Molecular cytogenetics of IGH rearrangements in non-Hodgkin B-cell lymphoma.

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin B-cell lymphomas (NHLs) and correlated to clinically relevant subgroups. However, the detection rate largely varied with the technique used. We analyzed the incidence of IGH rearrangements using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 96 patients with nodal NHL. An IGH rearrangement was identified in 71 cases (74%). A t(14;18)(q32;q21) was found in 37 of the 42 follicular lymphomas (88.1%) studied and a t(11;14)(q13;q32) in 12 of the 14 mantle cell lymphomas (85.7%). IGH rearrangements were identified in 21 of the 40 diffuse large B-cell lymphomas (52.5%), including seven t(14;18)(q32;q21) and four t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using 24-color FISH, chromosomal whole paints, telomeric probes and locus specific identifiers enabled us to characterize complex and/or masked IGH translocations in follicular lymphomas and mantle cell lymphomas and to identify all the chromosomal partners involved in IGH rearrangements in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe can be the first approach in NHL, after which chromosome painting and 24-color FISH can be used to identify the chromosomal partners involved in IGH rearrangements. The identification of these genes is of utmost importance for a better understanding of the molecular mechanisms involved in the genesis of lymphoma.

Characterisation of supernumerary chromosomal markers: a study of 13 cases.

Marker chromosomes are defined as 'structurally abnormal chromosomes in which no part can be identified' (ISCN 1995). Supernumerary marker chromosomes (SMC) are 'additional markers' whose origin and composition cannot be determined by conventional cytogenetics. Molecular cytogenetic methods are necessary to identify these additional chromosomal markers. In one third, the SMCs are clinically well-defined in the literature, the remaining two thirds present a major problem for genetic counselling in prenatal diagnosis. At present, different molecular cytogenetic methods are used to determine the origin of SMCs. In this work, we studied 13 SMCs detected by RHG-banding, completed by C-banding and/or NOR-staining. 24-color FISH was used as the primary technique when the chromosomal origin was unknown. Targeted FISH procedures with specific probes (whole chromosome painting, centromeric probe, locus-specific identifier, BAC, etc.) were then performed to confirm and/or specify the chromosomal material present in the SMC. Seven SMCs were found to be associated with phenotypic abnormalities. Five derived from autosomes and two from gonosomes; these are: der(12)t(4;12), dic(15), i(18p), r(19), der(22)t(11;22), r(X), and der(Y). Two markers, r(8) and idic(15), were identified during investigations of infertile couples. Three cases seemed to be phenotypically normal. Four were discovered prenatally: r(2) and r(19) referred for elevated maternal serum markers, der(13/21) referred for advanced maternal age. The fourth SMC, der(14/22), was found during familial investigation following the identification of the same marker in an infertile son. The precise characterisation of the SMCs is of utmost importance for genetic counselling, especially in prenatal diagnosis.

Fluorescence in situ hybridization analysis of 110 hematopoietic disorders with chromosome 5 abnormalities: do de novo and therapy-related myelodysplastic syndrome-acute myeloid leukemia actually differ?

A retrospective cytogenetic study of acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) was conducted by the Groupe Francophone de Cytogénétique Hématologique (GFCH) to evaluate the structural abnormalities of chromosome 5 associated with other chromosomal abnormalities, in particular of chromosome 7, in these pathologies. In all, 110 cases of AML/MDS were recruited based on the presence of chromosome 5 abnormalities under conventional cytogenetics and supplemented by a systematic fluorescence in situ hybridization study of chromosomes 5 and 7. The abnormalities of the long arm of chromosome 5 (5q) were deletions of various sizes and sometimes cryptic. The 5q abnormalities were associated with translocations in 54% of cases and were simple deletions in 46%. In 68% of cases, 5q deletions were associated with chromosome 7 abnormalities, and 90% of these presented a complex karyotype. Of the 110 patients, 28 had a hematopoietic disorder secondary to chemotherapy, radiotherapy, or both. Among 82 patients with de novo AML/MDS, 63 were older than 60 years. Chromosomal abnormalities often associated hypodiploidy and chromosome 5 and 7 abnormalities in complex karyotypes, features resembling those of secondary hemopathies. Systematic investigation of the exposure to mutagens and oncogenes is thus essential to specify the factors potentially involved in MDS/AML with 5q abnormalities.

Extramedullary relapse after favorable molecular response to donor leukocyte infusions for recurring acute leukemia.

Donor leukocyte infusions (DLI) have turned out to be an efficient way to re-establish complete remission (CR) in chronic myeloid leukemia (CML) patients relapsing after allogeneic bone marrow transplantation (BMT). In these patients, absence of PCR bcr-abl fusion transcripts confirmed the potency of donor leukocytes to induce molecular response in relapsed CML. This ensured sustained remission and long-term survival. In this study, the capacity of DLI to induce molecular remission in acute leukemia relapse after BMT was analyzed. The results showed that following DLI, leukemic cell eradication gradually occurred over a prolonged time period. The time to complete disappearance of the molecular marker of the disease was 30 weeks in RT-PCR analysis. A sustained and persistent elimination of an AML1/ETO-positive leukemic clone in an AML-M2 patient was observed. In contrast, an AML-M5 with t(11;19) and an E2A/PBX1-positive ALL achieving cytogenetic and molecular bone marrow CR developed following DLI unusual sites of extramedullary leukemia relapse, despite continued bone marrow remission. This study adds further proof of the benefit of donor cell therapy in acute leukemia but shows that complete leukemic cell eradication appears to require a critical interval in order to establish effective immune responses at all sites where leukemic cells persist.

FISH investigation of 5q and 7q deletions in MDS/AML reveals hidden translocations, insertions and fragmentations of the same chromosomes.

We have studied, by fluorescence in situ hydridization (FISH), chromosomes 5 and 7 in a series of 11 cases with 5q deletion, as sole anomaly (four cases), or in association with 7q deletion (seven cases), in MDS/AML patients. We found that, in some cases, a part of the so-called 'lost' chromosome 5 and 7 material, was actually translocated. These translocations may be either end-arm or whole-arm, as well as small insertions. Chromosomes 5 or 7 may be broken in more than two segments, defining 'fragmentation', giving rise to marker chromosomes. FISH allowed the identification of small material insertion, which is totally unidentified by classical cytogenetics. Chromosome 5 and 7 translocations occur irrespectively of the 'de novo' or 'secondary' type of myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML) patients.

Contribution of fluorescence in situ hybridization analyses to the characterization of masked and complex Philadelphia chromosome translocations in chronic myelocytic leukemia.

Bone marrow samples from 112 patients with chronic myelocytic leukemia were investigated using cytogenetic methods. Fluorescent in situ hybridization (FISH) with whole-chromosome paints and BCR-ABL probes was used to confirm and/or complete the banding findings when a variant or a masked Philadelphia chromosome (Ph) translocation was found. Eight variant Ph translocations were identified. Three-way Ph translocations were found in seven patients. Chromosome 4 was involved in two of these cases and chromosomes 3, 11, 14, 17, and 16 in one case each; in the patient with chromosome 16 involvement, a ring of the translocated chromosome 9 was identified, that is r(9)t(9;16;22). The eighth patient had a five-way Ph translocation: t(2;9;16;22;22). The BCR-ABL fusion gene was detected on the Ph chromosome in all eight cases; two cases presented also a deletion of the 5' ABL region on the derivative chromosome 9. In the five-way translocation, the 3' DNA sequence of the ABL oncogene was fused with the 5' DNA sequence of the BCR gene on the Ph chromosome and the 5' end of ABL was inserted into the other chromosome 22. A masked Ph chromosome was identified in one of the 112 patients; it involved the insertion of the 3' ABL into BCR on an apparently normal chromosome 22, resulting in the BCR-ABL fusion gene. In conclusion, FISH analyses allowed not only a more accurate characterization of complex Ph translocations with subtle abnormalities and the identification of cryptic rearrangements, but also the recognition of deletion of the 5' ABL region, which could carry with it a poor prognosis.

Chromosome 8 tetrasomies and pentasomies--a clonal abnormality closely associated with acute monocytic leukaemia.

We report four cases of polysomy 8 (one tetrasomy and three pentasomies) observed in acute monocytic leukemia (FAB M4 and M5). Three of them showed a rearrangement of 11q23 identified by conventional cytogenetic analysis and/or chromosome painting. Our cases as well as a review of the literature, suggest that polysomy 8 is preferentially associated with monocytic differentiation (24/31). These polysomies have been observed in 21 de novo leukemias and in 10 secondary hematological disorders. A 11q23 rearrangement has been detected in 9 out of 32 patients, by conventional cytogenetic techniques in 7 and by FISH in 2. We suggest that these cases should be analysed by FISH and molecular studies in order to detect a rearrangement of MLL/11q23. Monocytic differentiation is often associated with a change of the MLL gene and the polysomy 8 might be a particular clonal evolution secondary to 11q23 abnormality.