Hans-Heinrich Wacker

Lymphomas of the female genital tract: a study of 186 cases and review of the literature.

Malignant lymphomas in the female genital tract are rare, and those arising from this tissue system are extremely uncommon. Most pertinent reports lack clear references to the accepted classifications or failed to apply immunomarkers and molecular techniques for a reliable diagnosis. We analyzed a large group of patients with primary and secondary lymphomas of the female genital tract classified on the basis of the recent WHO consensus. A total of 186 patients with malignant lymphoma detected in the female genital tract were selected from the files of the Kiel Lymphoma Registry covering the period of 1974 to 2004. Stringent criteria were applied to separate systemic versus secondary lymphomas. All cases were reviewed on the basis of conventionally stained sections, relevant immunohistochemistry using the alkaline phosphatase/anti-alkaline phosphatase technique, and clinical information, as far as available. When required, gene rearrangement analysis was performed, including TCR-gamma chain gene and the three FR fragments of the IgG heavy chain gene. In addition, typical chromosomal translocations were detected by means of the FISH technique to verify the diagnosis, where needed. Thirty-seven percent of the cases were systemic lymphomas and 63% were mostly extranodal lymphomas primary to the female genital tract. The adnexa were involved in 87 cases, followed by uterine corpus in 23 cases, uterine cervix in 17 cases, portio in 9 cases, vagina in 11 cases, and vulva including clitoris in 8 cases. In 31 cases, two or more adjacent sites were involved. In both (primary and secondary) groups, the adnexa were the prevailing site of involvement. As expected, the overwhelming majority of cases were of B phenotype. The most frequent type of lymphoma proved to be diffuse large B-cell lymphoma, closely followed by follicular lymphoma, including all 3 grades of malignancy. Burkitt lymphoma showed a rather similar frequency. Marginal zone lymphoma occurred exclusively as primary lesions in the uterine mucosa. Lymphoplasmacytic lymphoma was restricted to the vulvo-vaginal area and occurred in women over 60 years of age. In conclusion, our study provides a thorough overview of various types of lymphoma affecting the female genital tract primarily or secondarily, which were classified on the basis of a widely accepted WHO classification. Although quite rare, our report should remind the pathologist of considering malignant lymphomas while reading biopsies taken from female genital organ.

Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the ‘Ki-67’proteins

Abstract. The monoclonal antibody (mab) Ki‐67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki‐S3 and Ki‐S5 detect proliferating cells in frozen and paraffin‐embedded tissues. They recognize proteins with the same molecular mass as Ki‐67 in Western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the Ki‐67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA‐deduced amino acid sequence already published for the Ki‐67 proteins.

Ki-M2R, a new specific monoclonal antibody, discriminates tissue macrophages from reticulum cells and monocytes in vivo and in vitro.

Utilizing rat peritoneal macrophages as the immunogen, a new monoclonal antibody enabling differential monitoring of the mononuclear phagocyte system (MPS) by immunohistochemistry has been raised. Designated Ki‐M2R, this antigen could be detected with the immune alkaline phosphatase reaction on all macrophages including those of bone marrow, lymphatic sinuses, lymphoid follicles, splenic red pulp, and von Kupffer cells of the liver, as well as on macrophages of connective tissue, renal interstitial tissue, serous cavities, and gastrointestinal tract. Langerhans cells—the MPS‐derived reticulum cells of the epidermis—interdigitating reticulum cells, and dendritic reticulum cells of lymphoid follicles were invariably negative. Blood monocytes were rendered positive only after evolving into macrophages upon appropriate stimulation. Thus, Ki‐M2R selectively labels monocytes after transformation into macrophages.

Prevalence, Clinical Pattern, and Outcome of CNS Involvement in Childhood and Adolescent Non-Hodgkin's Lymphoma Differ by Non-Hodgkin's Lymphoma Subtype: A Berlin-Frankfurt-Münster Group Report

PURPOSE We analyzed the prevalence, clinical pattern, and prognostic impact of CNS involvement in a large cohort of children and adolescents diagnosed with non-Hodgkins lymphoma (NHL), with special attention to differences according to NHL subtype. PATIENTS AND METHODS From October 1986 to December 2002, 2,381 patients (median age, 9.37 years; range, 0.2 to 23.8 years; female-to-male ratio, 1:2.7) from Germany, Austria, and Switzerland were registered. A total of 2,086 patients were eligible for the consecutive multicenter protocols NHL-Berlin-Frankfurt-Münster [BFM] -86, NHL-BFM-90, and NHL-BFM-95, and could be evaluated for outcome. RESULTS CNS involvement was diagnosed in 141 (5.9%) of 2,381 patients and was associated with an advanced stage of NHL. The percentage of CNS-positive patients was 8.8% for Burkitts lymphoma/Burkitts leukemia (BL/B-ALL), 5.4% for precursor B-lymphoblastic lymphoma (pB-LBL), 3.3% for anaplastic large-cell lymphoma, 3.2% for T-cell-LBL, 2.6% for diffuse large B-cell lymphoma, and 0% for primary mediastinal large B-cell NHL (P < .001). Most CNS-positive patients with pB-LBL, T-LBL, or BL/B-ALL had meningeal disease. The probability of event-free survival (pEFS; +/- SE) at 5 years was 85% +/- 1% for the 2,086 protocol patients (median follow-up, 6.5 years; range, 0.3 to 17.7 years). For the 112 CNS-positive patients, pEFS was 64% +/- 5%, compared with 86% +/- 1% for the 1,927 CNS-negative patients (P < .001). Although CNS disease had no impact on pEFS for advanced-stage T-LBL patients, CNS-positive patients with BL/B-ALL had a worse average outcome than CNS-negative patients with stage IV BL/B-ALL (60% +/- 5% v 81% +/- 3%; P < .001). In multivariate analysis, CNS disease was the strongest predictor for relapse in BL/B-ALL patients with advanced-stage disease. CONCLUSION Six percent of childhood/adolescent NHL patients were CNS positive. However, the prevalence, pattern, and prognostic impact of CNS involvement differed among NHL subtypes.

Tumor Sclerosis but Not Cell Proliferation or Malignancy Grade Is a Prognostic Marker in Advanced-Stage Follicular Lymphoma: The German Low Grade Lymphoma Study Group

PURPOSE Follicular lymphoma is an indolent lymphoma with a long median overall survival. However, a considerable number of patients die within the first 2 years after the onset of the disease. Because the treatment options vary with respect to antitumor effect and potential toxic adverse effects, the identification of high-risk patients would be helpful in directing therapeutic decisions in individual patients. Several histopathologic biomarkers for risk stratification have been suggested, but most markers have not been validated in patients treated in prospective trials. PATIENTS AND METHODS We report a comprehensive approach to evaluate histopathologic biomarkers, including WHO grade, histology, and proliferation and quantitation of immune bystander cells, in 158 patients with nodal advanced-stage follicular lymphoma treated first line within a randomized trial. RESULTS Tumor sclerosis was a significant prognostic marker of poor overall survival that was independent of the Follicular Lymphoma International Prognostic Index (FLIPI). WHO grade, proliferation, and total T-cell or macrophage content were not associated with overall survival. CONCLUSION The presence of sclerosis within the lymphoma is a marker of poor overall survival that is independent of the FLIPI. The quantification of macrophage or absolute T-cell content, grading, and proliferation are of no help in predicting the outcome of FL. Future studies need to identify surrogate markers for the prognostic immune signatures identified by gene expression profiling. Most importantly, new prognostic markers need to be confirmed in patients treated within prospective trials.

Reduced T‐cell receptor CD3ζ‐chain protein and sustained CD3ε expression at the site of mycobacterial infection

Control of mycobacterial infection by the cellular immune system relies both on antigen‐presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T‐cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T‐cell receptor signalling, we examined the protein expression of T‐cell signal transduction molecules (CD3ζ, ZAP‐70, p59fyn, p56l ck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3ζ‐chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3ε and CD3ζ expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3ε and CD3ζ expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3ζ compared to CD3ε. Using double immunofluorescence analysis, virtually no CD3ζ expression could be detected in comparison to the CD3ε expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3ζ‐chain expression, which may be restored by cytokines. IL‐2‐enhanced ζ‐chain expression and T‐cell effector functions, defined by interferon‐γ release, in M. tuberculosis‐specific and human leucocyte antigen‐DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3ζ is essential for CD3 signalling and for eliciting T‐cell effector functions, reduced CD3ζ protein expression could result in altered signal transduction and inefficient T‐cell effector functions. Alternatively, reduced CD3ζ‐chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils

SummaryThe aim of the present paper is to detect two different antigens simultaneously in a single slide. In cryostat sections of human tonsils, B-lymphocytes of follicle mantle-bearing surface IgD were immunostained with the alkaline phosphatase method using monoclonal anti IgD. The subsequent staining for T-lymphocyte subsets (T-helper and T-suppressor lymphocytes) was performed again with the alkaline phosphatase method using one of the monoclonal antibodies OKT 4, OKT 8, Leu 3a, Leu 2a. The best results with the alkaline phosphatase method were achieved using naphthol AS phosphate and Fast Blue BB for the revelation of the first antigen and naphthol AS-BI phosphate and diazotized New Fuchsin for the second.

Analysis of T-Cell Subpopulations in T-Cell Non-Hodgkin’s Lymphoma of Angioimmunoblastic Lymphadenopathy with Dysproteinemia Type by Single Target Gene Amplification of T Cell Receptor- β Gene Rearrangements

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin’s lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4+ and CD8+ T cells and proliferating Ki67+ cells of seven cases were micromanipulated from frozen tissue sections. TCRβ gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4+, and not among CD8+ T cells, indicating that the tumor clones in AILD usually derive from CD4+ T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRβ chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.

Immunophenotyping of dermal spindle cell tumors: diagnostic value of monocyte marker Ki-M1p and histogenetic considerations.

Various studies have reported the utility of anti-CD34 staining in the differential diagnosis of dermal spindle-cell tumors. To investigate whether monoclonal antibody Ki-M1p might add practical diagnostic information, we examined a total of 120 cutaneous spindle cell neoplasms using a panel of markers. Anti-CD34 antibody QBEnd/10 consistently stained dermatofibrosarcomas, Kaposis sarcomas, neurofibromas, and, to a lesser extent, hemangiopericytomas. A positive reaction was also found in > 18% of the dermatofibromas. Ki-M1p staining showed an intense immunoreaction in all dermatofibromas, whereas no reactivity was observed in dermatofibrosarcomas. In addition, a subset of cells was labeled in atypical fibroxanthomas and Kaposis sarcomas. Neurofibromas, spindle-cell hemangioendotheliomas, and hemangiopericytomas were negative. Dermatofibrosarcomas and atypical fibroxanthomas also moderately expressed smooth muscle-specific actin. Immunohistochemically, a discrimination between dermatofibrosarcomas and neurofibromas was possible only by means of an antibody against the nerve growth factor receptor. We conclude that the combination of several antibodies, in particular anti-CD34 and Ki-M1p, may improve the accuracy of diagnostic immunohistochemistry in the field of cutaneous spindle cell tumors. We speculate that dermatofibroma is primarily a macrophage-rich inflammatory lesion in which cytokine secretion induces a secondary proliferation of fibroblasts, whereas dermatofibrosarcoma is likely to issue from primitive dermal cells of uncertain origin.

Detection of Human Topoisomerase IIα in Cell Lines and Tissues: Characterization of Five Novel Monoclonal Antibodies

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the α-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase IIβ was ruled out by testing the antibodies on crude extracts from yeast cells expressing the β-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.