Comprehensive chronic lymphocytic leukemia diagnostics by combined multiplex ligation dependent probe amplification (MLPA) and interphase fluorescence in situ hybridization (iFISH)

Abstract

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.

Keywords: Chromosomal aberrations; Chronic lymphocytic leukemia (CLL); Fluorescence in situ hybridization (FISH); Multiplex ligation-dependent probe amplification (MLPA).

Figure 1

Number of aberrations present per sample as found in this study after application of all mentioned methods (banding cytogenetics, iFISH and MLPA) – values given in percent.

Figure 2

Suggestion how to proceed when doing MLPA as a primary test after GTG-banding: in case MLPA finds a tumor marker with adverse prognosis no further iFISH analyses is necessary. In case of an MLPA result suggesting intermediate, unclear or good iFISH for 3 to 6 target regions should be done. A probe for 6q may be also used; however, as case with a del(6q) are rare we would not recommend it at present as really indicated to be applied. According to the obtained results cases need to be regrouped. Finally, iFISH can be used to subclassify cases with good prognosis into such with favorable and unfavorable good prognosis.

Figure 3

Detection rates of cytogenetics, MLPA and iFISH as standalone approaches are depicted and compared with overall result combining all three tests as suggested in Figure 2 ; the corresponding results obtained in the 85 cases were aligned with and are expressed as the resulting prognostic relevance of the identified chromosomal aberrations.