Hans-Dieter Foss

Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis

BACKGROUND & AIMS Mechanisms of diarrhea in ulcerative colitis (UC) are still unknown. Functional and structural characterization of epithelial barrier and transport properties in ulcerative colitis (UC) was performed. METHODS Inflamed sigmoid colon epithelium from UC patients was studied by alternating current impedance analysis to determine the pure epithelial resistance as a measure of intestinal barrier function. Tight junction (TJ) structure was investigated by freeze-fracture electron microscopy. RESULTS Although total wall resistance was reduced in UC by 50%, impedance analysis uncovered a much more pronounced barrier defect. Epithelial resistance decreased from 95 +/- 5 to 20 +/- 3 omega3. cm2, which in conventional analysis is masked by an increase in subepithelial resistance from 14 +/- 1 to 36 +/- 3 omega3. cm2 caused by inflammation. This was paralleled by a change in epithelial cell TJ structure in UC. Strand count decreased from 6.94 +/- 0.25 to 4.76 +/- 0.47 at the surface and from 7.26 +/- 0.31 to 5.46 +/- 0.37 in the crypts. CONCLUSIONS The inflamed colonic mucosa in UC has an impaired barrier function that is much more pronounced than previously assumed. An altered TJ structure contributes to this barrier defect which, because of increased back leak, can reduce net ion transport. Thus, a leak-flux mechanism contributes to the diarrhea in UC.

Intestinal Non-Hodgkin’s Lymphoma: A Multicenter Prospective Clinical Study From the German Study Group on Intestinal Non-Hodgkin’s Lymphoma

PURPOSE Intestinal non-Hodgkins lymphomas are not well characterized. We therefore studied prospectively their clinical features and response to standardized therapy. PATIENTS AND METHODS Fifty-six patients with primary intestinal lymphoma were included in a prospective, nonrandomized multicenter study. Lymphoma resection was recommended and staging was performed according to the Ann Arbor classification. Patients were scheduled to receive six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) chemotherapy, and at stages EIII to EIV, they received additional involved-field radiotherapy. Corticosteroids were used in patients who could not receive chemotherapy. RESULTS Thirty-five patients had intestinal T-cell lymphoma (ITCL), 21 patients had intestinal B-cell lymphoma (IBCL; 18 diffuse large-cell lymphomas, two marginal-cell lymphomas, and one follicle-center lymphoma). Thirty-four patients at stages EI to EII (14 ITCL and 20 IBCL) and nine patients at stages EIII to EIV (all ITCL) received chemotherapy. No patient in stages EIII to EIV received radiotherapy, because death occurred in 12 of 14 patients. Two-year cumulative survival in patients with IBCL was 94% (95% CI, 82% to 100%) and higher than in patients with ITCL (28% [95% CI, 13% to 43%]; P <.0001), even when only stages EI to EII were considered (ITCL, 37.5% [95% CI, 16.5% to 58.5%]; P <.0001). IBCL patients compared with ITCL patients were at lower lymphoma stages (P <.01), had higher Karnofsky status (P <.005), had intestinal perforation less often (P <.05), required emergency operation less often (P <.05), received CHOP (P <.05) more often, and reached complete remission (P <.0005) more frequently. CONCLUSION IBCL patients at stages EI and EII respond well to chemotherapy, but the prognosis and treatment of ITCL patients is unsatisfactory.

Expression of vascular endothelial growth factor in lymphomas and castleman's disease

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines in human solid tumours and inhibition of VEGF‐induced angiogenesis suppresses tumour growth. Some groups of malignant lymphoma, including peripheral T‐cell lymphomas and Hodgkins disease, are characterized by a conspicuous proliferation of small vessels. To test the hypothesis that VEGF may also be involved in the angiogenesis in lymphomas and other lesions of the lymphoid system, VEGF expression was analysed in tissues, employing in situ hybridization with a 35S‐labelled RNA probe specific for this cytokine. Significant expression of VEGF transcripts was observed in Hodgkins disease and peripheral T‐cell lymphomas, particularly of the angioimmunoblastic type. In contrast, expression of this cytokine was minimal or absent in follicle centre lymphoma and chronic lymphocytic leukemia of B‐cell type. VEGF was mainly observed in reactive non‐lymphoid CD68‐negative cells, which probably represent fibroblasts or myofibroblasts. In normal and ulcerated tonsils, VEGF was expressed in the squamous epithelium but only rarely found in the lymphoid tissue. Although infectious mononucleosis tonsils contained high numbers of VEGF‐positive cells in the interfollicular zone, expression of this cytokine was not found in Epstein–Barr virus (EBV)‐infected cells, as determined by simultaneous in situ hybridization for VEGF and EBV‐encoded small nuclear RNAs (EBER). In 5/8 cases of Castlemans disease, germinal centres containing small vessels also showed expression of VEGF, in contrast to normal tonsillar germinal centres which are devoid of both vessels and VEGF transcripts. It is concluded that VEGF may be involved in the induction of the angiogenesis of both peripheral T‐cell lymphomas and Hodgkins disease, but not in low‐grade B‐cell lymphomas. In contradistinction to solid tumours, in which this cytokine is commonly secreted by the tumour cells themselves, in malignant lymphoma VEGF is not a product of neoplastic cells. Vascularization of germinal centres in Castlemans disease may also be a consequence of abnormal local expression of VEGF.

Overexpression or ectopic expression of MUC2 is the common property of mucinous carcinomas of the colon, pancreas, breast, and ovary

Mucinous carcinomas of the colorectum have been reported to overexpress the intestinal mucin MUC2. The purpose of this study was to determine whether this alteration is shared by mucinous tumours of the ovary, breast, and pancreas. A total of 40 breast carcinomas (22 of mucinous and 18 of ductal invasive type), 39 ovarian adenocarcinomas (16 mucinous, 23 serous), 47 colorectal carcinomas (25 mucinous and 22 non‐mucinous), and 41 pancreatic adenocarcinomas (14 mucinous, 27 non‐mucinous) were investigated by immunohistochemistry with the anti‐MUC2 monoclonal antibody 4F1 and the expression pattern was ranked. MUC2 mucin is expressed in the normal colonic epithelium; in the normal epithelium of the breast, ovary, and pancreas, it was not detectable by immunohistochemistry or by reverse transcriptase‐polymerase chain reaction (RT‐PCR). In agreement with previous reports, the colonic mucinous carcinomas differed significantly from the non‐mucinous carcinomas by strong MUC2 expression. In all mucinous carcinomas of the ovary, breast, and pancreas, de novo expression of the MUC2 gene was observed, which differentiated mucinous and nonmucinous carcinomas of these tissues (P<0·001). The overexpression or ectopic expression of the MUC2 gene exhibited by mucinous carcinomas of four organs indicates a common genetic lesion associated with the mucinous tumour phenotype.

Classical Hodgkin's Disease and Follicular Lymphoma Originating From the Same Germinal Center B Cell

PURPOSE Classical Hodgkins disease and non-Hodgkins B-cell lymphoma occasionally occur in the same patient. To clarify whether these different diseases share a common precursor cell, we analyzed the immunoglobulin rearrangements in tumor cells of the classical Hodgkins disease and the follicular lymphoma that developed in the same patient 2 years apart. PATIENTS AND METHODS Polymerase chain reaction (PCR) for the detection of rearranged immunoglobulin genes was carried out on single Reed-Sternberg cells and on whole tissue DNA extracted from the follicular lymphoma. PCR products were sequenced and compared with each other and with germ line immunoglobulin variable segments. Immunoglobulin heavy- and light-chain transcripts were analyzed by radioactive in-situ hybridization. RESULTS The same monoclonal immunoglobulin gene rearrangement was found in both neoplasms. The variable region of the immunoglobulin heavy-chain genes of the Reed-Sternberg and of the follicular lymphoma cells were differently mutated, but six somatic mutations were shared by both lymphoma cells. Although the coding capacity of the immunoglobulin genes was preserved in both neoplastic cell populations, immunoglobulin heavy- (mu) and light- (kappa) chain expression was restricted to the follicular lymphoma cells, except for small amounts of kappa light-chain mRNA in some Reed-Sternberg cells. CONCLUSIONS The neoplastic cells of the Hodgkins disease and the follicular lymphoma that occurred in this patient derived from a common precursor B cell. Its differentiation stage could be identified as that of a germinal center B cell. Thus, transforming events can be more important than the cell of origin in determining a disease entity.

Expression of the CD30 antigen in non-lymphoid tissues and cells.

Originally, expression of the CD30 antigen was shown to be typical of the tumour cells of Hodgkins disease (HD) and of anaplastic large cell lymphomas (ALCLs). In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non‐lymphoid tissues and malignancies, such as embryonal carcinomas (ECs), seminomas, cultivated macrophages, two histiocytic neoplasms, decidual cells, and mesotheliomas. As CD30 detection is important in the differential diagnosis of HD and ALCL, the expression of CD30 in different non‐lymphoid tissues was re‐evaluated by immunohistology and in situ hybridization. Extra‐lymphoid CD30 expression was found in 48/51 cases of EC or EC components of germ cell tumours, in decidual cells of 1/10 cases, in activated mesothelium in 16/28 pleural and peritoneal effusions, and in small foci of tumour cells in 2/8 mesotheliomas. CD30 expression was not confirmed in 27 germ cell tumours of the testis without an EC component nor in cultivated macrophages and 17 histiocytic malignancies. The knowledge of these CD30 expression patterns is important for the immunohistological differential diagnosis of anaplastic tumours. The absence of CD30 expression in reactive and neoplastic macrophages does not favour the concept that HD and ALCL are derived from these cells. Copyright

Differential Eµ enhancer activity and expression of BOB.1/OBF.1, Oct2, PU.1, and immunoglobulin in reactive B-cell populations, B-cell non-Hodgkin lymphomas, and Hodgkin lymphomas

It has previously been demonstrated that in cultured and in situ tumour cells of classical Hodgkin lymphoma (cHL), the immunoglobulin (Ig) promoter is inactive and its transcription factors Oct2 and/or BOB.1/OBF.1 are down‐regulated. In this study, the analysis of these transcription factors has been extended to a broad spectrum of B‐cell malignancies and the findings have been related to the situation in normal B‐cells of various differentiation stages and to the expression of Ig. Furthermore, an additional Ig transcription factor, PU.1, recently described to be absent from cHL, and a further regulatory element of the Ig gene, the intronic Eµ enhancer, have been studied. BOB.1/OBF.1 and Oct2 were present in all B‐cells expressing Ig, whereas PU.1 proved to be absent from late B‐cell differentiation stages and from a subset of germinal centre B‐cells. Interestingly, there were several normal (eg germinal centre centroblasts and monocytoid B‐cells) and malignant B‐cell populations (eg a proportion of diffuse large B‐cell lymphomas, DLBCLs) that were Ig‐negative, despite their BOB.1/OBF.1 and Oct2 expression. This study further shows that absence of PU.1 alone, as well as inactivation of the intronic Eµ enhancer, is not sufficient to down‐regulate Ig transcription. Taken together, the simultaneous absence of PU.1, Oct2, and/or BOB.1/OBF.1 is unique to Hodgkin and Reed–Sternberg (HRS) cells and cannot be detected in normal B‐cell subsets or B‐cell non‐Hodgkin lymphomas (B‐NHLs). This supports the concept that the down‐regulation of Ig in cHL does not reflect a physiological situation, but a defect probably closely linked to the pathogenesis of cHL. Copyright

Extranodal marginal zone B cell lymphomas of the uvea: an analysis of 13 cases.

The majority of primary lymphoproliferative lesions of the uvea represent low‐grade B cell lymphomas and often display a prominent plasmacellular differentiation. The purpose of the current study was to classify the uveal lymphoproliferations according to the REAL classification; examine the immune profile of the plasmacellular differentiated tumour cells using the plasma cell‐related antigens multiple myeloma oncogene‐1‐protein (MUM1), Vs38c, CD38 and CD138; and to compare this profile with that of mature reactive plasma cells. Following fixation, 13 lymphoproliferative lesions of the uvea were categorized on the basis of their morphology and immunophenotype according to the REAL classification. Included in the immunohistochemistry were B cell‐specific activator protein (BSAP), MUM1, Vs38c, CD38 and CD138. Nested polymerase chain reaction (PCR) was also performed on DNA extracted from paraffin sections for the detection of gene rearrangements of the heavy immunoglobulin chain (IgH). All of the 13 uveal tract lymphoproliferative lesions represented malignant lymphoma of B cell non‐Hodgkin type and could be diagnosed as ‘extranodal marginal zone B cell lymphomas’ (EMZL). The degree of plasmacellular differentiation varied between the tumours. In contrast to their non‐plasmacytoid counterparts, the ‘plasmacytoid’ EMZL tumour cells were negative for the B cell markers CD20 and BSAP, and demonstrated heterogeneous positivity for the markers MUM1, Vs38c, CD38 and CD138. The most consistent marker was MUM1, being observed in all tumours. Co‐expression of all plasma cell markers was observed in four (31%) uveal EMZL. Loss of CD138 expression was observed in six (46%) tumours, of Vs38c expression in five (38%) and of CD38 in one (7%) tumour. Although the diagnosis of malignant lymphoma was unequivocally based on morphological and immunophenotypical features, the molecular analysis was able to demonstrate clonal B cell populations in only one uveal EMZL. All uveal lymphoid proliferations investigated represented EMZL, with the corresponding morphology and immunophenotype as seen in EMZL in other extranodal locations. MUM1, followed by CD38 expression, were the most constant plasma cell antigens in the plasmacytoid EMZL tumour cells, with both Vs38c and CD138 positivity being lost in many tumours. Aberrant immune profiles of plasma cell‐related antigens may be of help in the establishment of malignancy in uveal lymphoproliferative lesions, particularly where interpretation of light chain expression and/or PCR results is difficult. Copyright

Neuroendocrine differentiation is a relevant prognostic factor in stage III-IV colorectal cancer

Objective To determine the prognostic relevance of neuroendocrine differentiation in colorectal cancer. Methods The survival of 116 patients with colorectal cancer of stages III (n = 59) and IV (n = 57) was correlated with the extent of neuroendocrine differentiation. Chromogranin A and synaptophysin were used as neuroendocrine markers. Based on the degree of immunoreactivity for these markers, tumours were classified as 0 (no expression of neuroendocrine markers), 1 (< 2% cells staining positive for neuroendocrine markers) and 2 (> 2% cells staining positive for neuroendocrine markers). Patients were followed up for more than 5 years or until death. Results Seven of 59 (11.8%) stage III cancers and 13/57 (22.8%) stage IV cancers belonged to group 2. The 96 patients of groups 0 and 1 lived for 48.9 months, whereas the 20 patients of group 2 survived for only 18.6 months (Kaplan–Meier survival curves, P < 0.001). The difference was most striking in stage III disease with 79.4 months’ survival for combined groups 0 and 1, and 38.9 months’ survival for group 2 (P < 0.01). Using the multivariate Cox regression model, the presence of more than 2% of cells with neuroendocrine differentiation was found to be an independent prognostic parameter for stage III and IV disease. No correlation was observed between neuroendocrine differentiation and tumour location, grade, depth of invasion or stage. Conclusion Neuroendocrine differentiation is often seen in colorectal cancer. It is an independent prognostic factor in stage III–IV colorectal cancer.

Frequent expansion of Epstein–Barr virus (EBV) infected cells in germinal centres of tonsils from an area with a high incidence of EBV‐associated lymphoma

Burkitts lymphoma (BL) and Hodgkins disease (HD) occurring in developing regions are frequently associated with Epstein–Barr virus (EBV) infection and have a high incidence in childhood. Recent genotyping studies indicate that the tumour cells of both neoplasms represent B cells that contain somatically mutated immunoglobulin heavy chain genes. This implies that the precursors of these neoplasms have participated in the germinal centre (GC) reaction. We therefore presumed that normal lymphoid tissues from children living in developing regions would harbour an increased number of EBV‐infected cells within the GC, when compared with children living in industrialized nations. To test this hypothesis, hyperplastic tonsils from 40 children living in Bahia (Brazil) and 40 from German children were analysed for the presence of EBV‐encoded small nuclear RNA (EBER) and EBV‐encoded proteins by in situ hybridization and immunohistology, respectively. Although the overall EBV infection rate was similar in both groups (50 per cent of Bahian vs. 45 per cent of German cases), a significantly higher number of EBER‐positive lymphoid cells were found in the GCs of 8/20 EBV‐positive tonsils from Brazil (9–89 cells/GC; mean: 14·5 cells/GC per case), while only 3/18 tonsils from Germany displayed a few EBER positive cells (1–9 cells/GC; mean: 0·5 cell/GC per case) in this compartment (p < 0·007). In addition, the EBV‐infected GC cells in Bahian samples resembled centroblasts, exhibited mitotic activity, and in two cases showed expression of EBV‐encoded latent membrane protein (LMP)‐1, findings not present in German samples. These data show that latently EBV‐infected cells participate more frequently in GC reactions in developing regions than in industrialized countries and may abnormally express the oncogenic protein LMP‐1. This could in part explain the higher incidence in this region of EBV association with lymphomas related to GC cells or their progeny, such as BL and HD. Copyright