Ras-associated autoimmune leukoproliferative disorder (RALD) is a chronic, nonmalignant condition that presents with persistent monocytosis and is often associated with leukocytosis, lymphoproliferation, and autoimmune phenomena. RALD has clinical and laboratory features that overlap with those of juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML), including identical somatic mutations in KRAS or NRAS genes noted in peripheral blood mononuclear cells. Long-term follow-up of these patients suggests that RALD has an indolent clinical course whereas JMML is fatal if left untreated. Immunophenotyping peripheral blood from RALD patients shows characteristic circulating activated monocytes and polyclonal CD10 + B cells. Distinguishing RALD from JMML and CMML has implications for clinical care and prognosis.

JMML is an aggressive malignant hematopoietic neoplasm of childhood with myelodysplastic and myeloproliferative features. Patients present with splenomegaly, fever, thrombocytopenia, monocytosis, and excess myelomonocytic cells that infiltrate skin and vital organs. JMML accounts for 20% to 30% of myelodysplastic/myeloproliferative disorders in the pediatric population. 5 The prognosis for JMML is poor with median survival of 1 year for untreated patients. Hematopoietic stem cell transplantation has become the standard of care for JMML. 6 , 7 CMML has a variable course in adults and often requires chemotherapy.

Ras-associated autoimmune leukoproliferative disorder (RALD) is a nonmalignant clinical syndrome initially identified in a subset of putative autoimmune lymphoproliferative syndrome (ALPS) patients. 1 Similar to patients with ALPS, RALD patients present with lymphadenopathy, massive splenomegaly, increased circulating B cells, hypergammaglobulinemia, and autoimmunity. 1-3 In contrast to ALPS, biomarkers such as CD4 – /CD8 – double negative T-cell receptor αβ (TCRαβ + ) T cells and serum vitamin B 12 levels are not always increased, and germline or somatic mutations in FAS, FASL, or CASP10 are absent in RALD. Persistent absolute or relative monocytosis is a cardinal feature of RALD. Bone marrow and peripheral blood smear findings overlap with those of juvenile myelomonocytic leukemia (JMML) in children or chronic myelomonocytic leukemia (CMML) in older patients. Activating somatic mutations that cause amino acid substitutions that affect codons 12 or 13 in KRAS or NRAS were identified in myeloid and lymphoid lineages. 2 In 2009, the revised classification and nomenclature of RALD was adopted to distinguish it from ALPS. 4

RAS genes (named for their role in forming rat sarcomas) were first recognized 50 years ago in tumor-initiating retroviruses (eg, Harvey sarcoma virus, Kirsten sarcoma virus, and Rasheed sarcoma virus), and their cellular homologs are implicated in myeloproliferative neoplasms8 and are found mutated in almost 30% of human cancers.9,10 How RAS proteins contribute to neoplasia and lymphoproliferative disorders remains to be fully elucidated.9,10 The RAS signaling proteins are ubiquitously expressed in all cells and serve as small guanosine triphosphatases (GTPases) that play diverse roles in cell cycle progression, proliferation, apoptosis, and cytoskeletal motility. In the immune system, they control B-cell tolerance and production of autoantibodies.11 Germline RAS mutations have been identified in nonmalignant conditions, including 5 neurodevelopmental dysmorphic syndromes termed “RASopathies” that carry an increased risk of autoimmunity and malignancy.12-14

Remarkably, the identical KRAS or NRAS mutations found in all RALD patients are also reported in up to 25% of JMML patients,15 suggesting a shared molecular etiology. Amino acid substitutions in codons 12 and 13 of KRAS or NRAS noted in our cohort of patients (Figure 1A) result in constitutive binding of GTP and activation of the NRAS or KRAS proteins thereby inducing the RAF/MEK/ERK signaling pathway. Increased signaling causes proliferation and downregulation of the proapoptotic protein Bim,1 resulting in attenuation of the intrinsic mitochondrial pathway of apoptosis. In vitro studies of T cells from RALD showed partial resistance to interleukin-2 withdrawal-induced apoptosis but sensitivity to other intrinsic apoptotic pathway stimuli. Farnesyltransferase inhibitors, which block the function of RAS, restored Bim levels and apoptosis in T cells from RALD patients.1,2

Figure 1 View largeDownload PPT (A) Simplified depiction of Ras protein with sites of mutation noted in Table 1. Combined structures of KRAS (blue) and NRAS (magenta). The mutation on the right (closest to bound guanosine diphosphate [GDP]) is G13, and G12 is on the left (farthest from bound GDP). (B) Peripheral blood monocytes from 8-year-old RALD patient 381.1 with leukocytosis, monocytosis, and lymphocytosis (×1000). (C) Bone marrow aspirate from same patient showing granulocytic hyperplasia with mild-to-moderate left shift in myeloid maturation with less than 5% blasts (×500). (D) Bone marrow from patient 104.1 showing pelgeroid granulocytes and left shift. (E) Healthy control peripheral blood (PB) with normal circulating monocytes that are CD16–. (F) Healthy control PB showing that only monocytes (turquoise) are positive for CD14 and granulocytes (green population) are negative for CD14. (G) Healthy control PB lymphocyte gate demonstrating that CD20+ B cells are largely CD10–. (H) PB CD14+ monocyte gate showing increased expression of CD16 on monocytes in RALD in comparison with monocytes from healthy control (E). (I) Prominent increased expression of CD14 on granulocytes (green) in RALD which is not seen in healthy controls (F). (J) Peripheral blood lymphocyte gate with B-cell lymphocytosis (polyclonal) with marked increase in CD10+ circulating late precursor B cells in comparison with healthy control (G). Figure 1 View largeDownload PPT (A) Simplified depiction of Ras protein with sites of mutation noted in Table 1. Combined structures of KRAS (blue) and NRAS (magenta). The mutation on the right (closest to bound guanosine diphosphate [GDP]) is G13, and G12 is on the left (farthest from bound GDP). (B) Peripheral blood monocytes from 8-year-old RALD patient 381.1 with leukocytosis, monocytosis, and lymphocytosis (×1000). (C) Bone marrow aspirate from same patient showing granulocytic hyperplasia with mild-to-moderate left shift in myeloid maturation with less than 5% blasts (×500). (D) Bone marrow from patient 104.1 showing pelgeroid granulocytes and left shift. (E) Healthy control peripheral blood (PB) with normal circulating monocytes that are CD16–. (F) Healthy control PB showing that only monocytes (turquoise) are positive for CD14 and granulocytes (green population) are negative for CD14. (G) Healthy control PB lymphocyte gate demonstrating that CD20+ B cells are largely CD10–. (H) PB CD14+ monocyte gate showing increased expression of CD16 on monocytes in RALD in comparison with monocytes from healthy control (E). (I) Prominent increased expression of CD14 on granulocytes (green) in RALD which is not seen in healthy controls (F). (J) Peripheral blood lymphocyte gate with B-cell lymphocytosis (polyclonal) with marked increase in CD10+ circulating late precursor B cells in comparison with healthy control (G).

Although both RALD and JMML share common RAS mutations, JMML cells apparently accumulate additional genetic abnormalities that contribute to the malignant phenotype. These include cytogenetic abnormalities and activating somatic mutations in PTPN11, c-CBL, ASXL1, and FLT-3.16-20 Additionally, germline mutations in neurofibromatosis type 1 (NF1) syndrome lead to a high risk of developing JMML in the first decade of life.16 NF1 encodes neurofibromin, which functions as a GTPase-activating protein that regulates the RAS/RAF/MEK/ERK signaling pathway.21 Noonan syndrome is also one of the so-called RASopathies characterized by germline mutations in genes involved in the RAS pathway, including PTPN11,22 KRAS, RAF1, and NRAS, resulting in an increased risk of developing JMML.14 On the basis of recent molecular analyses, more than 90% of JMML patients were found to harbor somatic mutations in NRAS, KRAS, NF1, PTPN11, and CBL, all of which are thought to act as driver mutations in the RAS signaling pathway.6,16 Somatic mutations in PTPN11 are indeed the most commonly identified mutations found in up to 35% of patients with JMML.17,22 Whole-exome sequencing has led to the detection of secondary mutations involving SETBP1 and JAK3 in 17% of JMML patients.23

The current accepted diagnostic criteria for JMML24,25 have been revised from the World Health Organization 2008 recommendations. Category 1 criteria (all of the following should be met) include persistent monocytosis with more than 1000 monocytes per microliter (1 × 109/L) in the peripheral blood, splenomegaly, less than 20% blasts in the bone marrow and/or peripheral blood, and absence of the t(9;22) BCR-ABL translocation. Category 2 criteria (at least 1 of the following conditions must be met) include somatic mutation in RAS or PTPN11, clinical diagnosis of NF1 or NF1 gene mutation, homozygous mutation in CBL, or monosomy 7. Category 3 criteria (at least 2 of the following must be met if category 2 criteria are not satisfied) include circulating myeloid precursors, white blood cell (WBC) count of more than 10 000/μL (10 × 109/L), increased hemoglobin F for age, clonal cytogenetic abnormality excluding monosomy 7, and granulocyte macrophage–colony-stimulating factor hypersensitivity of myeloid progenitors in vitro. Of note, 7% to 10% of JMML patients may not have splenomegaly at presentation; if the remaining category 1 criteria are met, in addition to 1 of category 2 criteria or 2 of category 3 criteria, a diagnosis of JMML may be made.