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Genome complexity in CLL: karyotype versus microarrays
not differ in the proportion of patients classified into each risk category. Notwithstanding, it should be pointed out that only moderate agreement was observed between them. Discordances in the risk assigned to nearly one third of the patients were found, including around 3% of patients classified in either high or low risk groups depending on the methodology employed for their study. We have demon- strated that most of these discordances are the consequence of known limitations intrinsic to each technique. In this regard, the clonal architecture in the sample could mask some alterations by GM, if present in low percentages below their limited sensitivity (~20%), while the CBA result would rely on the in vitro proliferative capacity of the altered clones.29,30 In addition, balanced abnormalities are only detectable by CBA and, on the contrary to expecta- tions, our FISH painting studies confirmed that not all the complex rearrangements described in the karyotype ulti- mately imply gain or loss of genomic material. On the con- trary, some abnormalities recorded as a single monosomy or unbalanced translocation in the karyotype turned out to be multiple CNA or even chromothripsis events when assessed by GM. Thus, our results suggested that discrep- ancies were not predictable by the type of abnormalities detected by any of the methods. Conversely, we discarded a global underestimation of the genomic complexity associ- ated with the application of the recommended filtering cri- teria for non-classical CLL CNA by GM.24 Small abnormal- ities (<5 Mb) were equally found by GM among concordant and discordant patients, and greater agreement in the num- ber of abnormalities could not be achieved when smaller CNA were also considered. Thus, we have confirmed that the present recommendations for GM analysis are robust for complexity assessment.24 The observed differences did not represent a poorer performance for CBA or GM in patients risk stratification. In both cases, the established risk groups showed significant differences in terms of TTFT, which were independent of TP53 and IGHV mutational status. Concerning OS, only high complex groups displayed a dismal evolution. In addition, the heterogeneity regarding the GM platforms employed could be a limitation of this study. However, all GM results were reviewed and uni- formly interpreted using the same criteria to filter CNA and similar findings were obtained among different GM compa- nies.
Regarding CBA data, previous publications have inves- tigated whether specific cytogenetic patterns not identifi- able by GM (presence of balanced or unbalanced rearrangements) may correlate with dismal outcome. Initial studies suggested that carrying chromosomal translocations was associated with poorer clinical course.31 More recently, this negative impact has been attributed to the presence of unbalanced rearrangements and its associ- ation with CK.2,32 Indeed, Rigolin et al. proved that CK car- rying unbalanced rearrangements constituted a very poor risk subset with particular features such as an increased proportion of TP53 aberrations and a lower frequency of 11q deletions. The presence of these aberrations has also been associated with a deregulated expression of genes involved in cell cycle control and DNA damage response.14 Visentin et al. showed that the combination of the pres- ence of CK and/or unbalanced rearrangements by CBA and IGHV mutational status improved their risk stratifica- tion.15 In our cohort, we observed a shorter TTFT for those patients with unbalanced rearrangements but the poor outcome was not confirmed within CK group.
Unexpectedly, GM were unable to detect CNA related to all the apparently unbalanced rearrangements. Indeed, the eight patients with high-CK classified as low-GC by GM carried this type of abnormality and showed a dismal evo- lution. On the other hand, our GM analyses identified patients with patterns of chromothripsis who showed a short TTFT. As previously reported, there was a high asso- ciation between chromothripsis and CK or TP53 aberra- tions.19,33,34 Our study is based in a retrospective cohort highly enriched in patients carrying CK, which was neces- sary to extensively compare both genetic methodologies in the detection of these prognostically relevant highly complex cases. Therefore, as it is not representative of a real-life CLL cohort, it hinders the development of more accurate genetic prognostic scores. Additionally, potential confounding effects of different therapeutic agents could be attributed to the retrospective and multicenter nature of the cohort enriched in treated patients. These could underlie the lack of statistical significance of genome com- plexity in the analyses for OS.
To date, most of the survival analyses of genomic com- plexity included in clinical trials have been reported using CBA data. Even though the prognostic/predictive value of CK for TTFT and progression-free survival in patients treated with chemoimmunotherapy has been extensively demonstrated,2-5,10 its clinical relevance in patients receiving the new treatment modalities has not been fully estab- lished. Initial data from clinical trials with novel agents, mainly developed in older relapsed/refractory and/or in high risk patients (TP53 del/mut, U-IGHV) suggested an adverse significance of CK.6-8,35 In contrast, a number of recent studies including extended follow-ups of older trials, pooled analyses or new drug combinations have not con- firmed its adverse significance.36-43 However, most of these studies have analyzed CK impact taking into account patients with ≥3 aberrations but not those with high com- plexity (≥5 aberrations), compared a low number of patients and showed relatively short follow-ups.44 Thus, additional analyses are needed to clarify the prognostic/pre- dictive impact of genomic complexity.
One particular finding of this study is that, even though overall concordance between FISH and GM is high (90%), GM do not detect around 20% of cases with TP53 deletion due to their low sensitivity.23,29 The presence of 17p13 dele- tions and/or mutations in TP53 predicts the poorest out- come and its assessment is currently mandatory in CLL study. Our results confirm that FISH should be maintained for the study of CLL patients complemented with one genome-wide technique to assess genome complexity for risk stratification. The choice between CBA and GM will depend on each laboratory, which should take into account the methods and equipment availabilities, personnel expertise and the economic costs, among others.
In conclusion, we have confirmed that both CBA and GM are valuable tools to assess the prognosis of CLL patients based on genomic complexity. Nevertheless, a con- siderable proportion of cases are discordantly classified depending on the technique employed. Consequently, pre- vious findings generated from CBA, currently the gold stan- dard for cytogenetic assessment, are not directly applicable to GM or other promising cytogenomic methodologies such as optical genome mapping. Additional validation studies are needed to establish the prognostic value of genomic complexity by GM in future prospective studies and clinical trials.
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