Comparison Between FISH and Immunostimulatory Oligonucleotide-Induced Metaphase Cytogenetics in Previously Untreated CLL: Impact on Prognosis
Authors: Tedeschi Alessandra , Mura Mariangela , Vismara Eleonora , Soriani Silvia , Ricci Francesca , Roncoroni Elisa , De Canal Gabriella , Nichelatti Michele , Morra Enrica , Montillo Marco 
Published: October 2019

Abstract
Abstract 4587

Introduction
Cytogenetic aberrations are considered the major prognostic indicators for predicting survival in CLL. FISH analysis results in the detection of clonal aberrations in more than 80% of cases and allows to prognostically categorize pts according to a hierarchical model. Karyotyping after stimulation with immunostimulatory CpG-oligonucleotide in combination with IL2 allows to detect aberrations in a higher proportion of cases when compared to chromosome banding analysis (CBA) after not stimulated cell cultures. It is not clear whether aberrations not detected by standard FISH have any impact on prognosis and disease progression. Few studies compared informations provided by CBA with interphase FISH and most of these series considered biological features assessed during disease course.

Material and Methods
In this study we analyzed 136 consecutive pts (median age 67y, range 40–88) in which biological features were assessed before any treatment. After giving informed consent pts provided blood specimens for FISH analysis and CBA with DSP30/IL2 culture. FISH was performed on interphase nuclei using probes for the detection of trysomy 12, deletions of 6q23, 11q22, 13q14 and 17p13. FISH for rearrangements of region 14q32 was also done. FISH and CBA results were compared and thereafter they were correlated with age, sex, Binet stage, presence of adenopathies, lymphocyte level, B2m, IgVH status, CD38, ZAP70 expression and time to first treatment (TFT).

Results
At least one abnormality was detected by FISH in 95 pts (70 %). Table 1 reports pts stratification according to the hierarchical risk model and the incidence of other abnormalities.
By CBA a normal karyotype was found in 81 pts (60%), in only 36 (45%) of them FISH analysis did not show abnormalities. However in 39 cases (48%) FISH analysis detected abnormalities that allowed to categorize pts according to hierarchical risk model as follows: del(17p) n=2, del(11q) n=1, del(13q) n=36. Further 6 pts showed an involvement of 14q as the sole abnormality. An abnormal karyotype was detected in 55 cases (40%). In 39 cases CBA allowed to stratify pts according to FISH hierarchical model. Karyotype was not informative regarding: del(17p) n=1, del(11q) n=1 and del(13q) n=6.
In 32 cases CBA revealed additional chromosome abnormalities that allowed to detect a complex karyotype in 9 patients; in the remaining cases balanced or unbalanced translocations were more frequently observed than numerical abnormalities (16 vs 7).
No differences in terms of age, lymphocytes level, B2m, CD38 were found among pts according to FISH aberrations. Even if not statistically significant unmutated IgVH and ZAP70 were more represented in del(11q) pts. However del(11q) correlated with Binet B stage (P=.022) and presence of adenopathies (P=.002). Furthermore pts with del(11q) showed a shorter TFT.
CBA revealed that complex karyotypes were more represented in the high risk group carrying del(17p). Complex karyotypes or karyotypes showing gains or losses of chromosomes significantly correlated with unmutated IgVH (P=.027;.0023;.0048 respectively). No correlations were found when analyzing biological features of karyotypes showing unbalanced or balanced translocations. As regards clinical characteristics chromosomal losses were more frequent in elderly patients (P=.028) and conferred a worse prognosis as TFT was shorter (P=.046).

Conclusion
Our data indicate that DSP30/IL-2 successfully stimulated for metaphase generation allowing to obtain a karyotype in 100% of pts. The low incidence of aberrations detected by CBA (40%) may be related to pts characteristics as in our series only untreated pts have been considered. CBA allowed to stratify pts according to the hierarchical model in only 55% of cases being the most common discrepancy related to 13q deletion. However offered important informations in addition to FISH identifying additional abnormalities in 23% of cases. A worst prognosis in terms of TFT was observed in pts showing chromosomes losses. We suggest that as FISH underestimate the complexity of chromosomal aberrations CBA should be used to complement FISH. Longer follow-up and larger numbers of pts are needed to clarify the prognostic impact of CBA.

Disclosures:
No relevant conflicts of interest to declare.