Demonstration that mast cells, T cells, and B cells bearing the activating kit mutation D816V occur in clusters within the marrow of patients with mastocytosis

Abstract

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

Figure 1

Representative laser capture microdissection. Mast cells, lymphocytes, B cells, and T cells were obtained from lesional and non-lesional areas of bone marrow biopsy tissue from a patient with mastocytosis. Micrographs shown before and after microdissection and cells captured on cap.

Figure 2

Detection of the D816V mutation in mast cells, lymphocytes, B cells, and T cells in patients with different categories of mastocytosis. A: Indolent mastocytosis. B: Smoldering mastocytosis. C: Mastocytosis with associated hematological non-mast cell disease. Analysis of T cells and B cells in lesional areas only. A non-lesional area is defined as an area where there are a sparse number of mast cells. L, denotes lesional area; N, denotes non-lesional area.

Figure 3

Sensitivity of detection of the D816V mutation by nested PCR. Results of HinfI restriction digestion of mixtures of mutant HMC1.2 (0,5,10,50,100) and wild-type HMC1.1 cells (100,95,90,50,0) with the corresponding gel fluorescence graph. Data are representative of three experiments.

Figure 4

Molecular analysis of clonality of B cells from bone marrow biopsies from a patient with mastocytosis. A: B cells obtained from lesional areas of a bone marrow biopsy from a patient with mastocytosis and from normal human appendix by LCM. B: Starting PCR product of B cells (400 to 500 cells) as well as cloned CDR3 obtained from microdissected B cells from a patient with mastocytosis and from normal human appendix. C: Corresponding IgH CDR3 VDJ sequences from the patient. The nucleotide and deduced amino acid sequences are shown with the segments encoded by D genes (regular font) and n nucleotides (bold) indicated. GenBank Accession Numbers for these and control human appendix cell sequences are given in Table 2.