Novel C-KIT Transcripts Identified in Mast Cell Leukemia: An Update of the Full Transcript and It’s Distribution.

Introduction: Mast cell leukemia (MCL) is the rarest but most fatal form of mast cell neoplasms. Several C-KIT activating mutations have been identified in systemic mastocytosis (SM), the most frequent one being D816V. Little is known about C-KIT mutations in MCL. In 2005, at ASH we presented a novel C-KIT transcript in two patients with MCL, accounting for the sole C-KIT transcript in one of the patients. The novel form had a deletion of exons 12 and 13, encoding the N-terminal portion of the bipartite kinase domain. Here, we describe the complete C-KIT transcript, its response to stem cell factor (SCF) stimulation in vitro, and its expression in non- MCL samples.

Materials&Methods: The entire C-KIT coding region was amplified by RT-PCR from cDNA prepared from bone marrow (BM) samples of 2 patients with MCL, 2 patients with SM associated with acute myeloid leukemia, isolated CD34+ cells from healthy individuals, and 2 sets of purified mast cells from patients with SM. Several clones from each sample were fully sequenced. Its expression in patient samples was verified by Western blotting. An expression construct was made, and the mutant C-KIT was expressed in COS-1 cells. Western blotting was used to test the ability of the mutant protein to undergo autophosphorylation upon SCF stimulation.

Results: Complete sequencing demonstrated that the mutant C-KIT contained several mutations: a large deletion of amino acids 59–437, corresponding to loss of the first three immunoglobulin-like domains in the extracellular region, deletions of amino acids 510–513, deletions of the amino acids encoded by exons 12 and 13, and novel single amino acid deletions unique to each patient. The mutant C-KIT transcript was present in normal CD34+ cells and in 2 BMs with SM associated with AML. However, it was absent in purified mast cells from 2 patients with SM. In contrast, these displayed the widely recognised D816V point mutation. Western blotting from patient samples revealed a truncated protein of the predicted 59KD size. However, the novel C-KIT did not autophosphorylate upon stem cell factor stimulation.

Discussion: We have identified a novel C-KIT isoform. It was exclusively present in MCL but completely absent in purified mast cells of SM. These findings indicate that it is a marker of immaturity and aggressiveness among mast cell neoplasms, which may serve as a diagnostic and prognostic parameter. Its presence in non-neoplastic samples in similar amounts with the wild type suggest that it is not malignancy specific. However, its predominace over the wild type may correlate with a pathologic state that remains to be explored in other hematopoetic neoplasms.