¹Division of Hematology, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People's Republic of China

²Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People's Republic of China

Correspondence to: S K Ma, Division of Hematology, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, People's Republic of China; Fax: 852 2817 7565

Leukemia (2002) 16, 953-955. DOI: 10.1038/sj/leu/2402442

TO THE EDITOR

Chromosomal deletions are recurrent abnormalities in leukemia, implicating the loss of tumor suppressor genes as a significant mechanism in leukemogenesis. Deletion of long arms of chromosomes 5 and 7, moreover, predicts for a very poor prognosis in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).^1,2 More recently, characterized deletions in myeloid malignancies include deletion 9q in AML,³ and deletion 20q in myeloproliferative disorder.⁴ Deletion 11q is a rare cytogenetic abnormality in the myeloid dis- orders, with a reported prevalence of 0.7% in de novo and secondary AML and MDS in one study.⁵ While the prognostic significance of del(11q) is uncertain, a previous report on deletion 11q23 showed that 15/16 AML and 5/12 MDS patients died with a median overall survival of 14.1 months.⁶ We performed molecular cytogenetics characterization of del(11q) abnormality based on archival material in AML and MDS patients diagnosed at our center. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) procedures were carried out in accordance with previously published protocols.⁷

A review of patient records revealed nine cases of del(11q) detected among 819 cases (1.1%) of AML and MDS diagnosed in the past 10 years (Table 1). The deletion existed both as a sole abnormality (n = 6) and in association with other changes (n = 3), including t(8;21) in case 2. Conventional cytogenetic analysis showed terminal deletion in six cases and interstitial deletion in three cases, respectively (Figure 1a). Based on morphological and CGH results (performed in cases 4, 6-9), the deletion involved proximal regions of chromosome band 11q23.

As the mixed lineage leukemia (MLL) gene is located at the 11q23 breakpoint, we performed interphase FISH on six cases (1-3, 7-9) in which cells in Carnoy's fixative were available, using a MLL dual-color break-apart rearrangement probe (Vysis, Downers Grove, IL, USA). It consisted of a 350 kb centromeric fragment and a 190 kb telomeric fragment labeled with different fluorochromes. Four cases showed a predominance of a single fusion signal (Figure 1b), consistent with preservation of one allele and deletion of the other. Case 7 showed MLL split signal in keeping with gene rearrangement (Figure 1c), which was confirmed with Southern blot hybridization using PS/4 probe on BamHI, EcoRI and SacI digests (Figure 1d). Review of the karyotype showed subtle elongation of chromosome 6q (Figure 1e). Further metaphase FISH using the relevant whole chromosome painting probes (Vysis) showed addition of chromosome 11 material to 6q27 (Figure 1g), and was confirmed by employing respective painting probes with reversed fluorochrome label (data not shown). Unbalanced changes such as addition of chromosome 11qter material to the telomere of chromosome 6 was ruled out by the presence of intact telomeres on both chromosomes as demonstrated by FISH (Figure 1h) using a PNA telomere probe set (Dako, Copenhagen, Denmark). While chromosomal rearrangement in this case might have resulted in fusion between MLL and a candidate gene most likely AF6 based on breakpoint location at 6q27, unfortunately no sample was available to decipher this possibility. Finally, there was a predomi

nance of two fusion signals in case 2, indicating the presence of two MLL alleles. Subsequent re-analysis of the karyotype showed the presence of 11q material on long arm of chromosome 15, so that del(11q) and add(15q) should be revised as t(11;15)(q21;q26) (Figure 1f). No metaphase, however, was available in the Carnoy's fixative for confirmation by chromosome painting experiments. The presence of three of four signals in this case indicated the existence of extra MLL alleles, most probably as a result of duplication of the derivative chromosome 15 in small numbers of cells.

Our study showed that, in agreement with a prior FISH study,⁸ del(11q) in myeloid malignancies uniformly involved band 11q23. It is tempting to speculate, based on our data, that monoallelic MLL deletion may play a role in initiation or progression of myeloid malignancies with del(11q) abnormality, especially in light of the hematological abnormalities displayed in MLL +/- mice⁹ indicative of haploinsufficiency. More importantly, we showed that del(11q) is heterogeneous at the molecular level and may be a pointer for cryptic rearrangements involving the chromosome 11q or the MLL gene. It follows therefore that the presence of del(11q) in the myeloid malignancies necessitates further characterization by molecular cytogenetics techniques, in particular to detect cryptic MLL translocations that are of prognostic significance.¹

Acknowledgements

The study was supported by a CRCG grant of the University of Hong Kong.

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Figure 1 (a) Conventional cytogenetics and CGH characterization of deleted segment at chromosome 11q. The black line denotes deletion at cytogenetics level in nine patients, whereas the red line denotes CGH results in five patients (cases 4, 6-9). In case 7, the absence of 11q abnormality on CGH most probably represents balanced rearrangement between chromosomes 6 and 11. (b) Interphase FISH with MLL probe, showing deletion of one MLL allele in three cells and preservation of two MLL alleles in one cell. (c) Interphase FISH with MLL probe in case 7, showing MLL split signal (separated red and green signals) in three cells, indicating MLL gene rearrangement. One cell shows two normal MLL alleles (fused red/green signals). (d) Southern blot analysis using PS/4 probe: P-DNA, placental DNA (negative control); RS4:11, cell line harboring t(4;11) (positive control). Case 7 showed rearrangement bands in BamHI, EcoRI and SacI digests, whereas case 6 was negative for MLL rearrangement. (e) Partial karyotype derivative chromosomes 6 and 11 in case 7. G-banding with trypsin/Giemsa. (f) Partial karyotype showing derivative chromosomes 11 and 15 in case 3. G-banding with trypsin/Giemsa. (g) Whole chromosome painting in case 7, showing addition of chromosome 11 material to 6q27. While the CGH result suggested balanced rearrangement, the presence of chromosome 6 material was not detected on chromosome 11q, most probably related to lack of sensitivity of chromosome painting probes to detect minute subtelomeric chromosomal material. (h) Intact telomeres on chromosomes 6 and 11 as detected by FISH using a PNA probe set for human telomeres.

Table 1 A summary of karyotype, interphase FISH results and clinical outcome in nine patients with deletion 11q