Clonal analysis of NRAS activating mutations in KIT-D816V systemic mastocytosis

Abstract

Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-D816V systemic mastocytosis. In this study, 44 patients with KIT-D816V systemic mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations. Activating NRAS mutations were identified in 2 of 8 patients with advanced disease. NRAS mutations were not found in patients with indolent systemic mastocytosis. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored both mutations. KIT-D816V was not detected in bone marrow CD34(+) progenitors, whereas the NRAS mutation was present. These findings suggest that NRAS mutations may have the potential to precede KIT-D816V in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity.

Trial registration: ClinicalTrials.gov NCT00001756 NCT00044122.

Figures

Figure 1.

(A) Activating NRAS mutations in 2 patients with severe forms of KIT-D816V systemic mastocytosis. KIT-D816V RT-PCR/RFLP: Detection of KIT-D816V (arrow) in the bone marrow mononuclear cells (BMMC) of the 2 patients. HMC1.2 cells were used as positive control. NRAS sequencing: Patient 1: Compared to control, a heterozygous missense mutation GGT→GAT at codon 13 was identified resulting in a Glycine to Aspartic Acid substitution (NRAS-G13D). Patient 2: A similar heterozygous missense mutation GGT→GAT at codon 12 was identified resulting in a Glycine to Aspartic Acid substitution (NRAS G12D). A wild-type sequence in the buccal germline DNA confirmed a somatic event. (B) Patient 2: Segregation of KIT-D816V and NRAS-G12D in flow sorted bone marrow cell populations. KIT-D816V is present in mast cells (arrow), but absent in the CD34+ cells. NRAS-G12D is present in both populations (asterisks). Results are representative of three separate flow sorting experiments and summarized in Table 1. (C) Mast Cell Immunophenotyping. Bone marrow mast cells were initially identified as CD117 bright positive, CD45 positive cells with characteristic forward and side scatter properties (circled). Compared to the isotype control, gated mast cells uniformly expressed the aberrant CD25 marker. (D) RAS gene expression in cultured human mast cells. HRAS, NRAS, KRAS and MRAS gene expression standardized to GAPDH is plotted on the Y axis. Mast cells on the X axis include CD34+ derived human mast cells (HuMC) harvested at weeks one, 2, 4 and 8; HMC1.1, HMC1.2 and LAD2 mast cell lines. Error bars are the Standard Error Mean (SEM) of three separate experiments each performed in triplicate. Different CD34+ donors were used for each HuMC experiment. *Note: KIT-D816V RT-PCR/RFLP gel lanes are cropped from the same gel without enhancement.

Figure 2.

Histopathological changes in bone marrow biopsies from 2 NRAS positive patients. Both patients have hypercellular marrow biopsies, but with markedly different mast cell burden, as assessed by % mast cell involvement of the biopsy sections. Representative H&E (A) and CD117 (B) immunostained sections show that Patient 1 has minimal involvement by mast cell aggregates, while Patient 2 has extensive marrow replacement by mast cells [(C) H&E; (D) CD117]. In addition, Patient 1 has myeloid hyperplasia with increase in monocytic precursors and blasts, while Patient 2 has normal trilineage maturation. (Magnification 40x for all photographs)