Jens Krugmann

IgVH mutational status and clonality analysis of Richter's transformation: diffuse large B-cell lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution.

Approximately 5% of B-cell chronic lymphocytic leukemia (B-CLL) patients develop a secondary aggressive lymphoma, usually of diffuse large B-cell type (DLBCL), termed Richters transformation (RT). Rarely, classic Hodgkin lymphoma (HL) is observed. Published small series suggest that tumor cells in DLBCL and HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. We describe the morphology, immunophenotype, and clinical features of 34 classic RT patients with DLBCL, 6 cases of B-CLL with HL, and 8 cases with scattered CD30-positive Hodgkin and Reed-Sternberg (HRS)-like cells. The clonal relationship of the 2 components was analyzed using sequencing analysis of immunoglobulin heavy chain variable region (IgVH) genes. In classic RT, 18/23 B-CLL cases (78%) showed clonal progression to DLBCL with identical IgVH sequences in both lymphoma components, whereas in 5 cases (22%) the DLBCL was clonally unrelated. Among clonally related RT samples, 73% carried unmutated IgVH genes, whereas 4/5 unrelated cases were mutated. Immunophenotypically, most cases of DLBCL irrespective of clonal relatedness showed significant differences in phenotype compared with the B-CLL, with common loss of CD5 and CD23. Using immuno-laser capture microdissection, sequencing of the IgVH CDR3 region of isolated HRS cells showed that 2/2 cases with HL were clonally unrelated, whereas they were clonally identical in 1/2 cases of B-CLL with scattered HRS-like cells. HRS or HRS-like cells in all 3 unrelated cases showed evidence of Epstein-Barr virus infection. Of interest, 5/6 cases of B-CLL with HL, and 5/6 cases of B-CLL with HRS cells showed mutated IgVH genes.

High-throughput tissue microarray analysis of G1-cyclin alterations in classical Hodgkin's lymphoma indicates overexpression of cyclin E1

Deregulation of G1‐cyclins (CCN) plays a key role in the pathogenesis of many human malignancies, including non‐Hodgkins lymphomas (NHLs). In contrast to NHL, little is known about phenotypic and genotypic changes in the regulation of the cell cycle in classical Hodgkins lymphoma (cHL). To facilitate analysis of aberrant gene expression in cHL, a lymphoma tissue microarray (TMA) containing 752 cores of 330 different cHL samples was constructed. Direct comparison of Epstein–Barr virus (EBV) latent membrane protein 1 (LMP‐1) expression in Hodgkins and Reed–Sternberg (HRS) cells on conventional full sections with the corresponding duplicate/triplicate tumour cores on the TMA showed a concordance of 100%, indicating that cHL‐TMA is a reliable and representative method for evaluating gene expression profiles in situ. Using TMA technology, protein expression and gene amplification of different G1‐CCNs in cHL were analysed. Among the G1‐CCNs analysed, cyclin E (CCNE) was expressed in 212/253 cases (84%). In most of the individual tumours, over 75% of the HRS cells stained positive for CCNE, suggesting that CCNE is overexpressed in cHL. This overexpression was not due to CCNE gene amplification, as judged by fluorescence in situ hybridization, and did not correlate with EBV infection, as assessed by the expression of LMP‐1. Thus, the overexpression of CCNE could be caused by profound changes in HRS cell‐cycle regulation that could contribute to the malignant phenotype. Copyright

Transendothelial Migration of Myeloma Cells Is Increased by Tumor Necrosis Factor (TNF)-α via TNF Receptor 2 and Autocrine Up-Regulation of MCP-1

The proinflammatory cytokine tumor necrosis factor (TNF)-α has been shown to facilitate leukocyte transendothelial migration. In multiple myeloma, TNF-α is an important factor in the promotion of growth and survival of the malignant cells. Studies have shown that enhanced TNF-α levels in myeloma patients correlated with aggressive disease. Therefore, we investigated the effect of recombinant human TNF-α on the migrational behavior of myeloma cells across the physiological barrier of the major disease compartment, i.e., human bone marrow endothelial cells. In the presence of TNF-α, we observed significantly increased migration both in established myeloma cell lines and in plasma cells from myeloma patients. Expression of TNF-receptor 2 (TNF-R2) but not TNF-receptor 1 (TNF-R1) was detected in myeloma cell lines. Myeloma cells of patients also showed expression of TNF-R2 but not TNF-R1. The effect of TNF-α could not be explained by altered expression of adhesion molecules or metalloproteases. Instead, we found an up-regulation of monocyte chemoattractant protein (MCP)-1 and confirmed that myeloma cells express the relevant receptor C-C chemokine receptor 2. Preincubation of myeloma cells with recombinant human MCP-1 also enhanced cell migration, and this effect, as well as the effect of TNF-α, was abolished by treatment with anti-MCP-1 antibody. In contrast, migration of myeloma cells in the direction of an MCP-1 gradient, i.e., chemotaxis, could not be observed in the cell lines investigated. Additionally, the mRNA level of TNF-α was up-regulated by the cytokine treatment, which points to an autocrine loop augmenting and/or stabilizing the TNF-α–MCP-1 pathway. In summary, our data clearly support additional investigations using anti-MCP-1 antibodies in myeloma progression.

High prevalence of a 30-base pair deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 gene and of strain type B EBV in Mexican classical Hodgkin's disease and reactive lymphoid tissue

Depending on geographic location and patient age Hodgkins disease (HD) is associated with Epstein-Barr virus (EBV), mostly type A EBV, in 20% to 100%. The highest prevalence occurs in children of developing countries. Molecular analysis of the oncogene coding for the latent membrane protein 1 (LMP-1) revealed a 30-base pair (bp) deletion in up to 46% of EBV-positive HD. We investigated the presence of EBV in a series of Mexican classical HD (n = 57) and reactive lymphoid tissues (n = 20) from a private and a public hospital with special emphasis on the prevalence of the 30-bp deletion and the type of EBV. EBV infection was analyzed at the cellular level by Epstein-Barr encoded early RNA transcripts (EBER) in situ hybridization (ISH) and by LMP-1 protein immunohistochemistry (IHC). Molecular analysis of the LMP-1 gene configuration was performed by polymerase chain reaction (PCR) with primers spanning the site of the deletion and subsequent Southern and/or dot blot hybridization using wild-type and deletion-specific probes. The prevalence of type A and type B EBV was investigated by PCR-analysis for divergence in the coding region of Epstein-Barr nuclear antigen (EBNA)-2. EBV was detected in Hodgkin- and Reed-Sternberg cells (H-RS) by LMP-1 IHC and/or EBER ISH in 35/57 (61%) Mexican HD including 18/32 (56%) with nodular sclerosis, 15/20 (75%) with mixed cellularity and 2/4 (50%) with lymphocyte depletion. In addition, LMP-1 gene sequences were detected by PCR in 9 cases of HD without LMP/EBER expression by H-RS cells and in 17/20 (85%) reactive lymph nodes, supposedly originating from rare latently infected B cells. Surprisingly, the 30-bp LMP-1 deletion was found in 28/35 (80%) EBV-positive HD. This deletion, however, was also found in all 9 (100%) HD with H-RS cells negative for EBV and in 10/17 (59%) reactive lymph nodes. Thus, the overall LMP-1 del prevalence in reactive tissue is 73% (19/26). Typing of EBV was successful in 26 cases of EBV-positive HD, 10 of these were infected by type B EBV (38%). Of the reactive lymphoid tissue, 9 (47%) were infected by type A, and 10 (53%) by type B; All 20 cases (100%) associated with type B, whether neoplastic or reactive, displayed the LMP-1 del variant compared with 18/25 (72%) infected by type A EBV. To our knowledge, this is the highest incidence for both the LMP-1 deletion variant and the infection by type B EBV in HD reported so far worldwide. Our data suggest that EBV infection contributes to the pathogenesis of the majority of Hodgkins disease cases in Mexico. The specific tumorigenic role of the LMP-1 deletion variant, however, is doubtful with regard to its high frequency in nonneoplastic lesions. Moreover, type B infection frequently occurs in Mexican HD and reactive lymphoid tissue and is consistently associated with the deletion variant pointing to a pathogenetic role of this combined genotype.

Combination of Cytology, Fluorescence In Situ Hybridization for Aneuploidy, and Reverse-Transcriptase Polymerase Chain Reaction for Human Mammaglobin/Mammaglobin B Expression Improves Diagnosis of Malignant Effusions

PURPOSE The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. The aim of this study was to improve tumor cell detection in effusions by molecular approaches. MATERIALS AND METHODS A total of 157 effusions from patients with tumors and 72 effusions from patients without a history or evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, fluorescence in situ hybridization (FISH) for aneuploidy, and reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of human mammaglobin (hMAM) and mammaglobin B (hMAM-B). RESULTS In effusions from patients with tumors, the sensitivities of tumor cell detection by cytology, FISH, and hMAM and hMAM-B detection were 46.2%, 53.3%, 36.4%, and 57.7%, respectively. The corresponding specificities were 94.4%, 97.0%, 87.1%, and 88.6%. Notably, a high percentage of effusions containing malignant cells were in fact transudates, indicating the necessity for molecular diagnostic work-up of transudates collected from patients with tumors. Dependent on the tumor type, the use of appropriate marker combinations improved tumor cell detection in effusions significantly. By combining all four diagnostic tests, a positive test result indicating the presence of malignancy was achieved in 81.1%, with a fairly good specificity of 70.1%. CONCLUSION Molecular techniques are definitely useful to detect malignancy in cytologically negative effusions. Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. This finding should help to improve tumor staging, prognostic assessment, and treatment monitoring.

Longer failure-free survival interval of Epstein-Barr virus-associated classical Hodgkin's lymphoma: a single-institution study.

We analyzed Epstein-Barr virus association in classical Hodgkin’s lymphoma from a single center in Austria with special emphasis on the latent membrane protein1 gene configuration and clinical outcome. All 119 (65 male, 54 female) patients were treated from 1974 to 1999 in the Division of Hematology and Oncology at the Department of Internal Medicine, University of Innsbruck, Austria. The mean follow-up time was 122 months (range, 3–333 mo). Epstein-Barr virus was examined by latent membrane protein1 immunohistochemistry and by in situ hybridization for Epstein-Barr virus–encoded early ribonuclein acid transcripts. For assessment of the Epstein-Barr virus subtype (A/B) and latent membrane protein1 gene configuration, the polymerase chain reaction was employed. Fifty-four reactive tonsils were used as the control population. These results as well as clinical parameters such as age, gender, tumor stage, risk factors, and B symptoms were correlated with failure-free and overall survival. Latent membrane protein1 was detected in 31/119 (26%) classical Hodgkin’s lymphoma, and Epstein-Barr virus subtyping was successful in 19 of the 31 virus-infected classical Hodgkin’s lymphoma cases, as well as in 28 of 54 reactive tonsils. Subtype A was observed in all classical Hodgkin’s lymphoma patients and in 26/28 (93%) tonsils. The 30–base pair latent membrane protein1 gene deletion was found in only 4/31 (13%) Epstein-Barr virus-associated classical Hodgkin’s lymphoma as well as in 20/54 (37%) reactive tonsils. Patients with Epstein-Barr virus–associated classical Hodgkin’s lymphoma showed a significantly longer mean time to first relapse of 99 months, as compared with 49 months for the Epstein-Barr virus–negative cases (P < .02), and were more frequent in those aged >45 years (P < .04). Epstein-Barr virus–associated classical Hodgkin’s lymphoma were predominantly of the mixed-cellularity subtype and occurred more frequently in male patients, in patients with Stage III and IV, and in patients with B symptoms as well as risk factors. However, overall survival did not correlate with Epstein-Barr virus association. The 30–base pair latent membrane protein1 gene deletion had no influence on overall survival and failure-free survival time. Although the number of patients with this specific mutation was low, it further shows that an increased oncogenic potential of the latent membrane protein1 deletion variant is unlikely. This large single-center study demonstrates a low prevalence of Epstein-Barr virus positivity in classical Hodgkin’s lymphoma in western Europe. In accordance with results of similar studies, the presence of Epstein-Barr virus has a beneficial effect on the length of failure-free survival despite the higher frequency of risk factors such as higher tumor stage or advanced age.

Epstein-Barr virus-associated Hodgkin's lymphoma and legionella pneumophila infection complicating treatment of juvenile rheumatoid arthritis with methotrexate and cyclosporine A.

We describe the case of a 53-month-old girl with juvenile rheumatoid arthritis (JRA), complicated by the occurrence of Hodgkins lymphoma and Legionella pneumophila infection during immunosuppressive treatment with methotrexate (MTX) and cyclosporine A (CSA). The girl had received variable anti-inflammatory combination therapy, including MTX for 28 months and CSA for 3 months. Thirty-six months after the onset of arthritis, the girl presented with an enlargement of the lymph nodes of the mediastinum, the hilum of the lungs, and the abdomen. Concomitantly, a diagnosis of Legionella pneumonia was rendered. Autopsy showed Epstein-Barr virus (EBV)-associated nodular sclerosing Hodgkins lymphoma. The neoplastic cells were positive for CD15, CD 30, and latent membrane protein 1 (LMP 1). The present case is the second reported to occur in a child, and it lends support to the hypothesis that immunosuppressive treatment may contribute to an increased risk of the development of EBV-associated lymphoproliferative disorders (LPD) in pediatric patients suffering from JRA.

Identification of the rare EGFR mutation p.G796S as somatic and germline mutation in white patients with squamous cell carcinoma of the head and neck

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are involved in tumorigenesis and response to targeted therapies in distinct cancer types. Squamous cell carcinomas of the head and neck (HNSCC) show an incidence of EGFR mutations varying from 7% in Asians to 0% to 4% in white patients. Mutational screening predominantly focuses on the analysis of hotspot regions of EGFR (exons 19 and 21).

Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib

Targeting the ubiquitin-proteasome pathway with the proteasome inhibitor bortezomib has emerged as a promising approach for the treatment of several malignancies. The cellular and molecular effects of this agent on colorectal cancer cells are poorly characterized. This study investigated the antiproliferative effect of bortezomib on colorectal cancer cell lines (Caco-2 and HRT-18). In order to define the proteins potentially involved in the mechanisms of action, proteome profiling was applied to detect the proteins altered by bortezomib. The in vitro efficacy of bortezomib as a single agent in colorectal cancer cell lines was confirmed. Proteome profiling with two-dimensional PAGE followed by mass spectrometry revealed the up-regulation of the major inducible isoform of heat shock protein 70 (hsp72) and lactate dehydrogenase B in both cell lines, as well as the induction of aldo-keto reductase family 1 member B10 (AKR1B10) in HRT-18 cells. Both AKR1B10 and hsp72 exert cell-protective functions. This study shows for the first time a bortezomib-induced up-regulation of AKR1B10. Small interfering RNA–mediated inhibition of this enzyme with known intracellular detoxification function sensitized HRT-18 cells to therapy with the proteasome inhibitor. To further characterize the relevance of AKR1B10 for colorectal tumors, immunohistochemical expression was shown in 23.2% of 125 tumor specimens. These findings indicate that AKR1B10 might be a target for combination therapy with bortezomib. [Mol Cancer Ther 2009;8(7):1995–2006]

Primary Gastrointestinal B-Cell Lymphoma - A Clinicopathological and Immunohistochemical Study of 61 Cases with an Evaluation of Prognostic Parameters

We hereby present a retrospective clinicopathological and immunohistochemical study of surgically resected primary gastrointestinal (GI) lymphoma with an analysis of parameters of potential prognostic relevance. From a larger series of 144 cases of primary GI lymphomas, we chose 61 cases with sufficient clinical follow-up (mean 60, range 1-219 months), classified either as extranodal marginal zone B-cell lymphoma of MALT type (MALT lymphoma) or diffuse large B-cell lymphoma (DLBCL), after having excluded other subtypes. In addition to conventional clinical and morphological parameters, the expression levels of Ki-67 (MIB-1), bcl-2 and p53 were evaluated for prognostic significance. Twenty-one (34.4%) cases were classified as pure low grade marginal zone B-cell lymphoma of MALT type, 12 (19.7%) cases as low grade MALT lymphoma with a high grade component (mixed type), and 28 (45.9%) cases as primary extranodal DLBCL. Most of the lymphomas (53/61; 86.9%) were localized in the stomach, 3 (4.9%) in the small bowel, 3 (4.9%) multifocal in both stomach and small intestine and 2 (3.3%) in the large bowel. MIB-1 expression in more than 30% of tumor cells was detected in 42 (68.6%), bcl-2 expression in 20 (32.8%) and p53 accumulation in more than 10% of neoplastic cells in 16 (26.2%) lymphomas. Both high Ki-67 expression and p53 accumulation were more prevalent in the DLBCL. 30 (49%) patients showed lymph node involvement at surgery, 14 (23%) patients suffered tumor recurrence, and 24 (38.5%) died during the follow-up period. Tumor recurrence occurred primarily in patients who had presented lymph node involvement (9/14, 64.3%). The 5-year survival rate was 66.1% for all patients. Important prognostic factors for overall survival were tumor stage (p < .004) and p53 accumulation (p < .05) in univariate analysis, and tumor stage in multivariate analysis (p < .001). Although p53 accumulation did not reach statistical significance in our small study group, it may be both important in the transformation of low grade MALT lymphoma and an indicator for aggressive behavior in high grade tumors.