Spectral Karyotyping (SKY) ANALYSIS APPLIED to CHRONIC LYMPHOCYTIC LEUKEMIA CELLS Immunostimulated with the COMBINATION DSP30/IL-2.
Authors: Oliveira Fabio Morato , Matos Daniel Mazza , Figueiredo-Pontes Lorena Lobo , Simoes Belinda , Rego Eduardo M. , Falcao Roberto P 
Published: October 2019

Abstract
Abstract 4692
Cytogenetic abnormalities play an important role as prognostic factors in CLL. The immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for chromosomal banding. This technique allows a more comprehensive chromosome analysis compared to FISH. On the other hand, spectral karyotyping (SKY) analysis, a recent molecular cytogenetic tool for the screening of the entire genome, has been shown to provide additional chromosome information. By using a combination of molecular cytogenetics strategies, the goal of this investigation was to use the SKY to identify masked chromosomal abnormalities in CLL cells stimulated by the combination of DSP30 and IL-2. In addition we compared the cytogenetic profile obtained (DSP30/IL-2) with FISH analysis from unstimulated cells and ZAP70 expression for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. One extra set of cell culture without any stimulant agent for iFISH analysis was performed for each patient. The iFISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction, in all patients. The ZAP70 expression was determined by flow cytometry analysis and the cut off value was 20%. In a group of 35 subjects studied, the cytogenetic analysis with DSP30/IL-2 showed chromosomal aberrations in 27. The following abnormalities were observed: +4, +5, +8(x2), +11, +12, +15(x2), -17, +18, +19, +21, del(6)(q24), del(11)(q13∼q23), del(12)(p13), del(13)(q31), del(14)(q24), del(17)(p13), t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Eight patients exhibited normal karyotype. The SKY analysis confirmed the abnormalities previously seen by G-banding (DSP30/IL-2), however, did not identify any new abnormality in subjects with normal karyotype. The iFISH analysis agreed with the cytogenetic profile obtained with DSP30/IL-2. The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL. Cytogenetic aberrations detected by G-banding in addition to FISH were heterogeneous. The limited panel used for iFISH analysis may contribute to underestimate the prognostic value, since others abnormalities may be present in patient's karyotype. In conclusion, SKY analysis did not reveal any masked abnormality beyond those showed by G-banding resolution. These results indicate that G-banding analysis (DSP30/IL-2) can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis.

Disclosures:
No relevant conflicts of interest to declare.