[Multiplexed methylation analysis--a new technology to analyse the methylation pattern of laser microdissected cells of normal breast tissue, DCIS and invasive ductal carcinoma of the breast]. / Multiplexierte Methylierungsanalyse--Eine neue Technik zur Analyse der Methylierung mikrodissezierter Zellen verschiedener Gewebekomponenten des duktalen Mammakarzinoms.

AIMS: DNA methylation has been shown to play an important role in breast cancer pathogenesis, but up until now it is not clear how the tissue components contribute to the overall methylation of the sample, because microdissection does not provide sufficient material for most standard methylation assays. METHODS: We developed a technology to analyse several methylation markers in a limited number of cells dissected from tissue sections. To evaluate the technology, we analysed, among others, the methylation markers PITX2, RASSF1A and TFF1 in 79 samples of three PITX2 methylation positive invasive ductal carcinomas. The microdissected samples were from the invasive part, the intraductal part, the stroma, tumor infiltrating lymphocytes, chest wall muscle, adipose tissue and healthy ducts. RESULTS: The multiplexed methylation analysis allows for the quantitative analysis of methylation patterns in microdissected samples with as few as 100 genome copies. In all analysed patients PITX2 and RASSF1A were highly methylated in invasive and intraductal carcinoma cells compared to other tissue components. TFF1 behaved inversely. PITX2 showed some methylation in normal adjacent breast tissue. The methylation of the individual markers varied little within one tissue type and between blocks. CONCLUSIONS: This technology is a powerful tool to analyse the methylation of multiple markers in different microdissected tissue components. Methylation patterns may differ significantly between different markers and tissue components. This technology may help to analyse different transitions states of breast and other cancers.

[DNA methylation: potential for diagnosis, prognosis and therapy--prediction in oncology]. / DNA Methylierung in der Molekularen Pathologie: Potential für die Diagnose, Prognose und Therapie--Prädiktion in der Onkologie.

In the last decades, a great variety of novel therapeutic regimens have become available for cancer. These therapies often are very specific and thus effective only in subsets of cancer patients. This has led to an increased need for the clinician to specifically choose the therapeutic strategy the patient profits most from, but at the same time avoiding over-treatment. This situation has a profound impact on the methods in diagnostic tumor pathology, since it requires precise pre-therapeutic tumor characterization to support the clinical management of the individual case. A common and early event in cancer is aberrant DNA methylation within gene regulatory regions which affects a variety of genes with different functions. Altered DNA methylation has been shown to carry prognostic as well as predictive information. As a DNA-based marker that can be analyzed in routine formalin fixed tissue, DNA methylation offers a series of technical advantages which allow for introduction into a routine lab. Here we review well established DNA methylation markers and discuss their potential for clinical use.

Expression of CD94/NKG2A and killer immunoglobulin-like receptors in NK cells and a subset of extranodal cytotoxic T-cell lymphomas.

Thirty-two natural killer (NK) and cytotoxic T-cell lymphomas and 14 noncytotoxic nodal T-cell lymphoma controls were immunostained with the use of monoclonal antibodies reactive against NK-cell receptor (NKR) molecules (CD94, NKG2A, p58.2, p58.1, p140, p70, p50.3). All NK-cell lymphomas (4 nasal/oral and 1 intestinal) expressed at least 1 NKR, the CD94/NKG2A complex. Two were positive for 1 or more killer immunoglobulin-like receptors. Of 15 extranodal cytotoxic T-cell lymphomas, 3 expressed CD94, including 2 intestinal and 1 hepatosplenic gammadelta T-cell lymphomas. In contrast, none of the nodal lymphomas were positive. Detection of NKRs may provide a useful tool to confirm the diagnosis of NK-cell lymphomas and to delineate a subgroup of cytotoxic T-cell lymphomas. Expression of NKRs only in extranodal cytotoxic T-cell lymphomas might reflect differences in the homing capabilities of cytotoxic T cells expressing NKRs in normal individuals and might be influenced in part by localized chronic immune reactions.

Oligoclonal expansions of T-cell repertoire in gastric mucosa associated lymphoid tissue type B-cell lymphoma and adjacent gastritis.

Local stimulation by Helicobacter pylori (HP), autoantigen, and a concurrent T-cell-mediated stimulation of B cells are believed to play an important role in gastric mucosa-associated lymphoid tissue (MALT) type B cell lymphomagenesis. Many autoimmune diseases have shown to lead to a skewed T-cell repertoire with autoantigen specific expansions and deletions. Characterization of lymphoma and gastritis areas of seven gastrectomy specimens using a T-cell receptor beta variable chain (TCR betaV) family-specific reverse transcriptase (RT)-polymerase chain reaction (PCR) assay and fluorescence-activated cell sorter (FACS) analysis revealed a local chronic and acute activation of T cells in lymphoma and an oligoclonal T-cell repertoire in gastritis and in lymphoma, partially sharing the same clones. Local activation and a partial identity suggest that an antigenic challenge caused by a common local pathogen may still continue to take place in MALT type lymphoma as in gastritis, consistent with the view that gastritis may be a precursor lesion of MALT type lymphoma. Expansions that were found only in one of the compartments suggest that also an immune hyperstimulation may contribute to the T-cell repertoire, possibly because of certain tissue antigens. Deletions of TCR betaV families found only in gastritis underline the view that autoantigen may play an important role in its pathogenesis.

Molecular analysis of rheumatoid factor (RF)-negative B cell hybridomas from rheumatoid synovial tissue: evidence for an antigen-induced stimulation with selection of high mutated IgVH and low mutated IgVL/lambda genes.

The mutational pattern of IgVH and IgVL genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgVH genes of the B cell hybridomas belonged to the VH3 family (DP42; DP47, n = 2; DP53), the VH1 family (DP75), the VH4 family (DP71) and the VH5 family (DP73); 7/7 IgVH genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgVH genes and the mean R/S ratio of all IgVH genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgVL/lambda genes belonged to the Vlambda1 family (DPL2, DPL5, DPL8nf), the Vlambda2 family (DPL11, n = 2) and to the Vlambda6 family (IGLV6S1); 6/6 IgVL genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgVL genes and the mean R/S ratio of all IgVL was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4+ follicular dendritic cells and Ki-67+ proliferating cells and a dominance of IgA+ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgVH and IgVL/lambda genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.

Most CD56+ intestinal lymphomas are CD8+CD5-T-cell lymphomas of monomorphic small to medium size histology.

The expression of the natural killer (NK) cell marker CD56 has been reported to occur in NK cell lymphomas/leukemias and a small group of peripheral T-cell lymphomas but has not been studied extensively in primary intestinal non-B-cell lymphomas. Normal human jejunal intraepithelial lymphocytes (IELs) are mainly T-cell receptor (TCR)-alphabeta+CD3+CD8+CD5low and include an approximately 15% fraction of CD56+ cells that could be the cells of origin for CD56+ intestinal T-cell lymphoma (ITL). To test this hypothesis, 70 cases diagnosed as ITL were immunophenotyped, and 15 CD56+ cases (21%) were identified. The majority of the CD56+ lymphomas was of monomorphic small to medium-sized histology, shared the common phenotype betaF1+/-CD3epsilon/cyt+CD8+CD4-CD5-CD57-TIA-1+ and had clonally rearranged TCR gamma-chain genes. In contrast, the CD56- lymphomas were mainly composed of pleomorphic medium and large cells or had a morphology most consistent with anaplastic large-cell lymphoma and were mostly CD8-. These findings suggest that the majority of CD56+ intestinal lymphomas are morphologically and phenotypically distinct T-cell lymphomas most likely derived from activated cytotoxic CD56+CD8+ IELs. Some overlapping histological and clinical features between CD56+ and CD56- ITLs indicate that the former belong to the clinicopathological entity of ITL. The consistent expression of cytotoxic-granule-associated proteins introduces ITL (both CD56+ and CD56-) into the growing family of usually aggressive extranodal lymphomas of cytotoxic T-cell and NK-cell derivation. In contrast to putative NK-cell lymphoma of the sinonasal region, intestinal NK-cell lymphoma seems to be very rare.

Intraclonal offspring expansion of gastric low-grade MALT-type lymphoma: evidence for the role of antigen-driven high-affinity mutation in lymphomagenesis.

Recent studies have shown that gastric mucosa-associated lymphoid tissue (MALT)-type lymphoma B cells are the malignant counterparts of hypermutated, postgerminal-center memory B cells. To further elucidate the role of antigen selection in the evolution of gastric low-grade MALT-type lymphoma, we analyzed intraclonal variations of the immunoglobulin heavy-chain variable region (Ig VH) genes expressed in three cases of lymphoma. The Ig VH genes expressed by tumor cells were amplified by PCR using primers for individual tumor-specific markers (complementarity-determining region 3 (CDR3)) and primers for six VH family leaders and then sequenced. The corresponding germ-line VH gene from these patients was also sequenced. The somatic mutations were highly concentrated in the CDR or framework region, with a clustering of replacement mutations in the CDR but only a few in the framework region. Each of the Ig VH genes of tumor cell clones of Cases 1 and 3 showed different mutations, whereas Case 2 showed no intraclonal variation. Although all three mutation pattern variants of Cases 1 and 3 occurred in postgerminal memory B cells, only one offspring from each case resulted in a dominant expansion. This finding suggests that antigen-driven high-affinity somatic mutation may play an important role in the expansion of intraclonal offspring from low-grade MALT-type lymphomas.

Receptor analysis of idiotype antibodies derived from MALT type B-cell lymphoma hybridomas.

The generation of human antibodies derived from extranodal MALT type B-cell lymphomas allows to evaluate steps in their pathogenesis as well as to establish potential immunological therapies. Intraclonal diversity and the existence of bystander nonmalignant B-cells outline the need for reliable identification of the tumor immunoglobulin representing hybridomas. Human heterohybridomas were generated from five cases of MALT type B-cell lymphomas (4 low grade, one high grade) by the fusion of lymphoma B-cells with the murine myeloma cell line NSO and tested for isotype identity with the tumor. RT-PCR using VH Fr1/JH primers was performed with RNA of tumors and hybridomas that share the same isotype with the tumor. PCR-products were sequenced directly. In each case lymphomas were hybridized with a comparable fusion efficiency. DNA sequence analysis of the immunoglobulin heavy chain identified one or more hybridomas derived from the tumor. However some other hybridomas which share the same isotype with the tumor, may be different in their VH family or their sequence. Hybridomas can be used as a tool for the research on the MALT lymphoma immunoglobulin receptor. For the identification of tumor immunoglobulin, secreting hybridomas sequencing and the check of molecular identity is indispensable after isotype determination.

Specific and sensitive two-step polymerase chain reaction assay for the detection of Salmonella species.

A polymerase chain reaction (PCR) assay was applied for the selective amplification of a characteristic sequence within a Salmonella-specific chromosomal fragment. A two-temperature PCR cycle enhanced both the speed and overall sensitivity of the amplification procedure. Twenty-one well-characterized Salmonella strains and a number of non-Salmonella strains were tested. With the exception of the rarely isolated Salmonella arizonae strain, the PCR-based approach enabled the specific identification of Salmonella with a detection limit of 10(3) organisms. In combination with a nested PCR assay, as few as ten organisms were detectable. Specificity was demonstrated as no distinct amplification products were detectable with any of the tested non-Salmonella strains. With a pre-enrichment step using paramagnetic anti-Salmonella beads, an increase in sensitivity was observed in the case of clinical samples while the amplification process was not influenced.

[Hybridomas as tools for research on MALT type lymphomas]. / Hybridome als Hilfsmittel bei der Untersuchung der Lymphome vom MALT Typ.

The antigen receptor of the MALT lymphoma cells provides crucial information, which may help to elucidate the pathogenesis of that tumor. Therefore hybridomas were produced from five MALT type lymphomas. The identity was proven by sequencing the VH chain of the tumor and the hybridomas. It could be shown the one step of subcloning and limiting dilution is necessary to obtain a monoclonal hybridoma in most cases. So it is indispensable to check the identity of the tumor antibody and the hybridoma antibody for each hybridoma. Otherwise it may be possible that bystander cells infiltrating the tumor, but not belonging to the malignant clone, get fused. Furthermore we provide evidence of an intra tumoral diversity within the MALT type lymphoma that was reflected by the different mutation pattern of the tumor derived hybridomas. Therefore the hybridomas are allowing investigation on the protein level about the hypermutation of the genes of the MALT lymphoma antibodies and the possible role antigenic selection within the tumor.

Autoantigen receptors in extranodal non-Hodgkin B-cell lymphomas.

Extranodal lymphomas of B-cell origin show a biologic behavior different from nodal lymphomas and an unexplained preference of a specific histologic type, the so called MALT-type. The lymphoma cells of this type show specific colonization of lymphoid follicle centers and subepithelial plasma cell differentiation suggesting that the tumor is immunologically responsive in vivo to as yet unidentified signals. Their tumor immunoglobulin has been shown to recognize autoantigens but lacks reactivity with bacterial antigens. Recent studies provide certain evidence, that antigen may play a role in the pathogenesis of gastric MALT-type lymphoma. The malignant B cells responded to antigen-triggering in vitro and have undergone somatic hypermutation probably in response to antigen selection. Therefore it is possible, that the local stimulation of lymphoma-precursor B-cells triggered either by exogenous (e.g. Helicobacter pylori) or endogenous (e.g. autoantigen) antigen in co-operation with the reactive inflammatory infiltrates establishes the local tumor growth until further mutagenic alterations guide tumor development and progression.