Cytogenetic and genomic characterization of cell line ARH77.

The cell line ARH77 is derived from a patient with plasma cell leukemia and has a complex and continually evolving karyotype. It is frequently used in biological studies of myeloma and plasma cell leukemia, so accurate characterization of the genome is valuable. Here we present a detailed cytogenetic investigation using G-banding and multicolor fluorescence in situ hybridization (M-FISH) in association with assessment of copy number alterations (CNAs) throughout the genome using array-based comparative genomic hybridization (aCGH). In addition to providing an accurate description of the karyotype, this complementary approach highlighted the relative merits of the individual techniques. Conventional cytogenetics and M-FISH indicated the location and types of the major chromosomal changes, whether balanced or unbalanced, and at the same time demonstrated the level of karyotypic evolution between cells. The aCGH profiles reflected the unbalanced chromosomal abnormalities detected by cytogenetics, providing refinement of their genomic breakpoint locations as well as the identification of novel genomic changes. Three aCGH platforms, comprising bacterial artificial chromosome (BAC) or oligonucleotide templates, were available for evaluation. Sixteen CNAs were consistently detected by all three platforms. Novel submicroscopic CNAs ( approximately 0.4 Mb) were detected by the highest resolution platform only, whereas the clones from the BAC arrays provided locus-specific FISH probes for confirmation of CNA.

Interphase molecular cytogenetic screening for chromosomal abnormalities of prognostic significance in childhood acute lymphoblastic leukaemia: a UK Cancer Cytogenetics Group Study.

Summary Interphase fluorescence in situ hybridization (iFISH) was used independently to reveal chromosomal abnormalities of prognostic importance in a large, consecutive series of children (n = 2367) with acute lymphoblastic leukaemia (ALL). The fusions, TEL/AML1 and BCR/ABL, and rearrangements of the MLL gene occurred at frequencies of 22% (n = 447/2027) (25% in B-lineage ALL), 2% (n = 43/2027) and 2% (n = 47/2016) respectively. There was considerable variation in iFISH signal patterns both between and within patient samples. The TEL/AML1 probe showed the highest incidence of variation (59%, n = 524/884), which included 38 (2%) patients with clustered, multiple copies of AML1. We were thus able to define amplification of AML1 as a new recurrent abnormality in ALL, associated with a poor prognosis. Amplification involving the ABL gene, a rare recurrent abnormality confined to T ALL patients, was identified for the first time. The use of centromeric probes revealed significant hidden high hyperdiploidy of 33% and 59%, respectively, in patients with normal (n = 21/64) or failed (n = 32/54) cytogenetic results. The iFISH contributed significantly to the high success rate of 91% (n = 2114/2323) and the remarkable abnormality detection rate of 89% (n = 1879/2114). This study highlights the importance of iFISH as a complementary tool to cytogenetics in routine screening for significant chromosomal abnormalities in ALL.

Three distinct subgroups of hypodiploidy in acute lymphoblastic leukaemia.

This study of children and adults with acute lymphoblastic leukaemia (ALL) is the largest series of patients with hypodiploidy (<46 chromosomes) yet reported. The incidence of 5% was independent of age. Patients were subdivided by the number of chromosomes; near-haploidy (23-29 chromosomes), low hypodiploidy (33-39 chromosomes) and high hypodiploidy (42-45 chromosomes). The near-haploid and low hypodiploid groups were characterized by their chromosomal gains and a doubled hyperdiploid population. Structural abnormalities were more frequent in the low hypodiploid group. Near-haploidy was restricted to children of median age 7 years (range 2-15) whereas low hypodiploidy occurred in an older group of median age 15 years (range 9-54). Patients with 42-45 chromosomes were characterized by complex karyotypes involving chromosomes 7, 9 and 12. The features shared by the few patients with 42-44 chromosomes and the large number with 45 justified their inclusion in the same group. Survival analysis showed a poor outcome for the near-haploid and low hypodiploid groups compared to those with 42-45 chromosomes. Thus cytogenetics, or at least a clear definition of the modal chromosome number, is essential at diagnosis in order to stratify patients with hypodiploidy into the appropriate risk group for treatment.

Cytogenetics of multiple myeloma: interpretation of fluorescence in situ hybridization results.

The cytogenetic picture in multiple myeloma (MM) is highly complex, from which non-random numerical and structural chromosomal changes have been identified. Specifically, translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and either monosomy or deletions of chromosome 13 have been reported in a significant number of patients from both cytogenetic and interphase fluorescence in situ hybridization (FISH) studies. Importantly, these abnormalities of chromosome 13 have recently been associated with a poor prognosis. In view of the highly complex nature of the karyotypes in MM patients, interphase FISH results may be difficult to interpret. In this study, cytogenetics and/or interphase FISH were carried out on bone marrow samples or purified plasma cells from 37 MM patients. Abnormal karyotypes, characterized by multiplex FISH (M-FISH) were found in 11 patients, all of which were highly complex. Interphase FISH revealed translocations involving the IGH locus in 16 (43%) patients. The IGH/cyclin D1 (CCND1) gene fusion characteristic of the translocation, t(11;14)(q13;q32), was seen in 12 (32%) of these patients and other rearrangements of IGH in four (11%) patients. Fourteen patients had additional copies of chromosome 11. Twenty patients (54%) had 13q14 deletions, 10 of whom also had t(11;14) or another IGH translocation. By comparing cytogenetic and FISH results, this study has revealed that significant chromosomal abnormalities might be hidden within highly complex karyotypes. Therefore, extreme caution is required in the interpretation of interphase FISH results in MM, particularly in relation to certain abnormalities, such as 13q14 deletions, which have an impact on prognosis.

Translocations of 14q32 and deletions of 13q14 are common chromosomal abnormalities in systemic amyloidosis.

Systemic monoclonal immunoglobulin light chain amyloidosis (AL) is associated with clonal plasma cell dyscrasias that are often subtle and non-proliferating. AL shares numerical chromosomal changes with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Illegitimate translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and deletions of the long arm of chromosome 13, [del(13q)], commonly occur in MM, MGUS and plasma cell leukaemia. In AL IGH rearrangements have been identified but, to date, there are no reports of del(13q). In this study of 32 patients with AL, 24 with systemic and eight with localized disease, translocations involving IGH and del(13q) were found using dual-colour interphase fluorescence in situ hybridization (FISH). IGH translocations were observed in 11 patients (37% overall and in 46% with systemic disease), of which nine had the IGH/CCND1 fusion from t(11;14)(q13;q32). Two showed IGH translocations other than the t(11;14) or t(4;14)(p16;q32). In one of these patients a breakpoint within the constant region of IGH between Calpha1 and Calpha2 was indicated. In the second a deletion covering Calpha1 and Calpha2 accompanied the translocation. Ten patients (27% overall and 33% of those with systemic disease) showed del(13q). The gain or loss of IGH and CCND1 signals provided evidence of numerical chromosomal changes in three patients.