Clemens H.M. Mellink

Whole-Genome Scanning by Array Comparative Genomic Hybridization as a Clinical Tool for Risk Assessment in Chronic Lymphocytic Leukemia

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene

We used BAC array-based CGH to detect genomic imbalances in 187 CLL cases. Submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. Oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb. The minimally deleted region included the ZNF280A, ZNF280B, GGTLC2, and PRAME genes. Quantitative real-time PCR revealed that ZNF280A, ZNF280B, and PRAME mRNA expression was significantly lower in the 22q11 deletion cases compared to non-deleted cases.

Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia

Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia

Translocation (2;11)(q37;q23) in therapy-related myelodysplastic syndrome after treatment for acute promyelocytic leukemia

Treatment of acute promyelocytic leukemia (APL) with a combination of anthracycline-based chemotherapy and all-trans retinoic acid (ATRA) leads to very high rates of complete remission and survival. There are only a limited number of publications on the development of therapy-related myelodysplastic syndrome (MDS) or acute myeloid leukemia during follow-up of APL. Although drugs targeting at DNA-topoisomerase II characteristically induce translocations involving 11q23, this was seldom seen in patients treated for APL. We report on a patient initially diagnosed with APL. Response to therapy was monitored by fluorescence in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction for the PML-RARalpha rearrangement. Consecutive samples showed a swift and complete reduction of PML-RARalpha rearranged cells. Twenty months after diagnosis, however, conventional cytogenetics revealed a complex karyotype with a translocation involving 11q23 and loss of chromosomes 7q and Xq. FISH analysis with the MLL probe identified 2q37 (harboring the SEPT2 gene) as the translocation partner of chromosome 11. We consider the rather unique t(2;11)(q37;q23) as the primary event causing therapy-related MDS in our patient. This case stresses the importance of conventional karyotyping to be performed on a regular basis in all treated APL patients for the early detection of chromosomal aberrations that indicate the development of therapy-related MDS or acute myeloid leukemia.

Expression pattern of the septin gene family in acute myeloid leukemias with and without MLL-SEPT fusion genes

Septins are proteins associated with crucial steps in cell division and cellular integrity. In humans, 14 septin genes have been identified, of which five (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) are known to participate in reciprocal translocations with the MLL gene in myeloid neoplasias. We have recently shown a significant down-regulation of both SEPT2 and MLL in myeloid neoplasias with the MLL-SEPT2 fusion gene. In this study, we examined the expression pattern of the other 13 known septin genes in altogether 67 cases of myeloid neoplasia, including three patients with the MLL-SEPT2 fusion gene, four with MLL-SEPT6 fusion, and three patients with the MLL-SEPT9 fusion gene. When compared with normal controls, a statistically significant down-regulation was observed for the expression of both MLL (6.4-fold; p=0.008) and SEPT6 (1.7-fold; p=0.002) in MLL-SEPT6 leukemia. Significant down-regulation of MLL was also found in MLL-MLLT3 leukemias. In addition, there was a trend for SEPT9 down-regulation in MLL-SEPT9 leukemias (4.6-fold; p=0.077). Using hierarchical clustering analysis to compare acute myeloid leukemia genetic subgroups based on their similarity of septin expression changes, we found that MLL-SEPT2 and MLL-SEPT6 neoplasias cluster together apart from the remaining subgroups and that PML-RARA leukemia presents under-expression of most septin family genes.

Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia

BackgroundA relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature.MethodsCytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient.ResultsWhen compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023)ConclusionWe found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.

The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia

BACKGROUNDnWhole-genome analysis by array-based comparative genomic hybridization (array CGH) is an emerging technique for the detection of recurrent unbalanced chromosomal aberrations in chronic lymphocytic leukemia (CLL). These chromosomal changes can be highly predictive of clinical course and are evaluated at present using classical cytogenetics and interphase fluorescence in situ hybridization. However, the significant limitations of these assays have resulted in efforts to move array CGH from use as a discovery tool in the research laboratory into the clinical laboratory as an alternative method for the evaluation of genomic prognostic markers in patients with CLL.nnnOBJECTIVEnThe HemeScan(™) array was developed as a clinical tool to provide prognostic marker identification with simultaneous diagnostic monitoring of the entire genome in CLL and other hematological malignancies.nnnMETHODSnThe authors review representative data from clinical testing of HemeScan for genomic aberration identification in CLL and present suggestions for the integration of array CGH genome scanning into the clinical laboratory.nnnRESULTS/CONCLUSIONnThe HemeScan assay for CLL precludes the need for G-banded cytognetics by simultaneously revealing prognostic marker status and the level of genomic complexity in > 85% of cases.