RESUMO
One-third of all splenic marginal zone lymphomas (SMZL) use the IgH VH1-02 gene. These cases are usually not associated with hepatitis C virus infection. Of interest, the rearranged VH1-02 genes display similar complementarity determining regions 3, a finding confirmed by our study. The latter suggests that these SMZL may produce antibodies with similar reactivity. We produced recombinant antibodies from 5 SMZL cases with VH1-02 gene rearrangement to study the binding reactivity of these antibodies. Surprisingly, the recombinant antibodies demonstrated poly- and self-reactivity as demonstrated by their reactivity with nuclear, cytoplasmic, as well as membranous antigens expressed by human cells and by reactivity with human serum. This polyreactivity was specific as demonstrated by ELISA. The antibodies did not react with proteins on the cell surface that are induced by apoptosis as shown for antibodies produced by chronic lymphatic leukemia with VH1-02 gene rearrangement. The results indicate that a common subset of SMZL arises from polyreactive B cells, a subset of marginal zone B cells that are important in the immunologic defense against infection.
Assuntos
Anticorpos/metabolismo , Rearranjo Gênico/imunologia , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/metabolismo , Linfoma de Zona Marginal Tipo Células B/imunologia , Proteínas Recombinantes/metabolismo , Baço/imunologia , Neoplasias Esplênicas/imunologia , Anticorpos/genética , Anticorpos/imunologia , Especificidade de Anticorpos , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/metabolismo , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Feminino , Células HEK293 , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Imuno-Histoquímica , Imunofenotipagem , Isoantígenos/imunologia , Isoantígenos/metabolismo , Cariotipagem , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/patologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Baço/metabolismo , Baço/patologia , Neoplasias Esplênicas/genética , Neoplasias Esplênicas/patologia , TransfecçãoRESUMO
BACKGROUND: Data regarding the role of flow cytometry (FCM) in the characterization of malignant effusions are limited to date. In the present study, we optimized the conditions for FCM immunphenotyping of effusions using a four-color analysis and investigated aspects related to the advantages and limitations of this method in this setting. METHODS: FCM analysis optimization for the study of epithelial cells was undertaken using five carcinoma cell lines, and subsequently applied to malignant pleural and peritoneal effusions using antibodies against epithelial and mesothelial markers (Ber-EP4 and EMA), CD138, and integrin subunits. FCM of frozen versus fresh specimens and the performance of FCM compared to immunhistochemistry were evaluated. RESULTS: FCM optimization was achieved and applied to clinical specimens, with resulting detection of epithelial markers and adhesion molecules on cancer cells. Frozen clinical specimens and cell lines showed reduced CD138 expression compared to fresh specimens, with conservation of the remaining epitopes. FCM generally showed comparable performance to immunhistochemistry. CONCLUSIONS: FCM is an effective method for characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and is comparable to immunhistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. The majority of epitopes are conserved in frozen cells, but a minority may be lost, suggesting that the thorough testing of each antibody in both conditions is mandatory.
