Genetic characterization of non-Hodgkin lymphomas using in situ hybridization

Abstract

Fluorescence in situ hybridization (FISH) has been shown to be a valuable and important technique in cytogenetics, as a complement to traditional banding analysis. This thesis focus on the characterization of chromosomal rearrangements in two hematological neoplasias, myelodysplastic syndrome (MDS) and non-Hodgkin lymphoma (NHL) using FISH.

The increased cytogenetic resolution obtained by FISH, made it possible to identify a true-whole arm translocation consisting of 17q and 18q, in a case of MDS. The derivative chromosome had a centromere with sequences from both chromosomes 17 and 18. Thus, a breakpoint on either of the p-arms could be excluded, and it could be concluded that the rearrangment resulted in monosomy of 17p and 18p without affecting expressed genes located in the centromeric region. Chromosome painting, FISH with chromosome specific libraries, on short term cultures of NHL showed that chromosomal rearrangements were often more complex than the banding analysis predicted. Painting analysis could contribute with new information, not only of translocations, but also of true deletions and amplification. This could significantly clarify the true karyotype of the malignant cell. A recurring rearrangement in NHL is additional material on the tip of the short arm of chromosome 1.

Using chromosome specific libraries it was possible to show that, in three out of nine such cases, the extra chromosomal material originated from the long arm of chromosome 2, resulting in a der(1)t(1;2). The three cases were all follicular NHL, although none of them had the t(14;18) found in 85-90% of follicular lymphomas. In addition, one of the cases, had the der(1)t(1;2) as the only rearrangement in one of two malignant clones. All three cases had the der(1)t(1;2) in addition to two normal chromosome 2 homologs, leading to a duplication of 2q31-qter. These findings exemplifies that new cytogenetic subgroups can be identified when banding analysis is complemented with chromosome painting.

Furthermore, we found a case of NHL with a der(22)t(2;22) as a sole aberration. This results in a cytogenetically identical duplication of 2q31-qter, as in the cases with der(1)t(1;2). These findings suggest that cancer relevant genes may be located at the distal 2q.LAZ3/BCL6 gene has been implicated in tumorigenesis of NHL. Therefore the expression pattern of the LAZ3/BCL6 gene was studied, using RNA in situ hybridization. It was shown that LAZ3/BCL6 is expressed in follicular center(FC) B-cells of both non-malignant reactive lymph nodes and FC-derived non-Hodgkin lymphomas. Furthermore, LAZ3/BCL6 is expressed in a number of different tissues such as skeletal muscle, spinal cord, trachea, and peripheral blood leukocytes. The data clearly show that the expression of the LAZ3/BCL6is not confined to the malignant cells, and thus its role in the malignant transformation must be reconsidered.