SNP Array Analysis in Refractory Anemia with Ringed Sideroblast Allows for Detection of a High Frequency of Previously Cryptic Chromosomal Defects with Possible Clinical Significance.

Among WHO low-risk categories of MDS, refractory anemia with ringed sideroblasts (RARS) can be more precisely identified based on typical findings in the iron stain preparations of marrow aspirates. Clonal hematopoiesis and chromosomal (chr) abnormalities are features that reveal a clear pathogenetic relationship to MDS. However, despite characteristic morphology, chr defects in RARS are heterogeneous, RARS shows considerable variability in the clinical course even for patients with identical cytogenetic defects. We analyzed the results of metaphase cytogenetics (MC) in a cohort of patients classified as RARS, RCMD-RS, or more advanced forms of MDS associated with ringed sideroblasts. Similar proportions of defects (around 50%) were found in RARS patients (n=68) as in all other remaining MDS forms studied (n=288). The most commonly occurring defects associated with ringed sideroblasts, compared to other forms of MDS, included those of chr 5 (14% vs 31%), 7 (11% vs 14%) and 8 (17% vs 20%), respectively. However, due to the low resolution of MC and its dependence on cell growth in vitro, this test is often negative or non-informative in MDS. High-density SNP arrays (SNP-A) allow for a precise identification of unbalanced genomic lesions. We hypothesize that cryptic chr aberrations do exist in most, if not all, patients with RARS. Their detection may help to improve prognostication, distinguish distinct subgroups of patients and point towards unifying pathogenic defects. Consequently, we have applied 250K SNP-A to analyze the marrow (n=14) or blood (n=15) in RARS. Pathologic lesions were defined based on a study of 36 normal marrow specimens (O’Keefe at al ASH 2006). By traditional MC, a defective karyotype was present in 18 of 29 patients (34%). Monosomy 7/7q- was found in 5 patients; deletions involving chr 5 and 11 in 2 cases; and 5 patients showed a complex karyotypes. However, when SNP-A was applied as a karyotyping tool (copy number and LOH analysis), we were able to detect lesions in 65% of patients (vs 34% by cytogenetics, p<.01). All but 2 aberrations found by MC were confirmed. New, previously cryptic lesions were identified, including losses of a portion of chr 8 (n=4) and deletions (n=2) as well as gains (n=2) within chr 17q. We have also detected segmental uniparental disomy (UPD) in 2 patients. This type of lesion cannot be detected using MC and provides an additional mechanism that can lead to LOH. When comparing MC to SNP-A, 1 lesion was found in 7/29 patients vs 12/29; 2 lesions in 0/29 vs 5/29; and ≥3 lesions in 3/29 vs 2/29 patients, respectively. Most remarkably, we have identified shared lesions, including an invariant deletion involving 4q31-21 (n=3, with 2/3 diagnosed as RARSt), loss of 8q24.23 (n=2); and a UPD involving 1p21.3-22.2 (n=2). These lesions contain important genes, including IL-15 and GAB1 (4p) or DR1 and TGFBR3 (1p). Our results indicate that if more precise techniques are applied, chr abnormalities can be detected in a higher proportion of patients with RARS. Analysis of clinical outcomes associated with defects identified by A-SNP is currently ongoing in our laboratory.