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Banding cytogenetics is still the gold standard in many fields of leukemia diagnostics. However, in chronic lymphocytic leukemia (CLL), GTG-banding results are hampered by a low mitotic rate of the corresponding malignant lymphatic cells. Thus, interphase fluorescence in situ hybridization (iFISH) for the detection of specific cytogenetic aberrations is done nowadays as a supplement to or even instead of banding cytogenetics in many diagnostic laboratories. These iFISH studies can be performed on native blood or bone marrow smears or in nuclei after cultivation and stimulation by a suitable mitogen. As there are only few comparative studies with partially conflicting results for the detection rates of aberrations in cultivated and native cells, this question was studied in 38 CLL cases with known aberrations in 11q22.2, 11q22.3, 12, 13q14.3, 14q32.33, 17p13.1, or 18q21.32. The obtained results implicate that iFISH directly applied on smears is in general less efficient for the detection of CLL-specific genetic abnormalities than for cultivated cells. This also shows that applied cell culture conditions are well suited for malignant CLL cells. Thus, to detect malignant aberrant cells in CLL, cell cultivation and cytogenetic workup should be performed and the obtained material should be subjected to banding cytogenetics and iFISH.

Deletions within chromosome 11q22-23, are considered among the most common chromosomal aberrations in chronic lymphocytic leukemia (CLL), and are associated with a poor outcome. In addition to the ataxia telangiectasia mutated (ATM) gene, the baculoviral IAP repeat-containing 3 (BIRC3) gene is also located in the region. BIRC3 encodes a negative regulator of the non-canonical nuclear factor.-light-chain-enhancer of activated B cells (NF-kappa B) protein. Disruption of BIRC3 is known to be restricted to CLL fludarabine-refractory patients. The aim of the present study was to determine the frequency of copy number changes of BIRC3 and to assess its association with two known predictors of negative CLL outcome, ATM and tumor protein 53 (TP53) gene deletions. To evaluate the specificity of BIRC3 alterations to CLL, BIRC3 copy numbers were assessed in 117 CLL patients in addition to 45 B-cell acute lymphocytic leukemia (B-ALL) patients. A commercially available multiplex ligation dependent probe amplification kit, which includes four probes for the detection of TP53 and four probes for ATM gene region, was applied. Interphase-directed fluorescence in situ hybridization was used to apply commercially available probes for BIRC3, ATM and TP53. High resolution array-comparative genomic hybridization was conducted in selected cases. Genetic abnormalities of BIRC3 were detected in 23/117 (similar to 20%) of CLL and 2/45 (similar to 4%) of B-ALL cases. Overall, 20 patients with CLL and 1 with B-ALL possessed a BIRC3 deletion, whilst 3 patients with CLL and 1 with B-ALL harbored a BIRC3 duplication. All patients with an ATM deletion also carried a BIRC3 deletion. Only 2 CLL cases possessed deletions in BIRC3, ATM and TP53 simultaneously. Evidently, the deletion or duplication of BIRC3 may be observed rarely in B-ALL patients. BIRC3 duplication may occur in CLL patients, for which the prognosis requires additional studies in the future. The likelihood that TP53 deletions occur simultaneously with BIRC3 and/or ATM aberrations is low. However, as ATM deletions may, but not always, associate with BIRC3 deletions, each region should be considered in the future diagnostics of CLL in order to aid treatment decisions, notably whether to treat with or without fludarabine.

In chronic lymphocytic leukemia (CLL), presence of acquired cytogenetic abnormalities may help to estimate prognosis. However, deletion of TP53 gene, which is associated with an aggressive course of the disease and poor prognosis along with a lack of response to treatment, is one of the alterations which may escape cytogenetic diagnoses in CLL. Thus, other techniques have emerged such as interphase fluorescence in situ hybridization (iFISH). Deletion of TP53 may but must not go together with the formation of an isochromosome i(17q); surprisingly this subgroup of patients was not in the focus of CLL studies yet. This study was about if presence of i(17q) could be indicative for a new subgroup in CLL with more adverse prognosis. As a result, TP53 deletion was detected in 18 out of 150 (12%) here studied CLL cases. Six of those cases (~33%) had the TP53 deletion accompanied by an i(17q). Interestingly, the cases with i(17q) showed a tendency towards more associated chromosomal aberrations. These findings may be the bases for follow-up studies in CLL patients with TP53 deletion with and without i(17q); it may be suggested that the i(17q) presents an even more adverse prognostic marker than TP53 deletion alone.

Background Banding-karyotyping and metaphase-directed-fluorescence-in-situhybridization (FISH) may be hampered by low mitotic index in leukemia. Interphase FISH (iFISH) is a way out here, however, testing many probes at the same time is protracted and expensive. Here multiplex-ligation-dependent-probe-amplification (MLPA) was used retrospectively in chronic lymphocytic leukemia (CLL) samples initially studied by banding cytogenetics and iFISH. Detection rates of iFISH and MLPA were compared and thus a cost-efficient scheme for routine diagnostics is proposed. Results Banding cytogenetics was done successfully in 67/85 samples. DNA was extracted from all 85 CLL samples. A commercially available MLPA probe set directed against 37 loci prone to be affected in hematological malignancies was applied. Besides, routine iFISH was done by commercially available probes for following regions: 11q22.3, 12p11.2-q11.1, 13q14.3, 13q34, 14q32.33 and 17p13.1. MLPA results were substantiated by iFISH using corresponding locus-specific probes. Aberrations were detected in 67 of 85 samples (~79%) applying banding cytogenetics, iFISH and MLPA. A maximum of 8 aberrations was detected per sample; however, one aberration per sample was found most frequently. Overall 163 aberrations were identified. 15 of those (~9%) were exclusively detected by banding cytogenetics, 95 were found by MLPA (~58%) and 100 (~61%) by routine iFISH. MLPA was not able to distinguish reliably between mono- and biallelic del(13)(q14.3q14.3), which could be easily identified as well as quantified by routine iFISH. Also iFISH was superior to MLPA in samples with low tumor cell load. On the other hand MLPA detected additional aberrations in 22 samples, two of them being without any findings after routine iFISH. Conclusions Both MLPA and routine iFISH have comparable detection rates for aberrations being typically present in CLL. As MLPA can detect also rare chromosomal aberrations it should be used as an initial test if routine cytogenetics is not possible or non-informative. Still iFISH should be used additionally to distinguish mono- from biallelic deletions and also to determine rate of mosaicism for 13q14.2 to 13q14.3. In case MLPA is negative the corresponding CLL samples should be tested at least by iFISH using the standard probe set to. Electronic supplementary material The online version of this article (doi:10.1186/s13039-014-0079-2) contains supplementary material, which is available to authorized users.