. Instead, it correlated strongly with the known clinical risk factors predictive of aggressive disease and poor outcome, especially older age and increased HbF level (**Table 3**). In fact, DNA methylation status is an independent prognostic factor and better predictor of clinical outcome than JMML genetic types. Frequent hypermethylation in 4 genes (*BMP4, CALCA, CDKN2B*, and *RARB*) were associated with a poor prognosis. Moreover, *RASA4* hypermethylation was related to poor prognosis and disease relapse after HSCT. DNA methylation could be used as a biomarker that can both identify patients who are predicted to fail HSCT as well as those who are most likely to experience spontaneous resolution and

with somatic *PTPN11* mutations and poor clinical outcome.

could be observed to avoid the acute and late side effects of HSCT [25].

aggressive biology with a high risk of relapse even after HSCT.

The heterogeneity of the disease is reflected by the varied clinical outcomes. Although the current standard of care for JMML is allogeneic HSCT, continued controversy exists about identifying those patients who need to be moved quickly to HSCT versus those rare patients who might be observed [26]. The majority of children with *CBL*-mutated JMML and some *NRAS*- mutated patients experience spontaneous disease regression. These children are clinically well with a low HbF. A careful watch-and-wait strategy may be indicated as the treatment of choice. On the other hand, JMML with somatic *PTPN11* mutations appear to represent cases with

**28**

**9. Treatment**

is not completely explained by the clinical and genetic markers which lent the epigenetic landscape of JMML a particular interest. There is hardly any pediatric oncology entity where research has benefited as much from epigenetics as JMML. Genome-wide interrogation of DNA methylation patterns has led to a classification of the disease into three distinct JMML subgroups that have clear pathogenetic and prognostic relationships. The DNA hypermethylation signature is associated with poor clinical outcome and increased risk for relapse following HSCT. Evidence suggests that DNA methylation changes could be used as a potential biomarker in a combined risk stratification algorithm in future clinical trials. Although the clinical activity of azacitidine as a DNA hypomethylating agent appears to be promising, it is unlikely that azacitidine alone have the potential to cure JMML [29]. However, azacitidine as monotherapy is safe and effective in controlling disease both in upfront and relapsed patients in order to proceed to HSCT [30]. In fact, azacitidine is currently the gold standard bridge to HSCT in JMML.

Despite the lack of associated mutations in the genes coding for the GMR, JMML is characterized by aberrant GMR signal transduction. This aberrant signaling is integral in the pathogenesis of JMML that underlines the dysregulated myelopoiesis of the disease. The resultant selective GM-CSF hypersensitivity was exploited as a diagnostic tool for JMML and it is especially useful for those diagnostically challenging cases with RAS pathway mutation-negative JMML. This entity represents the remaining 10% of cases where disease might be due to chromosomal translocations, other gene mutations, specific microRNAs (miRNA) or long noncoding RNAs (lncRNAs) [31]. GMR signal transduction is located upstream to both RAS signaling pathway and the JAK/STAT pathway. STAT5 is recruited to phosphorylated tyrosine residues on the activated β subunit of the GM-CSF receptor, indicating that STAT5 signaling profile reflects JMML hypersensitivity to GM-CSF. The p-STAT5 phospho-specific flowcytometry assay which largely replaced the traditional colony assay had also recently gained promising utility in monitoring the disease status. As disease worsens, a greater percentage of cells get hyper-responsive. Therefore, p-STAT5 phospho-flow signature could prove enticing as a marker of disease progression and indicator of relapse. As JMML lacks tractable markers, both DNA methylation and p-STAT5 phospho-flow signature are potential new armamentarium that could serve as futuristic checkpoints in order to promulgate uniform protocols designed to follow patients on therapy. Interestingly, the shared p-STAT5 phospho-flow signature in JMML, AML M4/M5 and CMML could explain the unique overlap of their clinical features.

So far HSCT represents the only therapy with a clear impact on the outcome of JMML patients. However, HSCT is replete with significant morbidities and deleterious late effects in this young population. The search for more effective and less toxic strategies coincided with advances in molecular oncology that opened up a realm of novel and targeted molecules capable of improving therapeutic tactics in JMML. In particular, scoping into the genetic basis of hyper-responsiveness in JMML could provide a means of assessing the efficacy of emerging kinase inhibitors such as those blocking the RAS (e.g., the oral MEK inhibitor trametinib) or JAK2-signaling pathways for the treatment of this disease. Notably, a combination of drugs targeting two different pathways might be more effective than a single drug. However, the choice of drugs used for treatment should be entirely based on patient's mutation status [32, 33].

**31**

**Author details**

Ashraf Abdullah Saad

Hospital (SQUH), Muscat, Oman

provided the original work is properly cited.

*Juvenile Myelomonocytic Leukemia (JMML): A Mimicker of* KMT2A*-Rearranged Acute…*

1.Dr. Bushra Mahmood Al-Abri (Hematopathologist) for her valuable contribution in illustrating and editing the peripheral blood and bone marrow figures.

2.Eng. Yasmin Adel (Architect) for designing the RAS pathway as a gear train.

The author declares no competing financial interests.

KMT2A Histone-lysine N-methyltransferase 2A

HSCT Hematopoietic stem cell transplantation

CFA hematopoietic colony formation assay

CMML chronic myelomonocytic leukemia

STAT signal transducer and activator of transcription factor

GM-CSF Granulocyte-macrophage colony-stimulating factor

CFU-GM colony-forming units -granulocyte/macrophage

p-STAT5 phospho-signal transducer and activator of transcription factor 5

JAK Janus-activated kinase

GMR GM-CSF receptor

PI 3-kinase phosphatidylinositol-3 kinase

CBL Casitas B-lineage lymphoma HHV-6 Human Herpesvirus 6 CMV cytomegalovirus

CML Chronic myeloid leukemia

Pediatric Hematology, Oncology and HSCT Unit, Sultan Qaboos University

© 2020 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,

\*Address all correspondence to: dr.ashraf123321@gmail.com

*DOI: http://dx.doi.org/10.5772/intechopen.94537*

**Conflict of interest**

**Abbreviations**

### **Acknowledgements**

I would like to express my deep gratitude for the following:

*Juvenile Myelomonocytic Leukemia (JMML): A Mimicker of* KMT2A*-Rearranged Acute… DOI: http://dx.doi.org/10.5772/intechopen.94537*

