Annette Zimpfer

Expression of B-Cell Markers in Classical Hodgkin Lymphoma: A Tissue Microarray Analysis of 330 Cases

Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma arise from B-lymphocytes. However, classical markers of the B-cell phenotype, such as CD20, are present only in about 25% of the cases. The aim of the present study was to assess expression of the B-cell–related antigens CD20, CD79a, and CD138 in classical Hodgkin lymphoma using a tissue microarray consisting of 330 classical Hodgkin lymphoma cases. Expression of CD15, CD20, CD30, CD79a, CD138, and latent membrane protein 1 of Epstein-Barr virus was assessed by immunohistochemistry, and the methodology was validated by direct comparison of CD20 expression on the tissue microarray cores with corresponding large sections. The influence of the number of arrayed sample cores on the obtained expression levels of CD20 was analyzed by comparing the results from single, duplicate, and triplicate cores. Two-hundred fifty-three (77%) of the 330 cases were morphologically representative. CD20 was expressed in 84 cases (33%), CD79a in 26 (10%), and CD138 in 2 (1%), respectively. CD20 and CD79a were co-expressed in 16 cases (P < .005), and expression of CD20 correlated inversely with CD15 (P < .01). Comparing the tissue microarray results with those from conventional sections for expression of CD20 yielded a concordance of 94% (63/67). Examining one, two, and three cores from individual cases revealed positivity for CD20 at 24% (61/253), 32% (82/253), and 33% (84/253), respectively. We conclude that B-cell markers are expressed in 38% of classical Hodgkin lymphoma in the following rank order: CD20>CD79a≫CD138. The use of two cores per tissue sample renders the tissue microarray technology effectively representative and thus very useful for high-throughput evaluation of heterogeneously expressed markers in classical Hodgkin lymphoma.

Tissue microarray technology: principles, pitfalls and perspectives—lessons learned from hematological malignancies

Detection, validation and incorporation into clinical use of new diagnostic, prognostic and therapeutic molecular targets in modern medical science should be time- and cost-efficient. Here, we discuss the principles, advantages, disadvantages and possible pitfalls of tissue microarray (TMA) technology, a powerful tool for high throughput large-scale morphological in situ analysis of molecular targets. Based on recent observations from molecular profiling of hematological malignancies, we review potential TMA applications assessing molecular targets in large collectives of tissue specimens.

Rare expression of T-cell markers in classical Hodgkin's lymphoma

Hodgkins and Reed–Sternberg cells of classical Hodgkins lymphoma are primarily of B-cell origin, although there are instances of T-cell antigen expression suggesting T-cell origin. We comprehensively analyzed expression of various T-cell antigens in 259 classical Hodgkins lymphoma cases using the tissue microarray technique. Expression of the T-cell antigens CD2, CD3, CD4, CD5, CD7 and CD8 was assessed by immunohistochemistry. Hodgkins and Reed–Sternberg cells of T-cell marker-positive cases were microdissected and analyzed by a multiplex polymerase chain reaction for clonal immunoglobulin heavy chain- and T-cell receptor γ gene rearrangements. In all, 12 cases (5%) expressed at least one T-cell marker in the following order: CD2 in 11 cases, CD4 in five, CD3 in two, and CD5 and CD8 in one case each; there were no CD7-positive cases, and five cases (2%) expressed more than one T-cell antigen. In positive cases, a mean fraction of 40% of the Hodgkins and Reed–Sternberg cells (range 20–100%) expressed the analyzed T-cell markers. Two cases (<1%) evidenced clonal T-cell receptor γ gene rearrangement. Phenotypic expression of T-cell antigens in Hodgkins and Reed–Sternberg cells of classical Hodgkins lymphoma is rare (5%), while genotypically, less than 1% of classical Hodgkins lymphomas are of possible T-cell origin. Therefore, T-cell antigen expression on Hodgkins and Reed–Sternberg cells is aberrant in the majority of cases and only infrequently classical Hodgkins lymphomas are of T-cell origin.

Prognostic significance of CD44 expression in diffuse large B cell lymphoma of activated and germinal centre B cell-like types: a tissue microarray analysis of 90 cases

Background: Gene expression profiling of diffuse large B cell lymphoma (DLBCL) revealed three disease types: germinal centre B cell-like (GC), activated B cell-like (ABC), and a “third” type. Expression of CD44 variant isoforms (CD44v) is associated with an unfavourable clinical outcome in DLBCL, but previous studies did not consider the clinicopathological heterogeneity of this disease. Aims: To analyse the expression and prognostic significance of CD44 in DLBCL types. Methods: A tissue microarray (TMA) comprising 90 DLBCLs was constructed. CD10, CD20, bcl-2, bcl-6, CD44 standard isoform (CD44s), and CD44v4, CD44v6, and CD44v9 were analysed immunohistochemically and correlated with clinical follow up. Results: TMA expression of CD10, CD20, bcl-2, and bcl-6 showed 100% concordance with results from conventional sections in 60 cases. Samples were segregated into 22 GC (bcl-6+/CD10+/bcl-2−), 25 ABC (bcl-6−/CD10−/bcl-2+), and 35 unclassifiable DLBCLs. Overall survival (OS) at 30 months was 89%, 44%, and 58% in GC, ABC, and unclassified types, respectively. CD44v6 was coexpressed with bcl-2, appeared predominantly on bcl-6 negative cases, and correlated with disease stage. Cases negative for CD44s could be separated into CD44v6 negative (OS, 82% at 70 months) and CD44v6 positive (OS, 58%). Conclusions: TMA technology is useful for immunophenotyping and clinicopathological analysis of large lymphoma populations. The GC phenotype of DLBCL is of independent prognostic significance for OS. Expression of CD44v6 correlates with disease stage, and might contribute to lymphoma dissemination. CD44v6 is expressed predominantly in ABC DLBCL, and in CD44 negative cases is associated with worse OS.

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

Aberrant expression of cell cycle regulators in Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma.

The characteristic Hodgkin and Reed–Sternberg cells of classical Hodgkins lymphoma, although highly positive for proliferation markers, do not accumulate to excessive cell numbers. These cells are characterized by abortive mitotic cycles, leading to multinucleation or cell death in mitosis. We have previously described high expression of G1-phase cyclins in classical Hodgkins lymphoma, which could explain the high percentage of cells staining for proliferation markers. To further our understanding of proliferation control in classical Hodgkins lymphoma, we extended our immunohistochemical analysis to the main S-phase cyclin, cyclin A, and its regulators p21CIP1 and p27KIP1. Expression of proliferating cell nuclear antigen (PCNA) was used as an additional marker for cells being in either S- or G2-phase. In 47% (112/239) of classical Hodgkins lymphoma cases p21CIP1 was detected within a mean frequency of 15% positive Hodgkins and Reed–Sternberg cells per case. Similarly, 47% (116/249) of the cases stained positively for p27KIP1 with a mean frequency of expression in Hodgkins and Reed–Sternberg cells of 12%. In contrast, 90% of the cells in all 246 evaluable classical Hodgkins lymphoma cases were positive for PCNA. In addition, 98% of Hodgkins and Reed–Sternberg cells in 99% (250/253) of the cases stained strongly positive for cyclin A. These findings further corroborate the hypothesis that Hodgkin and Reed–Sternberg cells exhibit a disturbed cell cycle with an abnormally short or even absent G1-phase. In contrast to other tumors, expression of PCNA or cyclin A had no prognostic value for patient survival.

High specificity of combined TRAP and DBA.44 expression for hairy cell leukemia

Because of marrow fibrosis, bone marrow aspirations are often nonconclusive in patients with hairy cell leukemia (HCL). Therefore, histologic examination is important in HCL but often difficult in cases with low numbers of tumor cells. A combined immunohistochemical positivity for DBA.44 and tartrate-resistant phosphatase was previously found in 100% of HCL and suggested to be specific for this diagnosis. To further assess the diagnostic specificity and sensitivity of this immunohistochemical approach in a higher number of cases, we analyzed 56 HCLs and lymphoma tissue microarrays, including 840 cases of the most frequent non-Hodgkin lymphomas. All HCLs showed combined positivity for these two proteins (100% sensitivity). Both antibodies were often positive in other lymphoma types. DBA.44 reactivity was especially frequent in follicular lymphomas (46%), whereas tartrate-resistant acid phosphatase (TRAP) expression was often seen in mantle cell lymphomas (57%), primary mediastinal large B-cell lymphomas (54%), and chronic lymphocytic leukemia/small lymphocytic lymphoma (41%). A combined DBA.44/TRAP positivity was seen in only 3% of non-HCL non-Hodgkin lymphomas, including cases of diffuse large B-cell lymphomas, follicular lymphomas, chronic lymphatic leukemia/small lymphocytic leukemias, and mantle cell lymphomas. Overall, these data confirm the utility of combined immunohistochemical DBA.44/TRAP expression analysis in confirming the diagnosis of HCL. However, combined positivity for these markers is highly sensitive but not absolutely specific for HCL.

Expression of minichromosome maintenance protein 2 as a marker for proliferation and prognosis in diffuse large B-cell lymphoma: a tissue microarray and clinico-pathological analysis

BackgroundMinichromosome maintenance (MCM) proteins are essential for the initiation of DNA replication and have been found to be relevant markers for prognosis in a variety of tumours. The aim of this study was to assess the proliferative activity of diffuse large B-cell lymphoma (DLBCL) in tissue microarray (TMA) using one of the minichromosome maintenance proteins (Mcm2) and to explore its potential value to predict prognosis.MethodsImmunohistochemistry for Mcm2 was performed on TMAs constructed from 302 cases of DLBCL. A monoclonal mouse antibody was used after heat induced antigen retrieval. Mcm2 expression was scored quantitatively. Positivity for Mcm2 was defined as presence of nuclear expression of Mcm2 in greater than or equal to 40 % of tumour cells. A statistical analysis was carried out of the association of Mcm2 and the clinico-pathological characteristics.ResultsMcm2 expression was clearly evident in the nuclei of proliferating non-neoplastic cells and tumour cells. Positivity for Mcm2 was found in 46% (98/211) of analysable cases. A significant correlation existed between Mcm2 expression and presence of bulky disease (p = 0.003). Poor disease specific survival was observed in patients with DLBCL positive for Mcm2 expression in the univariate analysis (p = 0.0424).ConclusionMcm2 expression can be used to assess tumour proliferation and may be useful as an additional prognostic marker to refine the prediction of outcome in DLBCL.

Construction and validation of a bone marrow tissue microarray

Background: The use of tissue microarrays (TMAs) is now a generally accepted method for the investigation of solid tumours. However, little is known about the applicability of the TMA technique for analysis of patients with acute leukaemia. A bone marrow (BM)-TMA analysis with 15 different immunohistochemical markers was performed. The TMA was validated by comparison with the corresponding full tissue sections. Materials and methods: A BM-TMA comprising 148 cases of acute leukaemia, including 115 acute myeloid leukaemia (AML) and 33 acute lymphoblastic leukaemia (ALL) cases, was constructed. Expression of CD3, CD10, CD15, CD20, CD34, CD61, CD68, CD79a, CD99, CD117, CD138, myeloperoxidase, haemoglobin A1, glycophorin and terminal deoxynucleotidyl transferase was immunohistochemically analysed. 50 cases of the TMA were directly compared with the corresponding full tissue section to validate the results. Results: Morphologically and immunohistochemically, 6 (4%) of 148 cases and 765 (11%) cores of 6912 individual analyses were not evaluable. A direct comparison of TMA cases with conventional full sections showed a concordance of the results of 100%. Conclusions: The small size of bone-marrow biopsies and the presence of bony trabeculae do not preclude construction and analysis of acute leukaemia TMAs. Acute leukaemia cases on TMA displayed the characteristic phenotypic profiles expected in different AML and ALL subtypes. Therefore, the TMA technique is also a promising method for high-throughput analysis of combined marker expression and clinicopathological correlations in patients with leukaemia.

Rare expression of KIT (CD117) in lymphomas: a tissue microarray study of 1166 cases

Aims : Imatinib mesylate specifically inhibits KIT tyrosine kinase activity, and has been proven to be effective in the treatment of gastrointestinal stromal tumours. Because other KIT‐expressing malignancies might benefit from Imatinib therapy, we evaluated the distribution and expression of KIT in 1166 cases of malignant lymphoma.