Sex Chromosome Loss May Represent a Disease-Associated Clonal Population in Chronic Lymphocytic Leukemia (CLL),

Abstract

3534 Sex chromosome loss is observed in the healthy population and increases with advancing age. It is also observed in hematological malignancies with variable clinical value. Its significance as a disease marker in CLL is not clearly defined. We undertook this study to evaluate whether Y or X loss is an age- and/or a CLL-associated abnormality, since it has not yet been reported. Out of 198 patients with stage B/C CLL analyzed by karyotype (K), 6 (3%) exhibited a loss of sex chromosome (Sutton et al, blood 2011). We also identified 14/745 (2%) additional patients in our CLL-database (2002–11). All patients had a Matutes score ≥ 4. K was performed on peripheral blood using TPA until 2005, and then CPG + IL2. FISH analyses were carried out to detect trisomy 12 and 11q22 (ATM), 13q14, 17p13 (TP53), X/Y deletions. IGVH gene mutational status was analyzed by PCR with a sequence homology cutoff of 98%. B- and T-cells were sorted by flow cytometry from peripheral blood of 5 patients (purity ≥ 94%) using CD19+ and CD3+ respectively. Polynuclear and mononuclear cells were enriched by Ficoll (purity ≥ 99%) for 4 patients. There were 13 males and 7 females (n =20). At the time of the K, there were 11 Binet stage A and 9 stage B, and median age was 64 (36–78). Regarding IGVH status, 9/12 (75%) cases were mutated. We observed 13 -Y and 7 -X, chromosome loss being the sole abnormality in 10 (50%) cases. The percentage of cells with -X or -Y ranged between 3 to 20/20 mitoses. K was complex (≥ 3 abnormalities) in 4 cases. When associated with another chromosomal abnormality, -X or -Y was the primary change in 2 cases, in the same clone in 7 cases, and as a sub-clone change in one case. FISH identified 16/20 (80%) del13q, 4/20 (20%) tri12, 3/20 (15%) delATM, 2/20 (10%) delTP53. Among del13q, there were 8 monoallelic, 7 concomitant mono- and biallelic, and 1 biallelic deletion. Sex chromosome loss was confirmed by interphase FISH in 14 patients, with percentage of loss between 5 to 84% (median: 68%). Co-hybridization with two probes including the X/Y probe was performed for 13 patients. The two analyzed abnormalities were present in independent clones in 4 cases, in the same cells in 2 cases; -X or -Y appeared as the primary change in 4 cases, and as a sub-clone change in 3 cases. The analyses of polynuclear and mononuclear populations in 4 patients showed a maximum of 2% of polynuclear cells with X or Y loss, whereas the mononuclear cells exhibited a significant higher loss frequency (range 6–87%, p =.03). The analyses of B- and T-cells in 5 samples showed a significant increase in losses frequency in B-cells (range 88–96%) compared to T-cells (2%, 2%, 3%, 4%, 6%) (p =.008). The incidence of -X in peripheral blood lymphocytes is about 3% of the cells in healthy women aged 16–50, rising to 5% in women aged over 85 years. The incidence of Y loss in men is distinctly lower, with a frequency less than 1% of the cells before 85 years (Wodja et al, J Appl Genet, 2003). The median of cells with sex chromosome loss in our series is 68%, with only 2 patients with low percentage: a 66-year old female (5%) and a 68-year old male (6%). However these percentages are higher than those observed in healthy people. Moreover, we never observed the loss of a sex chromosome in more than 2% in polynuclear cells of any of the patients, indicating that sex chromosome loss in CLL is associated with the tumor cells. It is not clear whether the low proportion of loss of sex chromosome in CD3+ cells that exceed in one case only slightly our laboratory threshold of 5% is due to contamination. Sex chromosome loss may occur during K evolution, as well as the acquisition of additional abnormalities may occur in cells with primary sex chromosome loss. Even if loss appears through an age-related effect in elderly cells, our data support an oncogenic property of this abnormality. One patient with –Y as the sole aberration in CLL, has developed a myelodysplastic syndrome upon treatment, with complex karyotype without -Y, suggesting that Y loss had been associated to CLL. In addition, del13q, especially when biallelic, appears to be closely associated to the loss of sex chromosome, as compared to our database (del13q: 16/20 (80%) vs 141/279 (50%), p =.01; biallelic del13q: 8/16 (50%) vs 24/141(17%), p =1×10−5). In conclusion, sex chromosome loss has to be considered as a clonal abnormality in CLL, is significantly associated with biallelic 13q deletion, and may participate to oncogenic transformation. Disclosures: No relevant conflicts of interest to declare.