Differences in the receptor status between primary and recurrent breast cancer--the frequency of and the reasons for discordance.

OBJECTIVE: Receptor discordances between primary and recurrent breast cancer have been described for years, but only a few analyses have elucidated the factors that influence receptor changes. METHODS: Explorative analyses of prospective data from a breast cancer database of a tertiary breast cancer unit. RESULTS: Recurrent tumours that had expressed oestrogen (ER) and progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2) as primary tumours were negative for the respective receptor in 22.8, 41.4 and 40.8% of cases. ER, PR and HER2 expression was found in 19.8, 16.7 and 11.5% of recurrent tumours, although no expression had been observed in primary tumours. Receptor discordances in recurrent disease leading to different therapeutic approaches were noted in 126 of 411 patients (30.7%). In patients with tumours expressing primary ER and HER2, independent factors associated with discordance were endocrine therapy and treatment with trastuzumab. CONCLUSION: High rates of receptor discordance were found. The impact of factors that influence receptor changes is small so that no subgroup of patients with recurrent breast cancer should be excluded from biopsy. Whenever possible, a biopsy should be taken to confirm the diagnosis of a possible relapse as well as the receptor status of patients with breast cancer.

Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.

Evaluation of proliferative activity is a cornerstone in the classification of endocrine tumors; in pulmonary carcinoids, the mitotic count delineates typical carcinoid (TC) from atypical carcinoid (AC). Data on the reproducibility of manual mitotic counting and other methods of proliferation index evaluation in this tumor entity are sparse. Nine experienced pulmonary pathologists evaluated 20 carcinoid tumors for mitotic count (hematoxylin and eosin) and Ki-67 index. In addition, Ki-67 index was automatically evaluated with a software-based algorithm. Results were compared with respect to correlation coefficients (CC) and kappa values for clinically relevant grouping algorithms. Evaluation of mitotic activity resulted in a low interobserver agreement with a median CC of 0.196 and a median kappa of 0.213 for the delineation of TC from AC. The median CC for hotspot (0.658) and overall (0.746) Ki-67 evaluation was considerably higher. However, kappa values for grouped comparisons of overall Ki-67 were only fair (median 0.323). The agreement of manual and automated Ki-67 evaluation was good (median CC 0.851, median kappa 0.805) and was further increased when more than one participant evaluated a given case. Ki-67 staining clearly outperforms mitotic count with respect to interobserver agreement in pulmonary carcinoids, with the latter having an unacceptable low performance status. Manual evaluation of Ki-67 is reliable, and consistency further increases with more than one evaluator per case. Although the prognostic value needs further validation, Ki-67 might perspectively be considered a helpful diagnostic parameter to optimize the separation of TC from AC.

Expression of activation-induced cytidine deaminase in malignant lymphomas infiltrating the bone marrow.

Somatic hypermutation of immunoglobulin genes and class switch recombination are pivotal processes in the germinal center (GC) reaction and have been implicated in the development of malignant B-cell lymphoma. Both processes require the enzyme activation-induced cytidine deaminase (AID). Expression of AID is largely restricted to GC B cells and B cells that undergo class switch recombination outside the GC. AID is also expressed in many B-cell lymphomas. This study investigates the expression of AID of malignant lymphomas infiltrating the bone marrow. Bone marrow trephines (n=130) with infiltration of Hodgkin lymphoma and non-Hodgkin lymphoma of B cell and T-cell type and trephines with reactive lymphoid follicles (n=16) were analyzed immunohistochemically for AID protein. AID is expressed in bone marrow infiltrates of malignant lymphomas. AID was generally detected in lymphomas of GC origin. Tumor cells of hairy cell leukemia are mostly AID. There is apparently no different expression of AID found in bone marrow infiltrates of malignant lymphomas compared with a control group with nodal malignant lymphoma infiltrates (n=105). These results suggest that the expression pattern of AID in lymphoma infiltrates in the bone marrow reflects that of extramedullary lymphoma infiltrates.

Frequent occurrence of B-cell lymphomas in angioimmunoblastic T-cell lymphoma and proliferation of Epstein-Barr virus-infected cells in early cases.

Secondary lymphomas occurring in the setting of angioimmunoblastic T-cell lymphoma (AILT) are considered to be rare. Their occurrence has been attributed to Epstein-Barr virus (EBV)-associated lymphoproliferations. A previous study detected a dysregulated hypermutation process in B-cells of AILT. The present study aimed at estimating the frequency of B-cell lymphomas in AILT. By studying the expression of EBV and activation-induced cytidine deaminase (AID) as an indicator of hypermutating cells, we assessed whether B-cell lymphoproliferations in AILT were strictly associated with EBV and whether hypermutation might contribute to lymphomagenesis. Among 161 cases of AILT, diagnosed between 1996 and 2005 at the lymph node registry, Frankfurt, Germany, 19 cases were detected that also had B-cell non-Hodgkin lymphoma (NHL) and two cases had classical Hodgkin lymphoma (HL). EBV was detected in tumour cells of 7/18 NHL and both HL, suggesting that factors other than EBV contribute to lymphomagenesis. AID was expressed in AILT in large cells disseminated in the tissue, implying that the process of somatic hypermutation is ongoing in AILT, although the GC architecture is disrupted. This might be relevant in the development of secondary lymphomas.

Somatic hypermutation of SOCS1 in lymphocyte-predominant Hodgkin lymphoma is accompanied by high JAK2 expression and activation of STAT6.

Aberrant activities of JAK/STAT signaling pathways have been observed in several hematologic malignancies. Here, we show high expression of JAK2 in the tumor cells of lymphocyte-predominant Hodgkin lymphoma in 85% of cases and activation of JAK2 in 39% of cases. STAT6, which is a target of JAK2, was activated in 50% of the cases. SOCS1 controls JAK2 activity and degradation. Mutations in SOCS1 of either somatic or germ-line origin were observed in micromanipulated tumor cells of 50% of cases. Most mutations truncated SOCS1 or caused replacement of amino acids in functional important regions. Activating mutations in exon 12 of JAK2, which are frequent in myeloproliferative diseases, were not observed. In lymphocyte-predominant Hodgkin lymphoma SOCS1 function may thus be frequently impaired by mutations, and this may contribute to high JAK2 expression and activation of the JAK2/STAT6 pathway.

The aberrant coexpression of several receptor tyrosine kinases is largely restricted to EBV-negative cases of classical Hodgkin's lymphoma.

The Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin's lymphoma (HL) aberrantly express up to 7 different receptor tyrosine kinases (RTK) with extensive heterogeneity regarding the number and combinations of expressed RTKs in individual cases and a more prominent coexpression in nodular-sclerosis (ns) than mixed-cellularity (mc) HL. To investigate whether RTK expression patterns are related to other pathogenetic mechanisms and clinical behaviour, we analysed a large collection of EBV(+) and EBV(-) cases of ns and mc subtype and cases with relapses for expression of the 7 RTKs. No specific relation of any RTK to a specific group of cases was observed. The analysis of average numbers of expressed RTKs per case as a measure for strength of overall RTK signalling revealed a relation with the histological subtype and the EBV-status. RTK coexpression was significantly higher in EBV(-) nsHL cases compared to both EBV(-) and EBV(+) mcHL cases. Among mcHL cases RTK coexpression was significantly higher in EBV(-) compared to EBV(+) cases. Coexpression of 3 and more RTKs was largely restricted to EBV(-) cases. The inverse correlation between strong RTK signalling and presence of EBV may indicate that RTK signalling can at least partially replace the role of EBV in HRS cell pathogenesis. For cases with aberrant coexpression of several RTKs inclusion of RTK inhibitors in therapy regimens may be a novel option.

Common features and differences in the transcriptome of large cell anaplastic lymphoma and classical Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVES: Anaplastic large cell lymphoma (ALCL) and classical Hodgkin's lymphoma (HL) are derived from different cell types, namely T cells and B cells, respectively. However, both lymphomas share a similar cytological and immunohistochemical tumor cell phenotype with little resemblance to their cells of origin. DESIGN AND METHODS: In this study, the transcriptional profiles of ALCL cell lines, primary ALCL tumor cells from peripheral blood and HL cell lines were compared to each other and to normal B-cell subsets, B non-Hodgkin's lymphomas (NHL) and B NHL- and Epstein-Barr virus (EBV)-transformed B-cell lines in order to establish their relationship at the transcriptional level and to identify genes with possible pathobiological impact. Expression of some of the genes identified was confirmed in microdissected primary tumor cells by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: HL samples clustered separately from ALCL samples, but HL and ALCL were found to be more closely related to each other than to any normal or malignant B-cell sample in the dataset. Their relationship was determined to a large extent, but not exclusively, by lack of expression of B-cell antigens and by the over-expression of mRNA encoding activation markers and structural proteins. Apart from established differences between HL and ALCL, further genes of interest could be identified that distinguish both entities from each other and from the other samples. The differential expression of PRAME, DDR2, SOCS3 and CEBPD in HL and ALCL was confirmed in primary tumor tissue by immunohistochemistry and/or RT-PCR. INTERPRETATION AND CONCLUSIONS: At a transcriptional level HL is more closely related to Alk+ ALCL than to the B-NHL or B-cell samples investigated, although it is a B-cell derived lymphoma. The newly identified genes discriminating HL and ALCL may be pathobiologically important and may serve as possible therapeutic targets.

Nodular lymphoid lesion of the liver with simultaneous focal nodular hyperplasia and hemangioma: discrimination from primary hepatic MALT-type non-Hodgkin's lymphoma.

Nodular lymphoid lesion (NLL) of the liver is a rare but unique entity and has also been termed reactive lymphoid hyperplasia of the liver. We describe the histological, immunohistochemical and molecular biologic findings of a case with NLL and two other tumors of the liver. The nodular lymphoid mass found in the liver was composed of heterogeneous small lymphocytes forming reactive follicles. Plasma cells, few immunoblasts, centroblasts, few macrophages, epithelioid cells, and giant cells were seen. The lymphoid infiltrate displaced the adjacent hepatic parenchyma. By immunohistochemistry and molecular studies, the lymphocytes were found to be polyclonal. The diagnosis of NLL was made. In addition to NLL, focal nodular hyperplasia and hemangioma were detected. The discrimination of NLL from primary hepatic malignant non-Hodgkin's lymphoma of mucosa-associated lymphoid tissue-type may pose diagnostic difficulties and may require the use of immunohistochemical and molecular techniques. The simultaneous occurrence of NLL with focal nodular hyperplasia and hemangioma in the liver has not been described before.

[Global gene expression analysis and novel signalling pathways in Hodgkin lymphoma]. / Globale Genexpressionsanalysen und neue Signalwege beim Hodgkin Lymphom.

To identify pathogenetic mechanisms in Hodgkin lymphoma (HL) we performed global gene expression profiling of four HL derived cell lines and compared the expression profiles with those of normal B cells and B cell non-HL. This analysis revealed a global loss of B-cell specific gene expression in Hodgkin-Reed/Sternberg (HRS) cells (21). Further analysis showed that ABF-1 and Id2, which are both negative regulators of the B cell master transcription factor E2A and likely also Pax-5, are aberrantly expressed in HRS cell lines (11, 17). For Id2, immunohistochemistry showed expression in the HRS cells of all cases, and E2A could be coimmunoprecipitated with Id2 from HRS cell lines, indicating that the aberrant Id2 expression may indeed contribute to the loss of the B-cell specific gene expression in HL (17). An analysis of the global gene expression data for aberrant expression of genes which are frequently involved in neoplastic transformation identified six receptor tyrosine kinases (RTK) aberrantly expressed in HRS cell lines. All RTKs were also (co)-expressed in primary cases and mostly activated by auto- or paracrine mechanisms (18). Analysis of greater number of cases identified a subgroup of HL which encompasses about 20 % of cases characterised by coexpression of at least four of the RTKs. An survey of several B cell lymphoma types with a pan-phospho-tyrosine specific antibody indicated that mediastinal large B-cell lymphoma is beside HL the only entity where aberrant TK activities cause an aberrantly high cellular phospho-tyrosine content in a significant fraction of cases.

Human splenic marginal zone B cells lack expression of activation-induced cytidine deaminase.

It has been speculated that somatic hypermutation of rearranged immunoglobulin variable (V) region genes does not only take place in the germinal center (GC) microenvironment, but also in the marginal zone (MZ) of the spleen, and that human peripheral blood IgM-positive B cells with somatically mutated V region genes may derive from mutating MZ B cells. As somatic hypermutation is strictly dependent on the enzyme activation-induced cytidine deaminase (AID), we used an AID-specific monoclonal antibody that is suitable for immunohistochemical staining to analyze human splenic MZ cells for AID expression. Analysis of tissue sections from 29 spleens revealed only very rare MZ cells (approx. 0.05%) showing AID staining, whereas in 25 of the spleen samples strong AID staining of GC B cells was observed. Thus, there are virtually no AID-expressing MZ B cells, indicating that somatic hypermutation does not take place at a significant level in the MZ. Consequently, it appears unlikely that the somatically mutated IgM B cells are generated in the splenic MZ. Moreover, the lack of AID-positive MZ B cells questions the recent speculation that B cell chronic lymphocytic leukemias with mutated V genes are derived from mutating MZ B cells.

Autocrine- and paracrine-activated receptor tyrosine kinases in classic Hodgkin lymphoma.

The pathogenesis of Hodgkin lymphoma (HL) is still largely unknown. Based on a search for footprints of pathogenetic mechanisms in global RNA expression data of Hodgkin/Reed-Sternberg (HRS) cell lines, we analyzed the expression and activation of 6 receptor tyrosine kinases (RTKs) in classic HL. Immunohistochemistry revealed that the RTKs platelet-derived growth factor receptor A (PDGFRA), DDR2, EPHB1, RON, TRKB, and TRKA were each expressed in HRS cells in 30% to 75% of patients. These RTKs were not expressed in normal B cells, the origin of HRS cells, or in most B-cell non-Hodgkin lymphoma (NHL). In the majority of patients at least one RTK was expressed, and in most patients several RTKs were coexpressed, most prominently in Hodgkin lymphoma of the nodular sclerosis subtype. Phosphotyrosine-specific antibodies revealed exemplarily the activation of PDGFRA and TRKA/B and an elevation of cellular phosphotyrosine content. Immunohistochemistry for RTK ligands indicated that DDR2 and TRKA are likely activated in a paracrine fashion, whereas PDGFRA and EPHB1 seem to be activated by autocrine loops. Activating mutations were not detected in cDNA encoding the RTKs in HRS cell lines. These findings show the unprecedented coexpression of multiple RTKs in a tumor and indicate that aberrant RTK signaling is an important factor in HL pathogenesis and that it may be a novel therapeutic target.

In angioimmunoblastic T-cell lymphoma, neoplastic T cells may be a minor cell population. A molecular single-cell and immunohistochemical study.

The significance of T-cell proliferations in angioimmunoblastic lymphoma (AILD) is still enigmatic. Although classified as a malignant T-cell lymphoma in the World Health Organisation lymphoma classification, some cases of AILD lack dominant T-cell clones. In a previous study, based on single-cell polymerase chain reaction (PCR), we obtained similar results as studies of AILD using Southern blot or conventional PCR: some cases of AILD contained large T-cell clones, and, in other cases, T-cell clones were undetectable. As in single-cell studies, only a limited number of cells could be investigated; thus, we wanted to gain more insight into the amount and distribution of tumour cells. By applying triple immunofluorescent staining with antibodies directed against T-cell receptor Vbeta-family-specific epitopes, we investigated T-cell populations in AILD and their localisation in the tissue in relation to B cells (CD20) and follicular dendritic cells (CD21). In two of five cases investigated, only a minority of the T-cells compartment belonged to the tumour clone. Neoplastic T cells were found throughout the tissue, including areas dominated by B cells.

T-cell variant of classical Hodgkin's lymphoma with nodal and cutaneous manifestations demonstrated by single-cell polymerase chain reaction.

The atypical cells of CD30(+) cutaneous lymphoproliferative disorders (CD30CLD) are commonly of T-cell origin and frequently have a similar morphology as Hodgkin or Reed-Sternberg cells of Hodgkin's lymphoma (HL). HL is one of the tumors associated with CD30CLD. Although most studies support a B-cell derivation of the tumor cells in HL, recently a few cases of classical HL with T-cell genotype have been reported. We report a patient who presented with CD30CLD whose lymph nodes showed classical HL of mixed cellularity subtype at presentation. By single-cell PCR, the same clonal gene rearrangements of the T cell receptor-beta gene locus could be assigned to the CD30(+) and CD15(+) cells of both skin and lymph node. In a lymph node biopsy specimen taken in relapse after several courses of chemotherapy, the CD30(+) tumor cells were abundant. The T cell-derived tumor cells displayed aberrant expression of the Pax-5 gene in all specimens. A common clonal origin of both CD30CLD and HL of the lymph node in the patient presented here suggests that HL with T-cell genotype exists in association with CD30CLD as well as in sporadic cases and may share clonal origin with the skin tumor.