E. Nacheva

Philadelphia-negative chronic myeloid leukaemia: detection by FISH of BCR-ABL fusion gene localized either to chromosome 9 or chromosome 22.

SUMMARY. Dual‐colour FISH has been used to study two patients with chronic myeloid leukaemia (CML) associated with a normal karyotype. Co‐localization of signals from BCR and ABL cosmids was observed in interphase nuclei from both patients. In one patient, analysis of metaphase spreads showed that the 3’region of the ABL gene was deleted from one chromosome 9 and inserted into chromosome 22. In a second patient 5’BCR sequences were missing from one copy of chromosome 22, and co‐localized with 3’ABL sequences on chromosome 9. These results demonstrate the molecular heterogeneity of Ph‐negative CML. In addition, they illustrate the potential usefulness of dualcolour FISH on interphase nuclei for monitoring the response to treatment of patients with Ph‐negative CML.

Characterization of 20q deletions in patients with myeloproliferative disorders or myelodysplastic syndromes

Deletions of the long arm of chromosome 20 are associated with several myeloid malignancies. We have analyzed the structure of the del(20q) in 30 patients and two cell lines. Twenty-one of the patients presented with a myeloproliferative disorder and nine with a myelodysplastic syndrome. Two categories of deletions were identified. Eighteen patients had a large deletion with loss of both G(+) bands from the long arm of chromosome 20. Twelve patients had small deletions with loss of one G(+) band from the long arm of chromosome 20. A chromosome paint was generated from a del 20q marker carrying a small deletion. This probe was hybridized to normal metaphases (reverse chromosome painting) and also to metaphases from patients with a del 20q (comparative reverse chromosome painting). All six small deletions analyzed were characterized by loss of the proximal G(+) band (q12) and retention of the distal G(+) band (q13.2). These data define a minimal deleted region extending from 20q11.2-20q13.1.

Does BCR/ABL1 positive acute myeloid leukaemia exist?

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi‐lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of ‘hot spot’ genome imbalances associated with BCR/ABL1 positive pre‐B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the ‘hot spot’ regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML.

Comparative genomic hybridization in acute myeloid leukemia. A comparison with G-banding and chromosome painting.

Comparative genomic hybridization (CGH) represents a new technique for global analysis of a whole genome for net loss or gain of chromosome regions. It offers several advantages over alternative techniques. It permits analysis of a whole genome in a single hybridization reaction, it does not require the generation of metaphases from tumor cells, and it only requires very small numbers of tumor cells. Most previous studies have concentrated on the application of CGH to the analysis of chromosome defects associated with solid tumors. In this paper we report the use of CGH to study bone marrow samples from a patient with acute myeloid leukemia and complex karyotypic abnormalities. The results obtained using CGH were compared with G-banding analysis. Both G-banding and CGH detected a 5q deletion, a 7q deletion, additional material derived from 8q, and an HSR on 11q. However, several apparently discrepant results were also obtained. Paints for chromosomes 3, 5, 7, 8, 11, 12, 14, 17, 22, and X were therefore used to resolve these differences. Our results demonstrate that CGH detected chromosome abnormalities associated with acute myeloid leukemia and that CGH provided information that was not obtained by G-banding analysis alone. These data suggest that CGH may prove a useful adjunct to conventional cytogenetic and molecular analysis of hematologic malignancies.

BCR rearrangement in apparent essential thrombocythaemia

Summary. The presence of the Philadelphia chromosome is a major determinant of the prognosis of patients with myeloproliferative disorders. We describe a case of apparent essential thrombocythaemia in whom cytogenetic analysis was normal. However, the presence of basophilia, the absence of abnormal megakaryocytes in a trephine biopsy and the female sex of the patient prompted Southern analysis of peripheral granulocyte DNA. This revealed a BCR rearrangement and the patient has therefore undergone allogeneic bone marrow transplantation. This case emphasizes the importance of both cytogenetic and molecular analysis of patients with apparent essential thrombocythaemia.

Fluorescence In Situ Hybridization Detection of Two Telomeres on the Short Arm of a Derived Chromosome 16 in an Infant with Thrombocytopenia

We report a case of severe thrombocytopenia with an abnormal bone marrow karyotype described by G-banding analysis as t(16;21)(p?13;q11). Using fluorescence in situ hybridization (FISH) analysis with whole chromosome paints, the chromosome rearrangement was shown to be more complex, with the additional cryptic involvement of the long arm of chromosome 3. The chromosome rearrangement involved the breakpoints 3q26, 16p13.3, and 21q11; this rearrangement has not been previously described. The size of genomic material translocated from the chromosome 16 homologue was too small to be detected by chromosome paint. A 16p-specific telomeric probe was hybridized to locate the translocated 16p material. The 16p telomeric unique sequence DNA was retained on the der(16) chromosome, indicating a more distal breakpoint. This study demonstrates that telomeric translocations can occur that would be undetected by telomeric-specific FISH probes.

Double Philadelphia masquerading as chromosome 20q deletion – a new recurrent abnormality in chronic myeloid leukaemia blast crisis

Summary. The most common abnormality of chromosome 20 in haematological malignancy is deletion of the long arm [del(20q)]. These interstitial deletions are variable in size and are seen in both premalignant haematological conditions and acute myeloid neoplasia. A commonly deleted region (CDR), mapped within the 20q11.2/q13.1 segment with an estimated size of 1·7 Mbp, is considered to present a primary genetic lesion marking a gene(s), the loss of which is responsible for the pathogenesis of these haematological disorders. While a small number of recurrent translocations involving chromosome 20 have also been reported, no recurrent aberration of this chromosome has been associated with myeloid disease progression. We present nine cases of Philadelphia (Ph)‐positive chronic myeloid leukaemia (CML) in which deletions of chromosome 20 were also detected by conventional karyotyping. In six cases, fluorescent in situ hybridization (FISH) mapping confirmed a del(20q) which corresponded to the myeloid CDR. In the remaining three cases however, the presumed del(20q) marker was shown to be the result of an unbalanced translocation between band 20p11 and a second copy of the Ph chromosome. This new abnormality, termed dic(20;Ph) for short, was identical to a del(20)q by G‐banding, and combined duplication of the breakpoint cluster region and Abelson murine leukaemia viral oncogene homologue (BCR‐ABL) fusion with loss of the 20p11‐pter segment. In all three cases, the dic(20;Ph) was associated with disease progression and therefore represents a new recurrent abnormality in CML blast crisis.