Spectral Karyotyping (SKY) Reveals a New Subset of MDS Patients with Clonal Chromosomal Abnormalities Not Detected by G-Banding Analysis

Abstract 1718

Cytogenetic findings in bone marrow cells of MDS patients are essential for a correct diagnosis and classification of the disease and constitute one of the most important independent prognostic factors. The classical cytogenetic analysis, however, often cannot be fully resolved by G-banding because of the presence of marker chromosomes, rings or unidentified material attached to chromosomes. Spectral karyotyping (SKY) has proven to be an important tool for the interpretation of complex karyotypes or identification of suitable abnormalities in hematological malignancies. By using SKY analysis in combination with G-banding were identified new clonal chromosomal abnormalities “masked” by the limited resolution of classical cytogenetic. As a consequence changes in IPSS score were observed. Bone marrow samples of 46 (forty-six) MDS patients were incubated in RPMI 1640 with 20% fetal calf serum for 72h at 37°C. Chromosome preparations were obtained by using standard procedures and the subsequent cytogenetic analysis and interpretation were made according to ISCN 2009. The patients studied were classified as refractory anemia (RA) and refractory anemia with ringed sideroblast (RARS), with less than 5% blast. Slides for SKY were prepared by using the same fixed chromosome preparations, stored at −20°C, as employed for G-banding analysis. Chromosome labeling was performed with the SKY fluorescent labeling kit (Applied Spectral Imaging, Migdal HaEmek, Israel) according to the manufacturer's protocol. A minimum of twenty metaphases were analyzed using the SkyView 5.5 software (ASI, Carlsbad, CA, USA). In a group of 46 subjects studied, the cytogenetic analysis (G-banding) showed chromosomal aberrations in 13 patients (54.2%) and normal karyotype was observed in 11 subjects (45.8%). The abnormalities observed were dup(1)(q21q32), inv(3)(q21q26), t(3;3)(q21;q26), +4, del(5)(q31), −7, del(7)(q22q36), +8, add(17)(p12), +i(17)(q10), del(20)(q11). The group with normal cytogenetic, SKY analysis revealed “masked” chromosomal abnormalities in 6 patients, being t(7;9)(q36;q34), ins(1;6)(q21;?), t(11;12)(p15;q24.1), ins(3;5)(p21;?), t(8;16)(q23;?) and ins(6;11)(q21;?). Among 13 cases studied with previous chromosomal abnormalities by G-banding analysis, SKY identified additional abnormalities in 8 patients. Some abnormalities found include t(6;9)(q27;q22), t(12;17)(p13;p12) and t(8;11)(p12;q12). For both groups with normal and altered karyotypes, the profile of masked chromosomal abnormalities seen were insertions and translocations involving small segments of chromosomes. In the majority of the cases the frequency of abnormal clones was less than 50%. However, in all patients the abnormalities identified by SKY were classified as clonal. All abnormalities identified were confirmed by FISH, by using a set of probes. SKY analysis has proved to be a promising and reliable method for identification of additional and complex chromosomal abnormalities usually present in a great number of human neoplasias. The contribution for the prognostic information of these new chromosomal abnormalities identified beyond the limited resolution of G-banding in MDS will require a detail analysis of the patients' evolution.

Financial Support: FAPESP (Proc. 07/52462-7)