Identical Novel C-Kit Transcripts in Two Patients with Mast Cell Leukemia.

Background: Mutations of c-kit have been well documented in mast cell neoplasms. The most common alterations found in human cutaneous and systemic mastocytosis are V560G and D816V. Mast cell leukemia is the most aggressive mast cell neoplasm, characterized by diffuse bone marrow and peripheral blood involvement. It has not been well studied due to its rarity. To date, no C-KIT mutational analysis of primary mast cell leukemia cells has been reported, although a mast cell leukemia cell line expresses the V560G and D816V mutations. Here we report on C-KIT cDNA analysis on two patients diagnosed with mast cell leukemia at the University of Chicago Hospitals. Both patients had peripheral blood involvement. Their diagnosis was confirmed on bone marrow biopsies with tryptase stains. Patient 1, diagnosed in 1989, died shortly after diagnosis. Patient 2 achieved a complete remission with allogeneic stem cell transplant. Upon relapse, treatment with Gleevec, resulted in an incomplete but significant response. This patient eventually died of sepsis following refractory cytopenias. Methodology: We retrieved liquid nitrogen frozen, pre-treatment bone marrow aspirate samples from both patients. We performed RT-PCR analysis of the entire coding region of C-KIT by amplifying 21 exons in smaller fragments. Each PCR product was cloned and sequenced. A thorough sequencing analysis was performed by screening several clones of each amplification product, with the purpose of being able to identify low abundance transcripts. Results: In both patients, the most abundant transcript was alternatively spliced and lacked exons 12 and 13. In addition, coexisting on the same allele, there were novel but not identical single amino acid deletions: deletion of L521 in patient 1 and of S715 in patient 2. A second novel alteration detected in both patients was the duplication of Q252. Patient 2 also demonstrated two independent, low abundance transcripts, one encoding the point mutation V559G and the other containing a deletion of exon 12. Discussion: Here we describe two novel and identical C-KIT alterations in two patients with mast cell leukemia (deletion of exons 12 and 13 and duplication of Q252). We hypothesize that both are key molecular changes underlying mast cell leukemia. Exons 12 and 13 encode the N-terminus of the kinase domain, which is thought to have modulatory effect on the kinase active site. Amino acid 252 is located in the fourth immunoglobulin-like domain of the extracellular region, which is thought to be critical for receptor dimerization and phosphorylation. We predict that these mutations result in the superactivation of C-KIT, based on the aggressive behaviour of mast cell leukemia. The response to Gleeevec seen in patient 2 indicates that at least some of these transcripts are Gleevec sensitive. Functional assays to test if the encoded C-KIT proteins are constitutively activated and whether Gleevec inhibits the novel C-KIT proteins are in progress.