CYTOGENETIC ABERRATIONS IN THE CD38 POSITIVE FRACTION OF CD38 NEGATIVE CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PATIENTS: A MARKER OF AGGRESSIVENESS?

Background. Chronic lymphocytic leukemia (CLL) is a disease with a high variability in clinical presentation and outcome. Even though many patients live for long periods with the disease, some cases may show a progression to a more aggressive leukemia which may be characterized by the acquisition of new genetic abnormalities either by clonal evolution or by an expansion of a clone with high risk aberrations. Prognostic parameters that correlate with worse clinical outcome include stage, the expression of CD38 and/or ZAP70, the unmutated configuration of the variable region of the immunoglobulin heavy chain gene (IGHV), the presence of specific chromosome aberrations and/or molecular mutations affecting TP53, NOTCH1, SF3B1 and BIRC3. However in patients with favourable prognostic features the biologic and molecular events leading to disease progression and the occurrence of new molecular cytogenetic lesions are largely unknown. Aims. We studied the biologic and clinical significance of minor cytogenetically abnormal clones in the CD38 positive fraction of untreated CLL patients with good prognostic parameters (CD38 negativity and normal karyotype or del(13)(q14) as single aberration). Methods. Twenty eight consecutive CD38 negative (Cd38 positive cells < 5%) CLL patients with normal karyotype or del(13)(q14) were evaluated in this study. CD38 positive and CD38 negative CLL cells were isolated by sequential immunomagnetic sorting (Dynabeads) following depletion of CD3, CD16, CD14 positive cells (purity > 99%). CD38 positive and negative cells were then analyzed by (i) FISH analysis using commercially available probes for the regions most frequently involved in CLL patients (13q14, 12q13, 11q22/ATM, 17p13/TP53, 14q32) and by (ii) micro-RNA expression. Data were then correlated with clinical parameters. Results. In 16/28 CD38 negative CLL patients, FISH analysis demonstrated the presence of minor (15-34% of the cells) cytogenetically abnormal clones within the CD38 positive fraction: 11q deletion in 7 cases, 17p deletion in 6, trisomy 12 in 5, 14q32 rearrangements in 1 case. According to FISH results patients were therefore classified as CLL with and without subclones in the CD38 positive fraction. By micro-RNA analysis we found that patients with subclones had a distinctive profile, when compared to patients without subclones, characterized by a downregulation of microRNA- 125a-5p (a tumor suppressor in various malignancies) both in the CD38 negative and positive populations. With a median follow-up of 36 months, patients with subclones showed a higher need of treatment ( 9/16 cases vs 1/11 in patients with and without subclones respectively, p=0.0159). Among CLL patients with subclones, 2 cases showed the emergence of a major clone in the peripheral blood sample (45-67% of the cells) with the same genetic lesions previously observed in the CD38 positive subpopulations.ConclusionsOur data showed that genetic instability within the CD38 positive fraction of CLL patients with favourable prognostic features (CD38 negativity and normal karyotype or del13q14) may favour the growth of small clones with poor prognosis cytogenetic aberrations which may be associated with microRNA-125a-5p disregulation, clonal expansion and, in some cases, disease progression.