Hodgkin and Reed Sternberg (H-RS) cells represent the malignant cells in classical Hodgkin's disease. Although derived from germinal center B cells, they do not express surface immunoglobulin. This has been explained by the presence of crippling mutations within the immunoglobulin genes in numerous cases of Hodgkin's disease. As immunoglobulin gene expression in B cells requires an interaction between octamer sites and the transactivating factors Oct-2 and Bob-1, this study addresses the expression of the transcription factors Oct-2 and Bob-1 in H-RS cells. In Hodgkin's disease-derived cell lines, low levels of Oct-2 transcripts but no Oct-2 protein were detected. Transcripts of Bob-1, a B-cell-specific co-factor of Oct-2, could not be observed in these cell lines. Absence of Oct-2 and Bob-1 protein expression in primary H-RS cells was demonstrated by performing immunohistochemistry in 20 cases of classical Hodgkin's disease. H-RS cells stained negative for both proteins in all of the cases analyzed. In conclusion, absence of functional Oct-2 and Bob-1 cells represents a novel mechanism for immunoglobulin gene deregulation in H-RS cells. Lack of Oct-2 and Bob-1 points to a defect in transcription machinery in H-RS cells and is associated with lack of immunoglobulin gene expression in these cells.
Almost all researchers agree on the lack of Bob-1 expression in Hodgkin/Reed-Sternberg (H/RS) cells in classic Hodgkin lymphoma (CHL), and utilize this marker as a diagnostic tool in conjunction with other markers to differentiate between lymphocyte predominance Hodgkin lymphoma (LPHL) and CHL.
Paraffin sections of randomly selected CHL were classified according to the WHO . A total of 18 CHL cases were studied. These included 2 cases of LRHL, 4 MCHL, 10 NSHL, 2 LDHL. The positive controls consisted of 2 reactive nodes selected with follicular hyperplasia and 2 cases of LPHL. The initial diagnostic panel included CD30, CD15, CD20 and CD3. EMA and ALK-1 were performed whenever indicated to rule out LPHL and anaplastic large cell lymphoma.
The expression of Bob-1 and EBV LMP-1 was evaluated by immunostaining (table 1). Following deparaffinization, endogenous peroxidase was inhibited by tissue sections incubation for 10 minutes at room temperature in 0.3% H2O2. Following antigen retrieval, slides were rinsed in distilled water and finally phosphate buffered saline (PBS). All incubations were performed at room temperature. After incubation with the primary antibody, sections were rinsed in PBS and incubated with the LSAB-2 detection kit and the steps were followed according to the manufacturer instructions (Dakocytomation). Diaminobenzedine tetrachloride (DAB) was applied for 10 minutes and lastly, sections were counterstained with Harris haematoxylin (Hx).
The ISH steps were performed according to the manufacturer's instructions (Novocastra, NCL-EBV-K). The slides were first dewaxed in xylene, hydrated in descending grades of alcohol and lastly immersed in water. One hundred μl of proteinase K in 0.05 mM Tris/HCL buffer pH 7.6 were applied for 10 minutes at 37C. This step was followed by slides immersion in water, dehydration and air drying. Depending on the tissue section, 20 to 50 μl of the probe hybridization solution were applied. Sections were coverslipped and incubated for 2 hours at 37C. The covers were allowed to drain off into a beaker; they were then washed in Tris containing 0.1% triton X-100. For the detection, 100 μl of the blocking solution was applied for 10 minutes followed by rabbit F (ab') anti-FTTC conjugated to alkaline phosphatase (AP) diluted 1:100 for 30 minutes. Slides were subsequently washed in TBS followed by the AP substrate buffer. The AP activity was demonstrated by incubation in dark overnight with a mixture solution of 5 bromo-4 choro indolyl phosphate, nitro-blue tretrazolium (BCNT). Finally, the slides were washed and counterstained with Mayer's haematoxylin. The control of the procedure included positive control sections and negative control probe supplied with the ISH kit.
The staining was considered positive when dark blue to black dots were encountered in the nuclei of the H/RS cells together with positivity of the positive control tissue and negativity of the duplicate sections hybridized with the negative probe.
What could be the other explanations of Bob-1+H/RS cells in the present study It has been speculated that EBV contributes to the transformation and maintenance of H/RS cells, by rescuing them from apoptosis. This has been attributed to the oncogenic potential of LMP-1 on B cells through upregulation of anti-apoptosis genes including bcl-2 , downregulation of p16INK4a  and activation of NFκB . What is the relation between EBV and Bob-1 NFκB and Bob-1 are transcription factors required for mouse B cell differentiation, serum IgM production, late B cell maturation and function . It seems that the activation of NFκB in EBV positive cases is associated with up regulation of Bob-1 since there is a close association between the expression of Bob-1 and EBV in the present study.
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Hodgkin's lymphoma is one of the most frequent lymphomas in western countries. There are two re-cognized forms: the classical Hodgkin's lymphoma form and the lymphocyte-predominant form. Morphologically, they are characterized by a few large tumour cells within a background of activated lym-phocytes and mixed inflammatory cells. It has been demonstrated, by molecular studies, that Hodgkin's lymphoma is, in most cases, a B-cell lymphoma that originates from germinal centre1 neoplastic cells that escape from FAS-mediated apoptosis by an unknown mechanism,2 probably involving c-FLIP overexpression.3 Neoplastic cells from classical and lymphocyte-predominant Hodgkin's lymphoma usually present potentially functional clonal immunoglobulin (Ig) gene rearrangements though carrying a high load of somatic mutations.1, 2
Transcription factors OCT.1 and OCT.2, and their coactivator BOB.1, are necessary for the octamer-dependent transcription of Ig and other important lymphoid-specific genes of B cells.9, 10, 11 Also, the activity of the cotransduced Ig promoter can be restored by transfection with BOB.1 and OCT.2 and/or OCT.1.7
The collaborating members of the Spanish Hodgkin's Lymphoma Study Group collected 355 cases of classical Hodgkin's lymphoma during the period between 1994 and 1998, of which 325 cases were suitable for this study (see below). Using standard tissue sections, according to the World Health Organization (WHO) classification,26 there were 184 nodular sclerosis, 112 mixed cellularity, 18 lymphocyte-rich, nine lymphocyte-depletion and two unclassificable classical Hodgkin's lymphomas. All patients were treated with standard polychemo-therapeutic regimens with or without adjuvant radiotherapy.
Tissue Arrayer device (Beecher Instrument, Silver Spring, MD, USA) was used to construct the tissue microarrays in CNIO.24 Briefly, the richest areas of HRS cells were marked in the paraffin blocks. In each case, two selected 1-mm-diameter cores from two different areas, were included along with 43 different controls. Thus, five different tissue microarray blocks were constructed, each containing 187 cores excepting block number 5 that contained 176 cores.
Interestingly, BSAP was strongly expressed in all the studied cases (Figure 1), with independence of the histological subtype. MUM.1 positivity was frequently detected (98% of cases), with a strong nuclear staining (Figure 2).
OCT.1 and OCT.2 were expressed in a notorious percentage of cases. Thus, when considering all the positive cases, with independence of the grade of staining, more cases expressed OCT.2 (Figures 3 and 4) than OCT.1 (62 vs 59%). Variable staining for BOB.1 was seen in the 37% of cases (Figures 5 and 6). The histological subtype that showed a higher percentage of OCT.1, OCT.2 and BOB.1 positivity, was lymphocyte-rich, and the subtype that most frequently was negative for some of these factors, was nodular sclerosis. There was not any statistical association between the histological subtype and OCT.1, OCT.2 and BOB.1 expression.
We found a direct association between OCT.2 and BOB.1 expression, regardless of the grade of positivity, OCT.2 expression was also statistically associated with all the B markers studied, excepting MUM.1.
The reviewed studies about the expression of transcription factors are based, mostly, on cellular lines and do not specify clearly the intensity of the positivity.4, 7, 8 In our study, when only the strongly positive cases were considered, independently from the histological subtype, no cases strongly expressing OCT.2 were found. The frequency of strong OCT.1 and BOB.1 positivity was poor and even lower if their simultaneous expression was analysed, as has been described elsewhere. Conversely, when we analysed our cases, with independence of the intensity of the staining, the percentage of expression was much higher than that described before: 62% for OCT.2, 59% for OCT.1 and 37% for BOB.1. Similarly, OCT.1, OCT.2, and BOB.1 expression without immunoglobulin production have been described recently by Pileri et al32 in a set of primary mediastinal B-cell lymphomas.
BSAP is a transcription factor encoded by PAX.5 gene, involved in the establishment and the maintenance of B-cell identity.29, 34 It is expressed in all B-cell stages of development from the pro-B-cell stage to the stage previous to plasma cell, where it is downregulated.35 Previous studies35, 36, 37 found a frequent BSAP expression in the HRS cells of classical Hodgkin's lymphoma. The percentage of BSAP positivity, in our cases, was of nearly 100%, which provides an evidence for the B-cell origin of this disease. Considering that PAX.5 regulates CD20, CD79a and OCT.2 expression, the low positivity rate of these markers found in our study is surprising. In order to explain a functional defect in BSAP protein of HRS cells, some studies have been attempted in search for mutations in PAX.5 gene,29 but the absence of such mutations points to a defect of other essential factors in the control of B lineage of classical Hodgkin's lymphoma. Most of our cases expressed MUM.1, a transcription factor that belongs to the interferon regulatory factor family (IRF).38 It has been demonstrated that MUM.1 is expressed in the final step of intragerminal centre B-cell differentiation and in subsequent steps of B-cell maturation.39, 40, 41 The high frequency of expression of MUM.1 in the HRS cells of classical Hodgkin's lymphoma in our cases, and in reported series,38 suggests a similar phenotype between plasma cells and HRS cells, although the presence of BSAP expression in the latter is inconsistent with a final stage of differentiation that is seen in plasma cells. 153554b96e