Detecting Clonal Evolution in CLL Patients Treated with Targeted Therapies Versus Chemotherapy Utilizing Cytogenetic Microarray Analysis
Authors: Martin James M. , Senchak Jordan , Ruth Karen , Testa Joseph R. , Al-Saleem Essel D. , Pei Jianming , Barta Stefan Klaus , Fisher Richard I. , Khan Nadia 
Published: October 2019

Abstract
Background
Chronic lymphocytic leukemia (CLL) is a common B-cell lymphoproliferative neoplasm. Although not required for the diagnosis of CLL, important prognostic information is obtained with fluorescence in-situ hybridization (FISH) testing for chromosomal aberrations including del(11q), trisomy 12, del(13q), and del(17p). Abnormalities involving TP53, the ATM gene, and cases constituting complex cytogenetics (3 or more abnormalities), among others, have been shown to impact patient survival and response to traditional chemoimmunotherapy (CIT). Cytogenetic microarray analysis (CMA) is a laboratory technique used to detect unbalanced chromosomal abnormalities. CMA allows for comparison of hundreds of discrete loci within a chromosome and has been validated in CLL, with greater sensitivity in detecting genomic alterations compared to FISH. The acquisition of new genomic alterations is termed "clonal evolution" and has been shown to have a detrimental effect on survival in patients with CLL (Shanafelt TD, et al. J Clin Oncol. 2006). CIT results in a higher proportion of complete responses (CR) and eradication of CLL clones compared to targeted therapies. While targeted therapies may successfully suppress CLL clones, a CR is rare with single-agent treatment (Byrd JC, et al. Blood. 2015). CMA may be a valuable tool in monitoring clonal evolution throughout a patient's disease course and may have a role in evaluation of patients on suppressive therapies due to the relatively high risk of clonal emergence.
Methods
This was a single-center retrospective study. Patients with pathologically-confirmed CLL were analyzed utilizing data obtained from the electronic medical record. Data was extracted from the CMA reports and clinic documentation. To be included in the study, at least two serial CMA evaluations were required, separated by at least four weeks. In the interval between CMA results, subsequent systemic treatment was recorded, if any. Treatments were categorized as ibrutinib/idelalisib ("I"), any cytotoxic chemotherapy ("C"), and no treatment/observation ("N"). Change in the number of abnormalities detected by CMA was calculated for each interval and evaluated as both continuous and categorical (increase/no change/decrease). Patients with over three abnormalities were deemed to have complex disease.
Genomic DNA of bone marrow or peripheral blood was extracted using Qiagen's Gentra Puregene kit. Chromosome microarray assay was performed using ThermoFisher Affymetrix CytoScan HD according to the manufacturer's protocol. Copy number and allele analysis was performed using Affymetrix Chromosome Analysis Suite using the default settings.
Results
There were 34 patients who met inclusion criteria, each with 2-4 CMA results, for a total of 81 CMA results. Forty-seven treatment intervals were analyzed. The median age was 61 years, and 64% of patients were male. Based on the first CMA result, the median number of detected abnormalities was 2 and mean was 2.6; 13 patients (38%) had complex disease. Complexity was not associated with a clinically significant change in the number of chromosomal abnormalities regardless of what treatment was received, if any (p=0.14).
Overall, 18% of patients (6/34) had a decrease in the number of abnormalities detected by CMA, while 21% (7/34) had an increase. Analyzing the data by treatment group, the mean change in number of abnormalities was -0.5, -0.6 and +0.3, in groups I, C and N, respectively (p=0.26) (Figure 1). Among the patients in group I, 23% (3/13) had a decrease in the number of abnormalities, while 23% demonstrated an increase. For those in group C, 25% (2/8) and 25% experienced increase and decrease, respectively. For patients in group N, 14% (3/21) had an increase and 0% had a decrease in CMA abnormalities. Among the two patients who experienced Richter's transformation, the complexity of their chromosomal abnormalities increased over time and preceded clinical emergence of transformation.
Discussion
CMA can sensitively identify the emergence or clearance of CLL clones and help detect clonal evolution throughout periods of treatment or observation. Though a relatively small number of patients had serial CMA data for evaluation, these data suggest that CMA should be prospectively analyzed in patients with CLL and may be used as a dynamic clinical tool to potentially modify treatments in patients with sub-clinical evidence of treatment failure.
Figure 1. Figure 1.

Disclosures
Barta: Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding.