**5. Genomics**

*Acute Leukemias*

monocytes. JMML morphologically resemble AML M4 and distinction must be

JMML is closely related to RAS-associated autoimmune leukoproliferative disorder (RALD) [9]. RALD is a non-malignant, non-infectious leukoproliferative disease that resembles the autoimmune lymphoproliferative syndrome (ALPS)

Similar to patients with ALPS, RALD patients present with lymphadenopathy, massive splenomegaly, increased circulating B cells, hypergammaglobulinemia, and

2.Germline or somatic mutations in *FAS, FASL, or CASP10* are absent in RALD.

RALD is a RAS-associated somatic disorder characterized by myelomonocytic and lymphoid hyperplasia that shares identical somatic *KRAS* or *NRAS* mutations found in up to 25% of JMML patients. It is thought that RAS activation itself can alter selection patterns of autoreactive B cells and antibody production leading to autoimmune manifestations. Overlap features of both JMML and RALD include:

2.Persistent absolute or relative monocytosis is a cardinal feature of RALD.

3.Bone marrow and peripheral blood smear findings overlap with those of JMML

4.Morphologic features compatible with dysplasia, such as hyposegmented pelgeroid neutrophils, can also be seen in RALD further obscuring the distinction

5.Autoimmunity can be noted in up to 25% of children with JMML, whereas

6.Nearly all patients with RALD meet the revised diagnostic criteria for JMML.

Distinguishing RALD from JMML can be impossible on clinical grounds alone. However, this distinction has an important prognostic value as RALD is characterized by an indolent clinical course whereas JMML can be fatal if left untreated. The most definitive diagnostic distinction between RALD and JMML occurs in the

hypergammaglobulinemia is present in more than 50% of cases.

/CD8−

) T cells and serum vitamin B12 levels are not always

double negative T-cell

Differentiating AML from JMML is vital for survival. Chemotherapy regimens for JMML are mainly cytoreductive as a bridge to hematopoietic stem cell transplantation (HSCT) rather than curative as for AML. Therefore, any delay in establishing the correct diagnosis and/or administration of wrong treatment can be lethal. Molecular diagnosis has become the mainstay to distinguish between AML and JMML. Cytogenetics and FISH should be immediately performed to detect *KMT2A*

made based on accurate blast and promonocyte counts.

gene rearrangements in every suspicious case.

caused by mutations affecting the FAS/FASL pathway.

1.In contrast to ALPS, biomarkers such as CD4−

1.Cytopenias, lymphadenopathy, and splenomegaly.

in children or CMML in older patients.

between RALD and JMML.

autoimmunity. However, RALD was separated from ALPS as:

**4.2 JMML masquerading as RALD**

receptor αβ (TCRαβ <sup>+</sup>

increased.

**18**

#### **5.1 The central role of the RAS signaling pathway**

JMML is an oncogenic RAS mutant cancer where approximately 90% of patients carry either somatic or germline gain of function mutations in *PTPN11, KRAS, NRAS, CBL, or NF1* genes that lead to constitutive activation of the RAS signaling pathway (**Figure 3**). The RAS signaling pathway is a component of multi-step signal transduction pathway that controls the cellular proliferation, differentiation,

#### **Figure 3.**

*Illustrative representation of the RAS signaling pathway portraying the transmission of extracellular signals in a gear train to the nucleus. Upon stimulation, a docking site for GRB2 is created which binds to GEF (SOS). The GRB2-SOS interaction converts RAS-GDP to RAS-GTP. The RAS-GTP initiates a cascade of phosphorylations on RAF, MEK and, lastly, ERK. The phospho-ERK then translocates into the nucleus. A counteracting force by GAP (neurofibromin) accelerates the conversion of RAS-GTP to RAS-GDP to restrain the activation of the RAS signaling pathway. Mutations of RAS pathway prevent the conversion of RAS-GTP to RAS-GDP to induce the constitutive activation of the RAS signaling pathway, the characteristic feature of JMML.*

and survival. It relays extracellular stimuli such as growth factors to the nucleus where the response to those stimuli is executed by an induced transcriptional program [11].

The RAS family include the proto-oncogenes *HRAS, NRAS and KRAS* which represent a subset of a superfamily of small membrane-localized GTPases. Like other small GTPases, RAS proteins function as molecular switches that cycle between an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound conformations. RAS is activated when the receptor tyrosine kinases (RTKs, a large superfamily of receptors for a wide array of growth factors) are activated by growth factor binding leading to RTK autophosphorylation and the creation of docking sites for adaptor molecules like growth factor receptorbound protein 2 (GRB2). The amount of GTP-RAS generated is regulated by two opposing regulatory mechanisms [12]:

a.Guanine nucleotide exchange factors (GEFs) are necessary for the conversion of GDP-RAS into GTP-RAS. The Son of Sevenless (SOS) is the GEF for the RAS signaling pathway. SOS is recruited by the adaptor protein GRB2 in response to RTK activation. The binding of SOS to GBR2 localizes it to the plasma

**21**

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

gene product of *PTPN11*, the most commonly mutated gene in JMML.

the gene implicated in Neurofibromatosis type 1.

kinase, resulting in a downstream proliferative effect.

activation of the RAS pathway.

**5.2 GM-CSF–dependent hypersensitivity**

b.GTPase activating proteins (GAPs), like neurofibromin act antagonistically to inactivate RAS by increasing their intrinsic rate of GTP hydrolysis to the inactive form (RAS-GDP) leading to the termination of the RAS signaling. Neurofibromin is the encoded gene product of the tumor suppressor gene *NF1,*

Cancer-associated RAS mutations typically alter amino acids G12, G13 or Q61. These mutant RAS proteins display impaired GTPase activity which renders them resistant to GTPase activating proteins (GAPs). Mutant RAS proteins are therefore locked in the signal-transmitting GTP- bound form (the cytosolic ratio of GTP is much higher than GDP at 10:1). The active GTP-RAS in turn activates the RAF

The *CBL* gene (11q23.3) is a proto-oncogene that encodes three distinct homologs which represent the mammalian CBL family of proteins. Members of the CBL protein family (Cbl/c-Cbl, Cbl-b, and Cbl-c/Cbl-3) are E3 ubiquitin ligases that functions as a negative regulator of many signal transduction pathways by promoting degradation by ubiquitination of activated RTKs (and nonreceptor tyrosine kinases). Studies in animal models and genetic analyses in human cancer have firmly established that CBL proteins function as tumor suppressors. The mutant CBL (lacking E3 ligase activity)- dependent oncogenesis is driven by loss of ubiquitination-dependent degradation of activated RTKs resulting in a synergistic increase in signaling. Mutant CBL proteins may indeed promote oncogenesis in JMML via the RAS signaling pathway. In addition, CBL is an adaptor protein that positively regulates signal transduction. Approximately 150 proteins are associated with or are regulated by CBL proteins. Among these proteins is the adaptor GRB2 that binds to CBL. The CBL-GRB2 interaction modulates the interaction between GRB2 and SOS and hence it is also possible that CBL-GRB2 interactions lead to

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the related cytokines interleukin 3 (IL-3) and interleukin 5 (IL-5) regulate hematopoietic cell survival, proliferation, differentiation, migration, and perform effector functions such as phagocytosis or reactive oxygen species release. Unlike other cytokine receptors, GM-CSF receptor (GMR) has a significant nonredundant role in macrophage-mediated acute and chronic inflammation, pulmonary homeostasis, allergic diseases, and myeloid haematologic malignancies [13]. GMR is found in myeloid cells and some non-hematopoietic cells, but it is not expressed by lymphoid cells such as T cells. GMR is composed of a ligand-specific α chain (GMR α) and a β common (βc) signaling subunit, which is shared with the IL-3 and IL-5 receptors. Both of which are members of the cytokine receptor family. The binding of GM-CSF to the ligand specific GMRα promotes the formation of a higher-order signaling complex that leads to the activation of non-receptor tyrosine kinases JAK2 and Src family kinases (c-Src and Lyn), which subsequently phosphorylate GMRβc to create docking sites for adapters and signal relay molecules (such as SHP-2) initiating downstream signaling. GM-CSF, IL-3, and IL-5 activate at least three downstream pathways: the JAK/STAT pathway, the RAS signaling pathway, and PI

membrane, where it can activate the membrane bound RAS. Gain of function of the Src-homology tyrosine phosphatase 2 (SHP-2) results in activation of the GEFs and in this way to a continuous activation of RAS. SHP-2 is the encoded

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

membrane, where it can activate the membrane bound RAS. Gain of function of the Src-homology tyrosine phosphatase 2 (SHP-2) results in activation of the GEFs and in this way to a continuous activation of RAS. SHP-2 is the encoded gene product of *PTPN11*, the most commonly mutated gene in JMML.

b.GTPase activating proteins (GAPs), like neurofibromin act antagonistically to inactivate RAS by increasing their intrinsic rate of GTP hydrolysis to the inactive form (RAS-GDP) leading to the termination of the RAS signaling. Neurofibromin is the encoded gene product of the tumor suppressor gene *NF1,* the gene implicated in Neurofibromatosis type 1.

Cancer-associated RAS mutations typically alter amino acids G12, G13 or Q61. These mutant RAS proteins display impaired GTPase activity which renders them resistant to GTPase activating proteins (GAPs). Mutant RAS proteins are therefore locked in the signal-transmitting GTP- bound form (the cytosolic ratio of GTP is much higher than GDP at 10:1). The active GTP-RAS in turn activates the RAF kinase, resulting in a downstream proliferative effect.

The *CBL* gene (11q23.3) is a proto-oncogene that encodes three distinct homologs which represent the mammalian CBL family of proteins. Members of the CBL protein family (Cbl/c-Cbl, Cbl-b, and Cbl-c/Cbl-3) are E3 ubiquitin ligases that functions as a negative regulator of many signal transduction pathways by promoting degradation by ubiquitination of activated RTKs (and nonreceptor tyrosine kinases). Studies in animal models and genetic analyses in human cancer have firmly established that CBL proteins function as tumor suppressors. The mutant CBL (lacking E3 ligase activity)- dependent oncogenesis is driven by loss of ubiquitination-dependent degradation of activated RTKs resulting in a synergistic increase in signaling. Mutant CBL proteins may indeed promote oncogenesis in JMML via the RAS signaling pathway. In addition, CBL is an adaptor protein that positively regulates signal transduction. Approximately 150 proteins are associated with or are regulated by CBL proteins. Among these proteins is the adaptor GRB2 that binds to CBL. The CBL-GRB2 interaction modulates the interaction between GRB2 and SOS and hence it is also possible that CBL-GRB2 interactions lead to activation of the RAS pathway.

#### **5.2 GM-CSF–dependent hypersensitivity**

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the related cytokines interleukin 3 (IL-3) and interleukin 5 (IL-5) regulate hematopoietic cell survival, proliferation, differentiation, migration, and perform effector functions such as phagocytosis or reactive oxygen species release. Unlike other cytokine receptors, GM-CSF receptor (GMR) has a significant nonredundant role in macrophage-mediated acute and chronic inflammation, pulmonary homeostasis, allergic diseases, and myeloid haematologic malignancies [13]. GMR is found in myeloid cells and some non-hematopoietic cells, but it is not expressed by lymphoid cells such as T cells. GMR is composed of a ligand-specific α chain (GMR α) and a β common (βc) signaling subunit, which is shared with the IL-3 and IL-5 receptors. Both of which are members of the cytokine receptor family. The binding of GM-CSF to the ligand specific GMRα promotes the formation of a higher-order signaling complex that leads to the activation of non-receptor tyrosine kinases JAK2 and Src family kinases (c-Src and Lyn), which subsequently phosphorylate GMRβc to create docking sites for adapters and signal relay molecules (such as SHP-2) initiating downstream signaling. GM-CSF, IL-3, and IL-5 activate at least three downstream pathways: the JAK/STAT pathway, the RAS signaling pathway, and PI

*Acute Leukemias*

**20**

program [11].

**Figure 3.**

*JMML.*

opposing regulatory mechanisms [12]:

and survival. It relays extracellular stimuli such as growth factors to the nucleus where the response to those stimuli is executed by an induced transcriptional

*Illustrative representation of the RAS signaling pathway portraying the transmission of extracellular signals in a gear train to the nucleus. Upon stimulation, a docking site for GRB2 is created which binds to GEF (SOS). The GRB2-SOS interaction converts RAS-GDP to RAS-GTP. The RAS-GTP initiates a cascade of phosphorylations on RAF, MEK and, lastly, ERK. The phospho-ERK then translocates into the nucleus. A counteracting force by GAP (neurofibromin) accelerates the conversion of RAS-GTP to RAS-GDP to restrain the activation of the RAS signaling pathway. Mutations of RAS pathway prevent the conversion of RAS-GTP to RAS-GDP to induce the constitutive activation of the RAS signaling pathway, the characteristic feature of* 

The RAS family include the proto-oncogenes *HRAS, NRAS and KRAS* which represent a subset of a superfamily of small membrane-localized GTPases. Like other small GTPases, RAS proteins function as molecular switches that cycle between an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound conformations. RAS is activated when the receptor tyrosine kinases (RTKs, a large superfamily of receptors for a wide array of growth factors) are activated by growth factor binding leading to RTK autophosphorylation and the creation of docking sites for adaptor molecules like growth factor receptorbound protein 2 (GRB2). The amount of GTP-RAS generated is regulated by two

a.Guanine nucleotide exchange factors (GEFs) are necessary for the conversion of GDP-RAS into GTP-RAS. The Son of Sevenless (SOS) is the GEF for the RAS signaling pathway. SOS is recruited by the adaptor protein GRB2 in response to RTK activation. The binding of SOS to GBR2 localizes it to the plasma

3-kinase activation pathway. These pathways should not be viewed as being mutually exclusive and may have substantial overlap [14].

RAS signaling pathway mutations might potentiate JAK/STAT signaling by stabilizing or directly activating the GM-CSF receptor or its associated signaling molecules. In particular, the dysregulated SHP-2 (a non-receptor protein tyrosine phosphatase) by JMML-associated mutations is normally essential for efficient STAT5 activation in myeloid cells that are stimulated with IL-3 (SHP-2 is recruited to phosphorylated tyrosine residues on the activated β subunit of the GMR). Elevated levels of RAS-GTP might also increase the degree and/or duration of JAK2 kinase activity. The disturbed GMR signaling in JMML leads to an aberrant response to GM-CSF which can be tested by 2 laboratory assays:

#### *5.2.1 CFU-GM assay (the traditional methylcellulose assay)*

The selective hypersensitivity of myeloid progenitor cells of JMML to the GM-CSF in hematopoietic colony formation assays (CFAs) results in spontaneous growth of colony-forming units -granulocyte/macrophage (CFU-GM) in the absence of exogenous growth factors. JMML cells are cultured in semisolid methylcellulose media producing excess number of monocyte–macrophage colonies in the absence of added exogenous growth factors. This spontaneous proliferation of JMML myeloid progenitor cells is primarily due to the striking hypersensitivity of progenitors to GM-CSF in vitro.

This phenomenon, and hallmark of the disease, was first described in 1991. The CFU-GM assay has 2 main pitfalls. The assay is not standardized across diagnostic laboratories and although sensitive it is rather a non-specific assay. Viral infections such as HHV-6 and CMV have also been reported to cause GM-CSF hypersensitivity. Although the hypersensitive pattern of CFU-GM colony growth in methylcellulose is neither necessary nor sufficient to establish a diagnosis of JMML, it used as a minor diagnostic criterion for those 10 percent of JMML patients who do not have identifiable molecular abnormalities affecting the RAS signaling pathway.

#### *5.2.2 p-STAT5 phospho-specific flowcytometry assay*

Low-dose GM-CSF can induce hyperphosphorylation of STAT5 in either CD33<sup>+</sup> / CD34+ (myeloid precursor cells) or CD33+ /CD34− /CD14+ /CD38low population (represents the more mature monocytic cells) in patients with JMML. In contrast to CD34<sup>+</sup> /CD33+ cells, neither CD34− /CD33+ (mature myeloid cells) nor CD34<sup>+</sup> / CD33<sup>−</sup> (non-myeloid precursors), demonstrated any STAT5 hyperphosphorylation in response to GM-CSF. These results indicate that the peculiar STAT5 hyperphosphorylation signature specifically resides in the myeloid compartment of hematopoietic progenitor cells [15].

After binding of GM-CSF to its receptor, Janus-kinase-2 (JAK-2) is recruited to the cytoplasmic domain of the β chain, and activation of JAK-2 occurs, which subsequently induces STAT-5 phosphorylation. This signaling pathway induces migration of STAT-5 dimers to the nucleus and promotes the transcription of various genes such as pim-1 and CIS to induce cell differentiation. This cytokine-specific response is exclusively present in JMML patients compared with healthy controls or other pediatric MPNs, indicating that it is critical for disease pathogenesis. The aberrant response of phospho-STAT5 (p-STAT5) to sub-saturating doses of GM-CSF is demonstrated by phospho-specific flowcytometry assay. This assay is superior to the traditional CFU-GM assay in the following aspects [16]:

**23**

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

colony assay but not by phospho-specific flowcytometry.

leukocytes, monocytes, and signaling associated mutations.

1.As compared to CFU-GM assay, p-STAT5 profiling is a rapid diagnostic tool for JMML. CFU-GM assay takes up to several weeks of culture as it requires

2.p-STAT5 profiling could discriminate JMML from diseases mimicking JMML, such as CMV infection and transient myeloproliferative disorder in Noonan syndrome (NS/MPD) that also exhibit GM-CSF hypersensitivity by in vitro

Interestingly, CMML (the adult equivalent of JMML) also displays GM-CSFdependent hypersensitivity. Primary CMML samples demonstrate GM-CSF– dependent hypersensitivity by hematopoietic CFAs and phospho-STAT5 (pSTAT5) flowcytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral

The p-STAT5 response to low doses of GM-CSF was also detectable in AML M4/ M5. JMML, CMML, and M4/M5 AML are related entities in which hyperactive RAS and aberrant JAK2/STAT5 signaling are early or initiating events. As such, M4/M5 AML might be distinct from other subtypes of AML, in which aberrant transcription factor fusions such as PML-RARA and AML1-ETO likely represent primary leukemogenic events. This has important therapeutic implications, as M4/M5 AML might be highly dependent on RAS and JAK2/STAT5 signaling, and therefore sensitive to inhibitors of these pathways. Because GM-CSF signaling is critical for monocyte differentiation and survival, targeting GM-CSF in the therapeutics of JMML in vitro and AML in vivo has been reported, with varying degrees of success [17].

The absence of the p-STAT5 phospho-flow signature in conditions that are phenotypically similar to JMML highlights the importance of the signaling disruptions that we observe specifically in JMML. The flow cytometric p-STAT5 profiling is a reliable, sensitive and specific diagnostic tool for identifying patients with JMML that was added in the 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia as a minor diagnostic criterion for those 10 percent of JMML patients who do not have identifiable molecular abnormalities

In fact, hyperphosphorylation of STAT5 does not only add to the genetic mapping of JMML but it has significant therapeutic implications. Kotecha et al. demonstrated that p-STAT5 phospho-flow signature may identify children with NRAS and PTPN11 mutations who will have a benign clinical course and can be observed closely without aggressive treatment. p-STAT5 phospho-flow signature was also absent in infants with NS/MPD which indicates that phospho-flow cytometry provides a more specific readout of the rewired signaling networks found in JMML. Remarkably, p-STAT5 signaling response can indicate disease status as the p-STAT5 responsive population was shown to disappear in remission and reappear during relapse. Differences in GM-CSF-stimulated phospho-STAT5 levels might be utilized to identify targeted agents with potential efficacy as well as to follow response to

As opposed to the somatic mutations found in cancer, germline mutations of genes that encode components or regulators of the RAS signal transduction pathway result in clinically defined group of human genetic syndromes collectively termed as "RASopathies" or "neuro- cardio-facio cutaneous syndromes (NCFCS) [19]. The RASopathies are one of the largest known groups of malformation syndromes with

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

monocyte depletion.

affecting the RAS signaling pathway.

treatment, relapse, or transformation to AML [18].

**5.3 JMML interlacing with RASopathies**

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


Interestingly, CMML (the adult equivalent of JMML) also displays GM-CSFdependent hypersensitivity. Primary CMML samples demonstrate GM-CSF– dependent hypersensitivity by hematopoietic CFAs and phospho-STAT5 (pSTAT5) flowcytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signaling associated mutations.

The p-STAT5 response to low doses of GM-CSF was also detectable in AML M4/ M5. JMML, CMML, and M4/M5 AML are related entities in which hyperactive RAS and aberrant JAK2/STAT5 signaling are early or initiating events. As such, M4/M5 AML might be distinct from other subtypes of AML, in which aberrant transcription factor fusions such as PML-RARA and AML1-ETO likely represent primary leukemogenic events. This has important therapeutic implications, as M4/M5 AML might be highly dependent on RAS and JAK2/STAT5 signaling, and therefore sensitive to inhibitors of these pathways. Because GM-CSF signaling is critical for monocyte differentiation and survival, targeting GM-CSF in the therapeutics of JMML in vitro and AML in vivo has been reported, with varying degrees of success [17].

The absence of the p-STAT5 phospho-flow signature in conditions that are phenotypically similar to JMML highlights the importance of the signaling disruptions that we observe specifically in JMML. The flow cytometric p-STAT5 profiling is a reliable, sensitive and specific diagnostic tool for identifying patients with JMML that was added in the 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia as a minor diagnostic criterion for those 10 percent of JMML patients who do not have identifiable molecular abnormalities affecting the RAS signaling pathway.

In fact, hyperphosphorylation of STAT5 does not only add to the genetic mapping of JMML but it has significant therapeutic implications. Kotecha et al. demonstrated that p-STAT5 phospho-flow signature may identify children with NRAS and PTPN11 mutations who will have a benign clinical course and can be observed closely without aggressive treatment. p-STAT5 phospho-flow signature was also absent in infants with NS/MPD which indicates that phospho-flow cytometry provides a more specific readout of the rewired signaling networks found in JMML. Remarkably, p-STAT5 signaling response can indicate disease status as the p-STAT5 responsive population was shown to disappear in remission and reappear during relapse. Differences in GM-CSF-stimulated phospho-STAT5 levels might be utilized to identify targeted agents with potential efficacy as well as to follow response to treatment, relapse, or transformation to AML [18].

#### **5.3 JMML interlacing with RASopathies**

As opposed to the somatic mutations found in cancer, germline mutations of genes that encode components or regulators of the RAS signal transduction pathway result in clinically defined group of human genetic syndromes collectively termed as "RASopathies" or "neuro- cardio-facio cutaneous syndromes (NCFCS) [19]. The RASopathies are one of the largest known groups of malformation syndromes with

*Acute Leukemias*

3-kinase activation pathway. These pathways should not be viewed as being mutu-

RAS signaling pathway mutations might potentiate JAK/STAT signaling by stabilizing or directly activating the GM-CSF receptor or its associated signaling molecules. In particular, the dysregulated SHP-2 (a non-receptor protein tyrosine phosphatase) by JMML-associated mutations is normally essential for efficient STAT5 activation in myeloid cells that are stimulated with IL-3 (SHP-2 is recruited to phosphorylated tyrosine residues on the activated β subunit of the GMR). Elevated levels of RAS-GTP might also increase the degree and/or duration of JAK2 kinase activity. The disturbed GMR signaling in JMML leads to an aberrant

The selective hypersensitivity of myeloid progenitor cells of JMML to the GM-CSF in hematopoietic colony formation assays (CFAs) results in spontaneous growth of colony-forming units -granulocyte/macrophage (CFU-GM) in the absence of exogenous growth factors. JMML cells are cultured in semisolid methylcellulose media producing excess number of monocyte–macrophage colonies in the absence of added exogenous growth factors. This spontaneous proliferation of JMML myeloid progenitor cells is primarily due to the striking hypersensitivity of

This phenomenon, and hallmark of the disease, was first described in 1991. The CFU-GM assay has 2 main pitfalls. The assay is not standardized across diagnostic laboratories and although sensitive it is rather a non-specific assay. Viral infections such as HHV-6 and CMV have also been reported to cause GM-CSF hypersensitivity. Although the hypersensitive pattern of CFU-GM colony growth in methylcellulose is neither necessary nor sufficient to establish a diagnosis of JMML, it used as a minor diagnostic criterion for those 10 percent of JMML patients who do not have identifiable molecular abnormalities affecting the RAS

Low-dose GM-CSF can induce hyperphosphorylation of STAT5 in either CD33<sup>+</sup>

(represents the more mature monocytic cells) in patients with JMML. In contrast

/CD33+

in response to GM-CSF. These results indicate that the peculiar STAT5 hyperphosphorylation signature specifically resides in the myeloid compartment of hemato-

to the cytoplasmic domain of the β chain, and activation of JAK-2 occurs, which subsequently induces STAT-5 phosphorylation. This signaling pathway induces migration of STAT-5 dimers to the nucleus and promotes the transcription of various genes such as pim-1 and CIS to induce cell differentiation. This cytokine-specific response is exclusively present in JMML patients compared with healthy controls or other pediatric MPNs, indicating that it is critical for disease pathogenesis. The aberrant response of phospho-STAT5 (p-STAT5) to sub-saturating doses of GM-CSF is demonstrated by phospho-specific flowcytometry assay. This assay is superior to the traditional CFU-GM assay in the

/CD34−

(non-myeloid precursors), demonstrated any STAT5 hyperphosphorylation

After binding of GM-CSF to its receptor, Janus-kinase-2 (JAK-2) is recruited

/CD14+

/CD38low population

(mature myeloid cells) nor CD34<sup>+</sup>

/

/

ally exclusive and may have substantial overlap [14].

response to GM-CSF which can be tested by 2 laboratory assays:

*5.2.1 CFU-GM assay (the traditional methylcellulose assay)*

*5.2.2 p-STAT5 phospho-specific flowcytometry assay*

(myeloid precursor cells) or CD33+

cells, neither CD34−

progenitors to GM-CSF in vitro.

signaling pathway.

/CD33+

poietic progenitor cells [15].

following aspects [16]:

CD34+

CD33<sup>−</sup>

to CD34<sup>+</sup>

**22**

an incidence of around 1 in 1000 individuals. They exhibit numerous overlapping phenotypic features such as neurodevelopmental dysmorphic features, an increased risk of autoimmunity and predisposition to cancer and abnormal myelopoiesis in infancy. The RAS signal transduction pathway plays an essential role in regulating the cell cycle and cellular growth, differentiation, and senescence, all of which are critical to normal development. Therefore, it is not surprising that RAS signal transduction pathway dysregulation has detrimental effects on both embryonic and later stages of development. RASopathies include:


Noonan syndrome (NS) is the most genetically diverse and most common RASopathy occurring in 1 of 1000 to 2500 births. NS is characterized by developmental disorders, short stature, a typical facial appearance (facial dysmorphism), congenital heart defects, and skeletal anomalies. So far, heterozygous mutations in nine genes (*PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MEK1* and *CBL*) have been documented to underlie this disorder or clinically related phenotypes. Germline mutations in the *PTPN11* gene have been described in 50% of the Noonan syndrome cases. Somatically acquired JMML-associated PTPN11 mutations are not seen in NS. Somatic mutations differ from germline PTPN11 mutations which are predicted to result in a weaker gain-of-function than the somatic mutations [20].

The most common hematopoietic disorder in NS is a transient myeloproliferative disorder (MPD) estimated to occur in up to 10% of all children with NS. Nearly all patients with NS/MPD have mutations in *PTPN11*. The transient MPD in Noonan syndrome is diagnosed in the neonatal period or early infancy. In contrast to JMML, NS/MPD is thought to be of polyclonal origin. It generally resolves spontaneously over months or years. However, leukocytosis and tissue invasion by monocytes and immature myeloid cells can have deleterious effects. An estimated 10% of cases of NS/MPD acquire a cytogenetic abnormality and progress to JMML. Thus, NS/ MPD is a tumor predisposition syndrome and affected patients should be followed closely. JMML in *PTPN11*-mutated NS seems to behave differently from sporadic JMML as it occurs at a very young age (infancy) and tends to regress spontaneously. Although the hematological picture can be indistinguishable from JMML in some cases, the myeloproliferation is polyclonal in most instances, and thus, the disorder has not been recognized by the World Health Organization (WHO) as a separate JMML category. Therefore, recognizing NS in a JMML patient is important in order to identify those patients who might benefit from a watch-and-wait strategy. Similar to *PTPN11*-mutated NS, a transient MPD was noted in some children with NS and *KRAS* or *NRAS* germline mutations.

*NF1* gene is a negative modulator that normally restricts RAS activation. Loss of heterozygosity (LOH) with loss of the normal NF1 allele in leukemic cells leads to activation of RAS signaling pathway. In most cases, the diagnosis of NF1 can be

**25**

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

established clinically by the presence of ≥6 caf'e au lait macules >0.5 cm in diameter. In addition, one-half of these children have a parent affected by NF1.The risk of developing JMML for the patient with NF1 is estimated 200- to 350-fold higher

Germ line mutations of the *CBL* gene cause CBL syndrome characterized by a high frequency of neurologic features/vasculopathy, mild NS-like features, and a high risk of JMML. The germline mutation represents the first hit, with somatic LOH being the second hit positively selected in JMML cells. Indeed, all children with JMML and *CBL* mutations were found to have a germline *CBL* missense mutation on one allele and acquired LOH on the other allele in leukemic cells. The development of vasculopathies occurs in the second decade of life unless allogeneic HSCT is successful. The most frequently observed vasculopathies are optic atrophy,

JMML cells show a normal karyotype in 65% of cases, sole monosomy 7 in about 25%, and other aberrations in 10%. A remarkable feature of many JMML cases with normal karyotype is a markedly increased synthesis of fetal hemoglobin (HbF). The likelihood of an abnormal karyotype is dependent on the genetic subtype, with monosomy 7 being noted most often in *KRAS*-mutated disease. Monosomy 7 is the most frequent chromosomal aberration in both MDS and JMML. It is likely that haploinsufficiency of genes located on chromosome 7 contributes to clonal

Deregulated RAS signaling is the main driving event in JMML. The 5 canonical

The concept that mutations associated with JMML are mutually exclusive within a given individual implies that a mutation in any one of these genes is sufficient to activate the signaling pathway and drive the proliferation of JMML cells. However, this does not mean that two or more genetic mutations cannot occur at the same time as one would comprehend from the term 'mutually exclusive'. In fact, coexisting mutations in *NRAS, KRAS, PTPN11, CBL and NF1* were found in 11% of JMML patients [21]. Analysis of single colonies for two patients with compound RAS pathway mutations demonstrated that both mutations occurred in the same colony. PTPN11 and NF1 lesions were the most frequent of these cooperative events. In

addition, one patient harbored two *NRAS* lesions (p.G13D and p.Q61K).

The five JMML genetic subtypes are key determinants for treatment adaptation in patients with JMML. However, their use in a risk stratification algorithm is hindered by their failure to independently prognosticate the clinical outcome. In fact, the prognostic relevance is owned by the number of mutations at diagnosis rather than the type of mutation itself. Patients with JMML who harbor two or

RAS pathway mutations (*PTPN11, KRAS, NRAS, CBL*, and *NF1* genes) represent five genetically and clinically distinct subtypes in JMML. Mutations in the respective genes can either occur as germline ("syndromic") or as somatic lesion in hematopoietic cells ("nonsyndromic"). *PTPN11-, NRAS-*, and *KRAS*-mutated JMML are characterized by heterozygous somatic gain-of-function mutations in nonsyndromic children, whereas JMML in neurofibromatosis type 1 and JMML in children with CBL syndrome are defined by germline RAS disease and acquired biallelic inactivation of the respective genes in hematopoietic cells. Their clinical

optic neuritis, hypertension, cardiomyopathy, and arteritis.

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

than in patients without NF1.

**5.4 Cytogenetic studies**

**6. JMML genetic subtypes**

features are presented in **Table 2**.

evolution.

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

established clinically by the presence of ≥6 caf'e au lait macules >0.5 cm in diameter. In addition, one-half of these children have a parent affected by NF1.The risk of developing JMML for the patient with NF1 is estimated 200- to 350-fold higher than in patients without NF1.

Germ line mutations of the *CBL* gene cause CBL syndrome characterized by a high frequency of neurologic features/vasculopathy, mild NS-like features, and a high risk of JMML. The germline mutation represents the first hit, with somatic LOH being the second hit positively selected in JMML cells. Indeed, all children with JMML and *CBL* mutations were found to have a germline *CBL* missense mutation on one allele and acquired LOH on the other allele in leukemic cells. The development of vasculopathies occurs in the second decade of life unless allogeneic HSCT is successful. The most frequently observed vasculopathies are optic atrophy, optic neuritis, hypertension, cardiomyopathy, and arteritis.

#### **5.4 Cytogenetic studies**

*Acute Leukemias*

2.Costello: HRAS.

an incidence of around 1 in 1000 individuals. They exhibit numerous overlapping phenotypic features such as neurodevelopmental dysmorphic features, an increased risk of autoimmunity and predisposition to cancer and abnormal myelopoiesis in infancy. The RAS signal transduction pathway plays an essential role in regulating the cell cycle and cellular growth, differentiation, and senescence, all of which are critical to normal development. Therefore, it is not surprising that RAS signal transduction pathway dysregulation has detrimental effects on both embryonic and

later stages of development. RASopathies include:

4.Neurofibromatosis type 1: NF1.

NS and *KRAS* or *NRAS* germline mutations.

anagen hair (SHOC2).

1.Noonan syndrome: PTPN11, KRAS, SOS1, RAF1, NRAS.

3.Cardiofacio-cutaneous syndromes (KRAS, BRAF, MEK1/2).

6.Legius syndrome (NF-1-like syndrome): SPRED1.

5.NS-like syndromes: CBL-syndrome (CBL) and NS-like disorder with loose

Noonan syndrome (NS) is the most genetically diverse and most common RASopathy occurring in 1 of 1000 to 2500 births. NS is characterized by developmental disorders, short stature, a typical facial appearance (facial dysmorphism), congenital heart defects, and skeletal anomalies. So far, heterozygous mutations in nine genes (*PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MEK1* and *CBL*) have been documented to underlie this disorder or clinically related phenotypes. Germline mutations in the *PTPN11* gene have been described in 50% of the Noonan syndrome cases. Somatically acquired JMML-associated PTPN11 mutations are not seen in NS. Somatic mutations differ from germline PTPN11 mutations which are predicted to result in a weaker gain-of-function than the somatic mutations [20]. The most common hematopoietic disorder in NS is a transient myeloproliferative disorder (MPD) estimated to occur in up to 10% of all children with NS. Nearly all patients with NS/MPD have mutations in *PTPN11*. The transient MPD in Noonan syndrome is diagnosed in the neonatal period or early infancy. In contrast to JMML, NS/MPD is thought to be of polyclonal origin. It generally resolves spontaneously over months or years. However, leukocytosis and tissue invasion by monocytes and immature myeloid cells can have deleterious effects. An estimated 10% of cases of NS/MPD acquire a cytogenetic abnormality and progress to JMML. Thus, NS/ MPD is a tumor predisposition syndrome and affected patients should be followed closely. JMML in *PTPN11*-mutated NS seems to behave differently from sporadic JMML as it occurs at a very young age (infancy) and tends to regress spontaneously. Although the hematological picture can be indistinguishable from JMML in some cases, the myeloproliferation is polyclonal in most instances, and thus, the disorder has not been recognized by the World Health Organization (WHO) as a separate JMML category. Therefore, recognizing NS in a JMML patient is important in order to identify those patients who might benefit from a watch-and-wait strategy. Similar to *PTPN11*-mutated NS, a transient MPD was noted in some children with

*NF1* gene is a negative modulator that normally restricts RAS activation. Loss of heterozygosity (LOH) with loss of the normal NF1 allele in leukemic cells leads to activation of RAS signaling pathway. In most cases, the diagnosis of NF1 can be

**24**

JMML cells show a normal karyotype in 65% of cases, sole monosomy 7 in about 25%, and other aberrations in 10%. A remarkable feature of many JMML cases with normal karyotype is a markedly increased synthesis of fetal hemoglobin (HbF). The likelihood of an abnormal karyotype is dependent on the genetic subtype, with monosomy 7 being noted most often in *KRAS*-mutated disease. Monosomy 7 is the most frequent chromosomal aberration in both MDS and JMML. It is likely that haploinsufficiency of genes located on chromosome 7 contributes to clonal evolution.

#### **6. JMML genetic subtypes**

Deregulated RAS signaling is the main driving event in JMML. The 5 canonical RAS pathway mutations (*PTPN11, KRAS, NRAS, CBL*, and *NF1* genes) represent five genetically and clinically distinct subtypes in JMML. Mutations in the respective genes can either occur as germline ("syndromic") or as somatic lesion in hematopoietic cells ("nonsyndromic"). *PTPN11-, NRAS-*, and *KRAS*-mutated JMML are characterized by heterozygous somatic gain-of-function mutations in nonsyndromic children, whereas JMML in neurofibromatosis type 1 and JMML in children with CBL syndrome are defined by germline RAS disease and acquired biallelic inactivation of the respective genes in hematopoietic cells. Their clinical features are presented in **Table 2**.

The concept that mutations associated with JMML are mutually exclusive within a given individual implies that a mutation in any one of these genes is sufficient to activate the signaling pathway and drive the proliferation of JMML cells. However, this does not mean that two or more genetic mutations cannot occur at the same time as one would comprehend from the term 'mutually exclusive'. In fact, coexisting mutations in *NRAS, KRAS, PTPN11, CBL and NF1* were found in 11% of JMML patients [21]. Analysis of single colonies for two patients with compound RAS pathway mutations demonstrated that both mutations occurred in the same colony. PTPN11 and NF1 lesions were the most frequent of these cooperative events. In addition, one patient harbored two *NRAS* lesions (p.G13D and p.Q61K).

The five JMML genetic subtypes are key determinants for treatment adaptation in patients with JMML. However, their use in a risk stratification algorithm is hindered by their failure to independently prognosticate the clinical outcome. In fact, the prognostic relevance is owned by the number of mutations at diagnosis rather than the type of mutation itself. Patients with JMML who harbor two or


**27**

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

**3- Increased fetal hemoglobin (HbF) for age.**

**/L.**

**3- DNA hypermethylation signature defines an aggressive JMML subgroup with** 

more somatic mutations at diagnosis have significantly worse event-free and overall survival rates than those with one or no event. In harmony with these findings, the known clinical risk factors predictive of poor outcome (**Table 3**) are only weakly associated with the type of index mutation but they would rather fit patients with

In addition to the mutations in RAS pathway genes (*PTPN11, NRAS, KRAS, CBL, or NF1*), other driver mutations thought to be the initiating events of JMML have been recently identified such as *RRAS, RRAS2*, or *SH2B3* mutations. *RRAS* and *RRAS2* genes are both members of the RAS GTPase family and hence expanded the spectrum of RAS pathway mutations in JMML. Activating mutations in *RRAS* underlie a phenotype within the RASopathy spectrum. Children with *RRAS*mutated JMML can have an atypical clinical course with rapid progression to AML [22]. Somatic *RRAS* mutations co-occurred with acquired *NRAS* lesions in atypical

In addition to the initiating canonical RAS pathway mutations, secondary clonal abnormalities were detected in about one-half of the patients. The importance of these mutations relies in how secondary mutations alter the behavior of cells in contrast to cells harboring only the primary lesion. Secondary mutations are often subclonal and may be involved in disease progression rather than initiation of leukemia. It appears that such mutations characterize patients with the highest risk of progression and poor outcome. The acquisition of the second mutation would thus also explain the continuum between RALD and leukemia. The clone harboring the secondary event frequently expands at the time of relapse post HSCT. This phenomenon has important therapeutic implications. Combination of therapies with agents that target the RAS pathway as well as the secondary genetic event could prove more efficacious in the correct genetic context than monotherapy alone.

The secondary mutational events occur inside or outside the canonical RAS pathway axis. They include second hits targeting the RAS pathway (so-called 'RAS double mutants') as well as mutations in *SETBP1, JAK3, SH283*, components of the polycomb repressive complex 2 (like *EZH2* and A*SXL1*), and occasionally, spliceosome genes. Some authors linked differential expression of key regulatory noncoding RNAs, such as let-7100 or miR-150-5p,101 to the various genetic subgroups

JMML characterized by late onset and rapid progression to AML as well.

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

**1- Age ≥ 2 years.**

**high risk of relapse. 4-** *PTPN11* **mutation. 5- Monosomy 7.**

**2- Platelet count ≤ 40 x 109**

**4- Male sex (in some references).**

**1- Two or more somatic mutations. 2- Secondary genetic mutations.**

Clinical risk factors:

Molecular risk factors:

*Poor prognostic factors.*

**Table 3.**

two or more underlying somatic mutations.

**7. Secondary genetic mutations in JMML**

#### **Table 2.**

*Clincal features of genetic subtypes of JMML.*

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

