Clonality Analysis And Mutational Status Of IGVH Gene In Richter's Syndrome

Background: B-cell chronic lymphocytic leukaemia (B-CLL) comprises 90% of chronic lymphoid leukaemias in Western countries. Patients with B-CLL have heterogeneous clinical courses. Approximately 3-10% of B-CLL patients encounter transformation to an aggressive lymphoma, mainly diffuse large B-cell lymphoma (DLBCL) or Hodgkin's lymphoma (HL) which has been defined as classical Richter's syndrome or HL variant of Richter's syndrome, respectively. Richter's syndrome is associated with a poor clinical outcome. The mutational status of immunoglobulin heavy chain variable region (IGVH) gene not only implies tumor cell origin in B-cell lymphoma but also constitutes an important prognostic factor in B-CLL since B-CLL patients with unmutated IGVH genes are correlated with a poor clinical outcome. Richter's syndrome occurs in B-CLL patients carrying mutated and unmutated IGVH gene, and tumor cells in DLBCLand HL can be donally identical to the B-CLL clone or arise as an independent lymphoma. But, the ration of clonally related cases to donally unrelated cases in Richter's syndrome,- the correlation between IGVH gene mutational status in B-CLL and clonal transformation to Richter's syndrome;the molecular characters of IGVH gene and involved molecular mechanisms are still unknown. However, on the one side of scientific theory, the transformation of B-CLL to Richter's syndrome connects two completely different lymphomas: NHL and HL;on the other side of clinical outcome, patients with Richter's syndrome have very poor outcome, thus, it has an important scientific significance and clinical value to investigate and find the molecular mechanism of Richter's syndrome and the possibly associated risk factors.Material: Paraffin embedded tumor specimens of 48 patients were identified in the archives of the Institute of the Pathology at Universities of Wuerzburg, Munich (both Germany) and Innsbruck (Austria) as well as in the files of the laboratory of Pathology, National Cancer Institute, NIH (USA). Cases were diagnosed based on histopathologic and immunophenotypic criteria as detailed in the current WHO classification. 34 cases were classical Richter's syndrome, 6 cases were Richter's syndrome with HL variant and additional 8 cases of B-CLL with CD30 positive HRS-like cells. Molecular analyses were performed in 26 cases of classical Richter's syndrome, 6 cases of Richter's syndrome with HL variant and 6 cases of B-CLL with CD30 positive HRS-like cells.Methods: Combined with menu microdissection, we isolated DNA of two components by standard phenol-chloroform extraction and then amplified IGVH gene complementarity-determining region 3 (CDR3) and CDR2 by PCR using fluorescence labelled primers. We analyzed the clonal rearrangement of IGVHgene CDR2 or CDR3 fragments and compared the sizes of products to find the correlation between two components. Furthermore we analyzed the mutational status, the families and usages of IGVH genes in Richter's syndrome after sequencing and tried to find its molecular characters and possibilities of pathogenesis. For HL variant of Richter's syndrome, we utilized laser capture microdissection (LCM) to pick up immuno-stained HRS cells and HRS-like cells;we used the semi nest PCR and sequencing IGVH gene CDR3 fragment, compared with that of B-CLL to confirm their relations. Moreover, we used immunohistochemical (IHC) staining to detect Zeta associated protein (ZAP70), p53, Interferon regulatory factor-4 (IRF-4) protein and latent member protein 1 (LMP1) expression in two components, combined with clinical outcome to find risk factors.Results: 1. for classical Richter's syndrome (DLBCL/B-CLL)? 1).Samples of 18 B-CLL/DLBCL (78%) were identical tumor cell clones, whereas DLBCL developed as a clonally independent neoplasm in 5 patients after sequencing were performed in 23 paired B-CLL and DLBCL cases;? 2). Among the clonally related pairs, 11/15 (73%) carried unmutated VH genes in both B-CLL and DLBCL component;? 3).VH3-23, VH3-74, VH1-2 and VH3-9 were overused in B-CLL transformation to DLBCL, the three former usages were also used as dominant genes in the clonally related B-CLL/DLBCL;? 4). Immunohistochemical staining showed that Richter's transformation DLBCL expressed CDS in 32.1%, CD23 in 14.3%, ZAP70 in 23.8%, p53 in 80.6% and IRF-4 in 82.6% cases;? 5).Follow-up data could be obtained from 17 classical Richter'ssyndrome patients, the median survival time of B-CLL transformation to DLBCL were 7 months whereas that of the Richter's transformation DLBCL at gastrointestinal was 28 months;? 6). No significantly different survival was found between clonally related or unrelated group, between IGVH gene mutated or unmutated group and the groups between expression and not expression of ZAP70, p53, IRF-4.2. for HL variant of Richter's syndrome and B-CLL with HRS-like cells (B-CLL/HL and HRS-like cells)? 1). 83.3% (5/6) B-CLL cases that showed transformation to HL carried mutated IGVH genes.? 2). HRS cells in 2 samples and HRS-like cells in 1 sample were clonally distinct to the B-CLL clone and infected by EBV, whereas 1 sample of HRS-like cells was related clone from the surrounding B-CLL cells and did not express latent membrane protein-1 (LMP1).? 3). VH4-34 and VH3-48 accounted for over half of the B-CLL cases transformation to HL.? 4). The median survival time of Richter's transformation HL and HRS-like cells were 21 and 33 months, respectively.? 5). The expression of ZAP70 was associated with IGVH gene unmutated status in total Richter's syndrome.Conclusions:1. for classical Richter's syndrome (DLBCL/B-CLL)? 1). Richter's transformation DLBCL evolves from the B-CLL clone in approximately 78% of patients. The ration of clonally related DLBCL to unrelated secondary Richter's transformation DLBCL is 2: 1;? 2). DLBCL clonal transformation predominantly (73.3%) occurs in B-CLL patients earring unmutated IGVH genes;? 3).The biased of IGVH gene usages increases the possibility of one or more known or unknown antigens involved in the Richter's transformation;? 4).Molecular differences of IGVH gene and very poor clinical outcome of Richter's transformation DLBCL indicate it may be regarded as a distinct subset of DLBCL.2. for HL variant of Richter's syndrome and B-CLL with HRS-like cells (B-CLL/HL and HRS-like cells)? 1). HRS-like cells might be the precursor of HRS cells and the B-CLL with HRS-like cells might be an intermediate stage of HL transformation.? 2). 83.3% B-CLL patients that show transformation to HL or CD30 positive HRS-like cells carry mutated IGVH genes.? 3). EBV-positive HL variant of Richter's syndrome develops as independent clonally"unrelated secondary malignancies, possibly as a consequence of the underlying immunodeficiency;The tendency that IGVH-unmutated B-CLL transforms to DLBCL whereas IGVH-mutated B-CLL transforms to HL implies the different transformation pathways in two subtypes of Richter's syndrome, and the involvement of different known or unknown antigens due to the differently restricted IGVH gene usages. This might be one of the reasons for different clinical outcomes of two subtypes of Richter's syndrome. ZAP70 expression is broadened as a marker of unmutated IGVH gene of B-CLL in Richter's syndrome;but the clonal relationship between two tumor components, the mutational status of IGVH gene and the expression of ZAP70, p53 and IRF-4 are not confirmed as a risk factor in our series.