Detection of c-kit mutation Asp 816 to Val in microdissected bone marrow infiltrates in a case of systemic mastocytosis associated with chronic myelomonocytic leukaemia

Abstract

Background/aims: The occurrence of myeloid leukaemia in patients with systemic mastocytosis is a well recognised phenomenon. However, the pathophysiological basis of such a coevolution has not been clarified. Recent data have shown that the c-kit mutation Asp 816 to Val is detectable in neoplastic mast cells in most patients with systemic mastocytosis, including those who have associated haematological disorders. The aim of this study was to study clonal disease evolution by analysing bone marrow cells from a patient with systemic mastocytosis and associated chronic myelomonocytic leukaemia (CMML) for the presence of this mutation.

Methods: The DNA of microdissected bone marrow cells from a patient with systemic mastocytosis and associated CMML was analysed for the presence of the c-kit mutation Asp 816 to Val by means of HinfI digestion and direct sequencing of semi-nested polymerase chain reaction (PCR) products.

Results: The two neoplasms could easily be identified and discriminated in paraffin wax embedded bone marrow sections by tryptase and chloroacetate esterase staining. A total number of 10 tryptase positive systemic mastocytosis infiltrates and 10 tryptase negative CMML infiltrates were removed by microdissection. As assessed by HinfI digestion and direct sequencing of semi-nested PCR products, the c-kit mutation Asp 816 to Val was detected in five of seven systemic mastocytosis infiltrates and four of six CMML infiltrates. By contrast, no c-kit mutation Asp 816 to Val was found in bone marrow infiltrates in patients with CMML without associated systemic mastocytosis (n = 20).

Conclusion: These data support a monoclonal evolution of systemic mastocytosis and concurrent CMML in the patient studied.

Figure 1

Systemic mastocytosis with associated chronic myelomonocytic leukaemia (CMML). (A) Focal compact aggregate of medium sized, slightly pleomorphic, spindle shaped cells; note the pronounced collagen fibrosis associated with the infiltrate. Tumour cells express tryptase strongly and thus can be clearly identified as mast cells. (B) Extremely hypercellular bone marrow with diffuse involvement by CMML. Tryptase staining only of loosely scattered mast cells. Note that focal mast cell aggregates are absent. (C) Overview of the bone marrow trephine with tryptase stained mast cell infiltrates (arrows). (D) Bone marrow trephine after microdissection of three mast cell infiltrates (arrows). Tryptase was detected using the AA1 antibody.

Figure 2

Molecular biological detection of of c-kit Asp 816 to Val point mutation. (A) Adenine/thymidine substitution at nucleotide (nt) 7176 of c-kit DNA (nt 2468 of c-kit mRNA) causing asparagine/valine substitution at residue 816 of the c-kit protein and creating a new restriction site for endonuclease HinfI. Note the pre-existing HinfI site at nt 7187. G!ANTC, HinfI recognition site; !, cutting position of the restriction endonuclease HinfI. (B) Electrographs obtained by direct sequencing of semi-nested PCR products. Sites of the point mutation are indicated by arrows. Note that the reverse PCR primer has been used for DyeTerminator cycle sequencing: complementary sequences are indicated on top of each electrograph; 3′ end, left; 5′ end, right. Nucleotide positions are indicated on top. The upper panel corresponds to fig 3 ▶, lane 2: a single peak indicates the mutated allele. The middle panel corresponds to fig 3 ▶, lane 3: a double peak indicates the heterozygous genotype. The lower panel corresponds to fig 3 ▶, lane 4: a single peak indicates the wild-type allele. Mut; mutation; loh, loss of heterozygosity; wt, wild-type.

Figure 3

Molecular biological detection of of c-kit Asp 816 to Val point mutation. Semi-nested PCR products from control DNA (lane 1) and three different microdissected mast cell infiltrates (lanes 2–4). In lane 1, the undigested full length PCR product is shown. Lanes 2–4 contain HinfI digested PCR products (table 2 ▶). In lane 2 only the mutated allele is present, most probably indicating loss of heterozygosity (LOH). Signals from both the wild-type and mutated allele are present in lane 3 (heterozygous genotype). Lane 4 contains the wild-type sequence (homozygous genotype). In lane 5 only water was amplified as a negative control. M, HaeIII digested φX DNA.