Minimal residual disease in vitro of a pre-B-lymphoma.

We have studied the survival of clonogenic neoplastic cells of a murine pre-B-lymphoma (BCl-1) of the spleen in culture. We have found quantitative deficiencies such as reduced surface adherence of stromal cells and impaired CFU-F (colony forming units-fibroblast) and pre-CFU-F colony and layer formation in stromal cultures of lymphoma bearing spleen, as compared to cultures from normal spleen. There are two populations of clonogenic BCl-1 lymphoma cells surviving in culture: one population is surface adherent, and the other is non-adherent. Both populations transmit the lymphoma to healthy indicator mice. We hope that this model will be helpful in studying minimal residual leukemic disease [1] in culture.

[Lymphoplasmocytoid immunocytoma of the skin]. / Lymphoplasmozytoides Immunozytom der Haut.

A 68-year-old male patient presented with numerous red-brown papules on the trunk and neck. Cutaneous lymphoplasmocytoid immunocytoma was diagnosed following histological and electron microscopical detection of atypical lymphoid cells in the dermis. Immunohistochemistry revealed a monoclonal proliferation of IgG-kappa-positive B-lymphocytes. There was no extracutaneous manifestation of the lymphoma. Complete remission was affected by total-body irradiation with an electron beam.

[Case reports on the importance of the locoregional radiotherapy of Merkel cell tumor]. / Kasuistische Beiträge zur Bedeutung der lokoregionären Strahlentherapie des Merkel-Zell-Tumors.

The Merkel cell tumor is becoming an increasingly diagnosed primary neoplasm of the skin. This subepidermal tumor is commonly located on the head and neck or extremities of elderly patients. Occasionally misinterpreted as cutaneous metastases, they show a high rate of local recurrence (27 to 52%) and distant metastatic spread (18 to 52%). The definitive diagnosis can be made with immunohistochemistry. Wide surgical excision with postoperative irradiation to the local site and regional lymphatics is the therapy of choice. In seven patients we describe management strategies and discuss their clinical results.

Heterogeneity of tumour necrosis factor production in renal cell carcinoma.

Endogenous tumour necrosis factor (TNF) production was investigated by in situ hybridisation and immunohistochemistry in 8 renal cell carcinoma (RCC) patients at different stages of disease. Analysis of frozen sections of tumour biopsy specimens revealed variable degrees of macrophage infiltration and great heterogeneity in TNF gene expression. Two metastatic tumours investigated showed abundant TNF protein production and marked macrophage infiltration. Based on morphological criteria, these TNF-positive cells most likely belong to the macrophage lineage. Two years after nephrectomy the individual survival time was recorded; however, the small numbers did not yet allow any correlation of TNF production to the clinical course of disease. Further studies will be required to eventually reveal the role of TNF in renal cell carcinoma development.

Frequency and structure of t(14;18) major breakpoint regions in non-Hodgkin's lymphomas typed according to the Kiel classification: analysis by direct DNA sequencing.

We have examined 165 unselected cases of non-Hodgkin's lymphomas for rearrangements involving the t(14;18) major breakpoint region using a polymerase chain reaction (PCR) and direct sequencing of amplified major breakpoint region bcl-2/JH junctional regions. The lymphomas, diagnosed according to the updated Kiel classification, consisted of 33 centroblastic-centrocytic, 37 centroblastic, 27 immunocytic, 10 immunoblastic, 10 centrocytic, 2 lymphoblastic, 2 Ki-1-positive anaplastic large cell, 14 peripheral T-cell, and 4 unclassified lymphomas. In addition 18 chronic lymphocytic leukemias, 2 hairy cell leukemias, and 6 plasmacytomas were studied. In 17 cases a bcl-2/JH gene fusion sequence was amplified by PCR. A bcl-2/JH gene fusion was detected only in three lymphoma subgroups: 13 of 33 centroblastic-centrocytic (39%), 2 of 37 centroblastic (6%), and 2 of 27 immunocytic (8%) were positive. In two cases, major breakpoint region bcl-2 rearrangements verified by genomic Southern analysis were not detected by PCR. Direct sequencing of all 17 PCR-amplified, previously uncharacterized t(14;18) junctional regions provided corroborating evidence for the specificity of the assay. The procedure gave sequencing results even from limited amounts of lymphoma cells as obtained by fine needle aspiration of lymph nodes or from clinically uninvolved sites. Clone-specific sequences were identified due to the involvement of different JH segments, the variations among the exact JH and bcl-2 breakpoint positions, and the extensive incorporation of junctional region (D-) N-nucleotides. These clone-specific sequences allow accurate identification of clinically occult lymphoma cells and reduce the threat of false positive results. The finding of exceptionally long intervening sequences in some of the junctions and the partial homology with published DH segments in three cases support the view that some of the putative N-regions harbor DH regions.

Clinical characteristics of high-grade lymphomas with immune genes in germline configuration.

In a prospective study of 42 high-grade lymphomas which were categorized according to the Kiel classification, the clinical significance of immune genotyping was studied. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were investigated. In 33 cases the immune genotype confirmed the phenotype. In one case with equivocal phenotype a TCR beta-chain rearrangement proved the T-cell origin of the lymphoma. None of the cases showed a bigenotype. There were eight lymphomas with immunoglobulin and TCR beta-chain and gamma-chain genes in germline configuration, which were divided into a group of immature lymphomas and a group of lymphomas with a more mature phenotype. The immature lymphomas had widespread disease, rapid progression, and favorable prognosis after intensive chemotherapy. The group of T-cell and Ki-1 lymphomas with null-cell genotype was clinically heterogeneous. Three of four cases were secondary lymphomas after lymphomatoid papulosis, lymphomatoid granulomatosis, or Hodgkin's disease. All cases presented with extranodal involvement. Only one of these patients is in continuous remission. In conclusion, the lack of immunoglobulin and TCR beta-chain and gamma-chain gene rearrangements does not exclude the diagnosis of high-grade malignant lymphoma, especially in cases with unusual extranodal involvement. However, the DNA analysis identifies a null-cell genotype subset of high-grade lymphomas which may have clinical significance.

High incidence of Epstein-Barr virus genomes in Hodgkin's disease.

Tissue specimens from 198 cases of Hodgkin's disease and 151 non-Hodgkin lymphomas, as well as 34 nonmalignant lymph node biopsies were examined for the presence of Epstein-Barr virus (EBV) DNA by polymerase chain reaction. Epstein-Barr virus-specific DNA sequences were detected in DNA extracts from frozen and/or paraffin-embedded tissues of 58% of Hodgkin's disease cases. High and low grade non-Hodgkin lymphomas as well as chronic lymphocytic leukemia biopsies contained EBV DNA in 26%, 14%, and 7% of the cases, respectively. Ten percent of the control lymph nodes with normal histology were EBV positive. In Hodgkin's disease biopsies, subsequent in situ hybridization revealed an exclusive localization of the viral DNA in the tumor cells. These findings suggest an involvement of EBV in the pathogenesis of Hodgkin's disease in a substantial proportion of cases.

Heterogeneity of immunoglobulin gene rearrangements in B-cell lymphomas.

We have examined 69 B-cell non-Hodgkin's lymphomas (NHL) for rearrangements of the immunoglobulin (Ig) or T-cell antigen receptor (TCR) genes. The lymphomas were assigned to the categories of the Kiel classification and their B-cell nature was confirmed by immunostaining. Only 2 cases (with CLL) displayed clonal T beta-chain TCR gene rearrangements together with rearranged heavy- and light-chain Ig genes. The remaining 67 lymphomas had a germline beta-chain TCR-gene configuration. Three different patterns of Ig gene rearrangements were identified; (A) presence of both heavy- and light-chain rearrangements (H+L+); (B) rearrangement of heavy-chain gene only (H+L-); (C) heavy- and light-chain genes in germline configuration (H-L-). All the 45 low-grade NHLs and the 4 immunoblastic lymphomas exhibited pattern A and all had their kappa gene rearranged or deleted. Of 24 low-grade lymphomas tested, 13 (54%) had an addition rearrangement of the lambda light-chain gene. In contrast, the 19 high-grade centroblastic (cb) B-NHLs had distinct patterns of Ig-gene rearrangement: 12 with pattern A, 4 with B and 2 with C. In this group only 2 of 17 (12%) cases analyzed had evidence of lambda light-chain rearrangement whereas 12 of 18 (67%) had a kappa gene rearrangement or deletion. In one case expressing sIgM/lambda and with heavy chain Ig-rearrangement, no DNA was available for Ig light-chain analysis.

[Primary cutaneous Ki-1-lymphoma]. / Das primäre kutane Ki-1-Lymphom.

With the help of immunohistochemical techniques and new antibodies developed in the last few years, we were able to identify 2 cases of nodular tumors of the skin as highly malignant Ki-1 lymphomas. The clinical course was characterized by the development of multiple skin tumors without any evidence of systemic involvement. After the excision of several skin tumors; we performed chemotherapy with the MACOP-B regimen. In one of our patients, however, this treatment could not prevent the occurrence of further tumors. Possibly, the cutaneous anaplastic Ki-1 lymphomas presented here are closely related to or even identical with mycosis fungoides d'emblée.

[Epstein-Barr virus and malignant lymphomas--studies on incidence and localization of viral DNA]. / Epstein-Barr Virus und maligne Lymphome--Untersuchungen zur Inzidenz und Lokalisation des Virus.

Tissue specimens from 198 cases of Hodgkin's disease and 151 non-Hodgkin lymphomas as well as 34 non-malignant lymph node biopsies were examined for the presence of Epstein-Barr virus (EBV) DNA by polymerase chain reaction. EBV-specific DNA sequences were detected in DNA extracts of 58% of Hodgkin's disease biopsies. Non-Hodgkin lymphomas and benign lymph node lesions were associated with EBV in far smaller proportions. In Hodgkin's disease biopsies, subsequent in situ hybridization revealed an exclusive localization of EBV DNA in the tumour cells, suggesting an involvement of EBV in the pathogenesis of Hodgkin's disease in a substantial proportion of cases.

Polymerase Chain Reaction Analysis of t(14;18) Junctional Regions in B-Cell Lymphomas.

The polymerase chain reaction (PCR) procedure was used for rapid and highly specific amplification of the t(14;18) bcl-2/JH DNA junctional regions in B-cell lymphomas. By using Taq-polymerase and relatively long oligonucleotide primers-a 33-mer for bcl-2 and an universal 25-mer complementary to the JH consensus sequence-the primer annealing and primer extension steps could be carried out at the same temperature (70°C), thus markedly reducing the reaction time and significantly improving the specificity of the reaction. The specificity of the amplification allowed visual identification of the bcl-2/JH PCR-products in ethidium bromide stained agarose gels. DNA-sequence analysis of PCR-amplified, previously uncharacterized t(14; 18) junctional regions, confirmed the specificity of this assay. Moreover, preliminary data show that the procedure is capable of documenting the presence of occult lymphoma cells in both the peripheral blood and bone marrow.

Regulation of tumor necrosis factor gene expression in colorectal adenocarcinoma: in vivo analysis by in situ hybridization.

Tumor necrosis factor (TNF) produced by macrophages is thought to contribute to the host defense against development of cancer. However, since tumor cells themselves are able to produce TNF, it is conceivable that TNF may also play an adverse pathological role in carcinogenesis. To better understand the functional significance of TNF in neoplastic disease, we have determined the cellular source of TNF activity produced in 10 patients with colorectal cancer. Northern blot analysis of RNAs extracted from fresh biopsy specimens revealed detectable TNF mRNA levels in all instances. By using in situ hybridization of frozen sections, scattered cells expressing TNF mRNA could be discerned. Based on morphological criteria, these TNF-positive cells most likely belong to the macrophage lineage. Macrophages in normal tissue surrounding the tumor did not express TNF mRNA, suggesting that macrophage activation occurs locally at the site of neoplastic transformation. Immunohistochemistry using anti-TNF monoclonal antibodies revealed that less than 1% of tumor-infiltrating macrophages synthesize TNF protein. Thus we present evidence that in colorectal cancer only a small proportion of tumor-infiltrating macrophages produces TNF, indicating that the microenvironment of the tumor provides adequate, yet suboptimal, conditions for macrophage activation.