Original articles
Trisomy 4 Leading to Duplication of a Mutated KIT Allele in Acute Myeloid Leukemia with Mast Cell Involvement

https://doi.org/10.1016/S0165-4608(99)00221-6Get rights and content

Abstract

A G→T transversion at nucleotide 2467 of the c-KIT gene leading to Asp816→Tyr (D816Y) substitution in the phosphotransferase domain has been previously identified in a patient with rapidly progressing AML-M2 and mast cell involvement; the patient's blasts had a 47,XY,+4,t(8;21)(q22;q22) karyotype. Herein we confirm the simultaneous presence of both major chromosomal changes by multicolor fluorescence in situ hybridization (FISH) on interphase CD34+ mononuclear cells. By setting up culture leukemic blasts, spontaneous differentiation of adherent cells with mast-cell like features was proved by histochemical and immunoenzymatic analyses. Fluorescence in situ hybridization evidence of trisomy 4 confirmed the origin of differentiated cells from the leukemic blasts. Semiquantitative polymerase chain reaction (PCR) and phosphoimage densitometry of wild-type and mutated KIT alleles on bone marrow blasts made it possible to demonstrate that chromosome 4 trisomy led to a double dosage of the mutated KIT allele. This finding, and that of trisomy 7 and MET mutation in hereditary renal carcinoma represent the only cases of human tumors in which an increased number of chromosomes carrying an oncogene activated by point mutation have been detected.

Introduction

Aberrations in chromosome numbers are widespread in tumors; however, unlike the structural rearrangements that have often led to the identification of causal genes and then been used as specific diagnostic markers, their pathogenetic significance is in most cases elusive. Some numerical changes recur in specific neoplasms, in which they may represent the sole anomaly: in hematological malignancies this is the case of trisomy 8 in myeloid disorders [1], trisomy 4 in M2 or M4 acute myeloid leukemia [2], trisomy 9 in myeloproliferative disorders, trisomy 11 in stem cell leukemia and in acute myeloid leukemia 3, 4, trisomy 21 in acute lymphoblastic leukemia [5], trisomy 12 in chronic lymphocytic leukemia [6], and the recently reported trisomy 6 as a primary karyotypic aberration in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) 7, 8. Despite these and other significant associations, the gain of a specific chromosome has mainly suggested the presence of a dominantly acting growth-regulatory gene without providing any evidence of a link between its dosage and the generation of trisomy by chromosome nondisjunction [9].

The first studies of trisomy as an oncogenetic mechanism made use of a mouse model consisting of chemically-induced skin papillomas and squamous cell carcinomas. Recurrent trisomy 7 in these tumors led to molecular studies that allowed the cytogenetic finding to be interpreted as a major mechanism by means of which a mutated HA-RAS-1 allele is over-represented [10].

Germline and somatic mutations of the MET protooncogene have recently been identified in papillary renal carcinomas, which are characterized by trisomy of chromosomes 7, 16, and 17 [9]. Nonrandom duplication of the chromosome 7 bearing the mutated MET has been demonstrated in independent tumors from patients with hereditary renal carcinomas, and this event has been implicated in tumorigenesis [11].

We have previously reported a novel somatic mutation (D816Y) of the KIT protooncogene in blasts from a patient with AML characterized by mast cell differentiation and rapid disease progression [12]. It has been demonstrated that the corresponding D814Y mutation in the murine gene results in ligand-independent activation of receptors by promoting dimerization of the c-KIT kinase 13, 14. Interestingly, a t(8;21) and chromosome 4 trisomy were detected by conventional cytogenetics as being the only deviations from the normal karyotype in the patient's bone marrow blasts [15]. We report here interphase fluorescence in situ hybridization (FISH) results confirming trisomy 4 in immunosorted CD34+ bone marrow blasts and adherent cells from bone marrow cultures. Phosphoimage densitometry directly quantitated a 2:1 imbalance between the mutated and the normal KIT allele demonstrating the nonrandom duplication of chromosome 4 bearing the mutated KIT allele in leukemic blasts.

Section snippets

Cell Culture

Bone marrow mononuclear cells (BMMNC) were obtained from the patient at diagnosis, and then cultured (5 × 105/mL) at 37°C and 5% CO2 without cytokines in RPMI/10% fetal calf serum (FCS) (Life Technologies, Inc., Gaithersburg, MD, USA) on coverslips in six-well microculture plates for up to 8 weeks. The coverslips were washed with phosphate buffered saline (PBS), fixed in 10% paraformaldehyde for 24 hours, and then either stained with Alcian blue (BDH, Poole, England) for 5 minutes or processed

Morphology and Interphase FISH of Leukemic Blasts

The clinical and immunophenotypic aspects of the AML-M2 patient harboring a novel c-KIT mutation affecting spontaneous mast cell differentiation have been previously reported [12]. The mutation is a G→T transversion at nt 2 467 (cDNA), leading to an aspartic-to-tyrosine change at codon 816 of the KIT receptor. A few blasts with mast cell-specific metachromatic granules and a typical morphological pattern were apparent in the bone marrow biopsy taken at diagnosis (Fig. 1a). Expansion of tumor

Discussion

The detection of D816Y KIT mutation in blasts carrying trisomy 4 from a patient with M2 myeloid acute leukemia and mast cell involvement raised the issue of the putative correlation between the mutated KIT allele and chromosome 4 gain.

The c-KIT gene is located on chromosome 4q12 [19], and is expressed in about half of the CD34+ precursors and mast cells [20]. Activating mutations of c-KIT are found in mast cell disease 21, 22, 23, 24, but also in AML affecting mast cells 12, 15, 25, 26. In the

Acknowledgements

This investigation was supported by a grant from the Italian Association for Cancer Research (AIRC) and by MURST 1998 (to L. L.). We thank Dr. R. DiLernia (Milan) for providing access to the Axiovert 25 CFL used for acquiring CCD images of cell cultures, and Dr. S. Duga (Milan) for access to the GS-700 Imaging Densitometer.

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