Chromosome Aberrations In Chronic Lymphocytic Leukemia Research Articles

Normal Fish Cytogenetics and 13q Deletions Unveil Marked Biological and Clinical Heterogeneity In Chronic Lymphocytic Leukemia

Abstract Abstract 2692 Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of chronic lymphocytic leukemia (CLL) patients (pts) [Dohner et al, 2000]. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. Interphase FISH (I-FISH) is a rapid and sensitive technique for analysis of chromosome aberrations in CLL, but the limited number of DNA probes available to screen B-CLL results in a high number of cases presenting normal cytogenetics. Moreover, deletion 13q14 on FISH analysis which is the most common cytogenetic abnormality in CLL is a favourable prognostic biomarker when detected as a sole abnormality, but a higher percentage of 13q- nuclei was found to be associated with significantly shorter time to treatment (P<0.001), [van Dike et al, 2010]. On this line, the primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) within the normal FISH cytogenetics subset on the basis both of ZAP-70, CD38, CD69 expression by flow cytometry and IgVH status; 2) to correlate percentages of 13q- nuclei (<50% or ≥50%) with PFS and OS and finally 3) to confirm I-FISH as an independent prognostic factor. We investigated 400 pts, median age 65 years (range 33–89), 235 males and 165 females. With regard to modified Rai stages, 115 pts had a low stage, 266 an intermediate stage and 19 a high stage. One hundred forty-two pts (35.5%) exhibit a normal karyotype, 133 pts (33.2%) showed an isolated 13q-, 69 pts (17.3%) presented trisomy 12, 30 pts (7.5%) 11q deletion, 20 (5%) 17p deletion and 6 (1.5%) other chromosome abnormalities. Clearly, pts with intermediate/poor cytogenetic abnormalities (trisomy 12, del11q-, del17p-) showed significant shorter PFS and OS (8% vs 33% at 16 years, P<0.0001 and 41% vs 65% at 16 years, P=0.0001) in comparison with normal or del13q- pts. Therefore, in order to better define the apparently homogenous subset of the patients with normal cytogenetics, we analysed ZAP-70, CD38, CD69 and IgVH status within this subgroup. Interestingly, we observed significant longer PFS and OS for ZAP-70 negative pts (60% vs 29% at 10 years, P<0.0001 and 92% vs 23% at 16 years, P=0.0006), for CD38 negative pts (54% vs 22% at 10 years, P=0.0008 and 83% vs 0% at 14 years, P=0.03), for CD69 negative pts (60% vs 23% at 10 years, P=0.0003 and 83% vs 63% at 14 years, P=0.02) and finally also for IgVH mutated cases (64% vs 35% at 10 years, P=0.0002 and 95% vs 45% at 14 years, P=0.04). Moreover, pts with isolated 13q- in <50% of nuclei (60 pts) showed a longer PFS and OS (68% vs 6% at 16 years, P=0.0003 and 92% vs 0% at 18 years, P=0.002; Figure) compared to those with ≥50% of nuclei (72 pts). No significant correlation was found between 13q- percentages and CD38 or ZAP-70 or IgVH status, while it was found with CD69 expression (P=0.01). In multivariate analysis of PFS, FISH cytogenetics was confirmed to be an independent prognostic factor (P=0.002) together with Rai stages (P=0.005), ZAP-70 (P=0.0001), IgVH status (P=0.0002) and CD69 (P=0.007). Therefore, in the clinical practice, ZAP-70, CD38, IgVH status and CD69 are irreplaceable in order to define prognosis and treatment within the large series of CLL pts with normal cytogenetics, probably presenting abnormalities undetectable by FISH. Besides, the percentage of nuclei exhibiting 13q- has to be considered an important predictor of the outcome and the clinical implications of 13q- in CLL seem to appear more complex than originally considerated. In conclusion, a comprehensive scoring prognostic system based on a combination of clinical, genetic, phenotypic parameters and IgVH status improves the separation of prognostic subgroups in CLL already early in the course of the disease. Disclosures: No relevant conflicts of interest to declare.

Cytogenetic characterisation in Chinese patients with chronic lymphocytic leukemia: A prospective, multicenter study on 143 cases analysed with interphase fluorescence in situ hybridisation

Chronic lymphocytic leukemia (CLL) is infrequent in Chinese people. Conventional cytogenetic analysis underestimates the frequency of chromosome aberrations in CLL due to the low rate of spontaneous mitoses. The aim of this study was to prospectively explore the frequency of chromosomal abnormalities in Chinese patients with CLL using interphase fluorescence in situ hybridisation (FISH) and probes for 12 centromere, 13q14, 14q32, 17p13, 11q22 and 6q23 on 143 patients with CLL. Molecular cytogenetic aberrations were found in 104 patients (72.7%) and 40 patients (28.0%) with more than two abnormalities. The most frequent abnormality was del(13q14) (47.6%), followed by trisomy 12 (21.7%), 14q32 translocation (19.6%), del(17p13) (12.6%), del(11q22) (11.9%) and del(6q23) (4.9%), respectively. The percentages of patients with aberrations by FISH were 75.4%, 72.3% and 67.7% for Binet stages A, B and C, respectively. In early stage (Binet A), del(13q14) aberration was more frequent than in Binet B and C (61.5% vs. 31.9% and 41.9%) (P = 0.021). Patients with advanced stage (Binet C) had more frequent del(17p13) aberration than in Binet A and B (32.3% vs. 9.2% and 4.3%) (P = 0.008). It was showed that the frequencies of the chromosomal abnormalities in our study population were similar to the frequencies in Western countries.

Cryopreserved chronic lymphocytic leukemia cells analyzed by multicolor fluorescence in situ hybridization after optimized mitogen stimulation.

We investigated the utility of multicolor in situ fluorescence hybridization (mFISH) on cryopreserved blood cells from 11 chronic lymphocytic leukemia (CLL) patients. The results demonstrate that an individually chosen optimized mitogen combination induces proliferation of neoplastic B-cells after cryopreservation. Abnormal cells were detected in eight samples by mFISH, and, in six samples, the abnormality could be verified by comparative genomic hybridization or interphase FISH. In addition to typical CLL abnormalities, such as del(11q) or +12, several balanced translocations and single-cell abnormalities were found. Thus, mFISH can reveal new prognostically relevant chromosome aberrations in CLL.

Genetic features of B-cell chronic lymphocytic leukemia.

The genetic features of B-cell chronic lymphocytic leukemia (CLL) are currently being reassessed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). Conventional cytogenetic studies by chromosome banding are difficult in CLL mainly because of the low in vitro mitotic activity of the tumor cells, which leads to poor quantity and quality of metaphase spreads. Molecular genetic analyses are limited because candidate genes are known for only a few chromosomal aberrations that are observed in CLL. FISH was found to be a powerful tool for the genetic analysis of CLL as it overcomes both the low mitotic activity of the CLL cells and the lack of suitable candidate genes for analysis. Using FISH, the detection of chromosomal aberrations can be performed at the single cell level in both dividing and non-dividing cells, thus circumventing the need of metaphase preparations from tumor cells. Probes for the detection of trisomies, deletions and translocation breakpoints can be applied to the regions of interest with the growing number of clones available from genome-wide libraries. Using the interphase cytogenetic FISH approach with a disease specific set of probes, chromosome aberrations can be found in more than 80% of CLL cases. The most frequently observed abnormalities are losses of chromosomal material, with deletions in band 13q14 being the most common, followed by deletions in 11q22-q23, deletions in 17p13 and deletions in 6q21. The most common gains of chromosomal material are trisomies 12q, 8q and 3q. Translocation breakpoints, in particular involving the immunoglobulin heavy chain locus at 14q32, which are frequently observed in other types of non-Hodgkin's lymphoma, are rare events in CLL. Genes affected by common chromosome aberrations in CLL appear to be p53 in cases with 17p deletion and ataxia telangiectasia mutated (ATM), which is mutated in a subset of cases with 11q22-q23 aberrations. However, for the other frequently affected genomic regions, the search for candidate genes is ongoing. In parallel, the accurate evaluation of the incidence of chromosome aberrations in CLL by FISH allows the correlation of genetic abnormalities with clinical disease manifestations and outcome. In particular, 17p abnormalities and deletions in 11q22-q23 have already been shown to be among the most important independent prognostic factors identifying subgroups of patients with rapid disease progression and short survival. In addition, deletion 17p has been associated with resistance to treatment with purine analogs. Therefore, genetic abnormalities may allow a risk assessment for individual patients at the time of diagnosis, thus giving the opportunity for a risk-adapted management.