Molecular cytogenetic studies in the Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma

Hodgkin lymphoma (HL) is a malignant lymphoma that largely affects young adults and is the second most common malignancy in this age group. Overall, around one third of cases are associated with the Epstein-Barr virus (EBV) and this association is believed to be causal. In young adults less than 20% of cases are EBV-associated and there are few clues to the aetiology of the EBV-negative form of the disease. Studies on the molecular genetics of HL have been hindered by the scarcity of the tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, within tumours and the lack of an animal model. Recently developed techniques for the enrichment and micromanipulation of HRS cells from cell suspensions and frozen sections have, however, opened up new avenues for the investigation of this enigmatic disease. Diagnosis of HL is often straightforward but in some cases differentiation from nonHodgkin lymphoma (NHL) can be problematic. In a recent epidemiological study of 575 consecutive cases of HL in Scotland and the Northern Region of England, expert histopathological review suggested that -5% of cases had been wrongly classified as Hl (Jarrett et al. 2003). Although there has been enormous progress in the treatment of HL around 25% of cases do not respond to conventional treatment, and there is a need for better prognostic markers to identify such patients at the time of diagnosis in order to institute more aggressive therapy, Cytogenetic analysis has been extremely productive in the investigation of NHLs where the identification of non-random chromosomal abnormalities has led to the disicovel-Y of numerous oncogenes. In follicle centre cell lymphoma (FCL), tumour ::':[;;~Il;' carry the t(14:18) translocation which juxtaposes the immunoglobulin (Ig) chain gene on chromosome 14 with the bcl-2 gene on chromosome 18, in deregulation of BCl-2. Investigation of this translocation has led to an understanding of the biology of both FCL and apoptosis, and the delmonstration of t(14;18) has proven useful both diagnostically and in clinical :':',X/',\>.' > follow-up. Similarly, the identification of the t(2:5) translocation in anaplastic large cell lymphoma (AlCL) allowed production of antibodies to the fusion protein NPMAlK and the recognition of a discrete subgroup of patients with AlK-positive lymphoma or 'ALKoma'. contrast to the situation with NHL, conventional cytogenetic studies in HL have been less numerous and have been hindered by the scarcity and low mitotic rate of HRS cells. Normal karyotypes have been found in almost half the cases studied but it is questionable whether such karyotypes actually correspond to the HRS addition, chromosomal abnormalities have been detected in the reactive component of lesions further complicating interpretation. alternative approach to studying genetic abnormalities in HL is to use , ;;~ ':,.:~mparative genomic hybridisation (CGH). This technique provides a global approach for screening clinical specimens for genetic imbalances and can be :performed when only very small amounts of tumour deoxyribonucleic acid (DNA) are available. CGH involves labelling tumour DNA and DNA from a normal source with two different fluorescent dyes. The labelled DNA samples are mixed at an .equal ratio and used as a probe for in situ hybridisation on normal metaphase The hybridisation profile of test and control DNA along the length of each 'hr·nrrl.....c:nmIP is then analysed, the fluorescent ratio of the two fluorochromes is ·'A',\;·v··/··, calculated and regions of chromosomal loss or gain determined. Two groups have recently applied CGH to the study of HL. Using slightly different methodologies, both found consistent chromosomal losses and gains, however, the results from the two studies differed, emphasising the need for additional studies. To determine whether recurrent genomic imbalances are a feature of HL, CD30positive HRS cells were laser microdissected from 20 cHL cases and 4 HL-derived cells lines and SUbjected to analyses by CGH. In primary tumours, the most frequently involved chromosomal gains were 17q (70%), 2p (40%), 12q (40%), 17p (40%), 22q (35%), 9p (30%), 14q (30%), 16p (30%), with minimal overlapping regions at 17q21, 2p23-13, 12q24, 17p13, 22q13, 9p24-23, 14q32, 16p13.3 and 16p11.2. The most frequent losses involved 13q (35%), 6q (30%), 11q (25%) and 4q (25%), with corresponding minimal overlapping regions at 13q21, 6q22, 11q22 and 4q32. Statistical analysis revealed significantly more gains of 2p and 14q in the older adult cases; loss of 13q was associated with a poor outcome. The results suggest that there is a set of recurrent chromosomal abnormalities associated with cHL and provide further evidence that cHL is genetically distinct from nodular lymphocyte predominance Hodgkin lymphoma (NLPHL). Abnormalities of 17q are infrequent in other lymphoma or NLPHL; this finding, coupled with current knowledge of gene expression in cHL, suggests that genes present on 17q may play an important role in the pathogenesis of cHL. Combined immunophenotype and interphase cytogenetic (FICTION) studies· were used as techniques for follow-up studies. High expression of both STAT3 and STAT5a has been described cHL (Chen et al. 2001; Hinz et al. 2002) and their genes are located in 17q21.2. Constitutive activation of NF-KB has been found to be a feature of the HRS cells in cHL and has been shown to facilitate escape from apoptosis (Bargou et al. 1996). The c-rel gene encodes for a subunit of NF-KB and is located on chromosome 2p16. REL amplification has been shown in some cHL cases (Joos et a!. 2002) and therefore REL was selected as another candidate gene for further study. Non-functional inhibitor proteins of NF-KB, such as IKBa, has been described as an alternative mechanism for the aberrant activation of NF-KB (Wu et al. 1996). As part of the follow-up studies, /kBa gene mutation status and loss of heterozygosity (LOH) in the HRS cells were determined by sequence analysis and SNP assays.EThOS - Electronic Theses Online ServiceGBUnited Kingdo