Production and characterisation of an antimelanoma monoclonal antibody KBA.62 using a new melanoma cell line reactive on paraffin wax embedded sections.

AIMS--To generate new monoclonal antibodies directed against melanoma associated antigens using a new melanoma cell line, KAL. METHODS--The melanoma cell line was established in culture from a lymph node metastasis of malignant melanoma. Normal Balb/c mice were immunised with KAL cells. Splenocytes were used for fusion experiments using standard techniques. Hybridoma supernatants were tested for antibody binding activity using an indirect immunoperoxidase method on frozen sections from KAL tumour cells xenografted onto nude mice and human tonsils. KBA.62 was selected because of its reactivity with melanocytic proliferations on both frozen and paraffin wax sections. RESULTS--On immunoblotting, KBA.62 reacted with three bands of 140, 135 and 128 kD and two weak bands of 88 and 73 kD. In normal human tissues basal melanocytes in the epidermis did not react with this antibody and only occasional labelling of endothelial cells was noted. Of the human tumours, KBA.62 reacted strongly and uniformly with the majority of benign (21/21) and malignant (75/86) melanocytic proliferations. Staining was localised predominantly to the cell membrane with little or no cytoplasmic reactivity. Negative staining was observed in the majority of human non-melanocytic neoplasms, the exceptions being some carcinomas (11/89), particularly the well differentiated squamous cell type. This, however, was not thought to present a diagnostic problem. CONCLUSIONS--KBA.62 appears to be potentially useful in ascertaining the immunomorphological diagnosis of malignant melanoma in routinely processed paraffin wax sections.

Production of monoclonal antibodies to human estrogen-receptor protein (ER) using recombinant ER (RER).

Two monoclonal antibodies (MAbs), IC5 and ID5, were produced using spleen cells from BALB/c mice immunized with recombinant estrogen-receptor protein (RER). On immunoblotting, both MAbs reacted with the 67-kDa polypeptide chain obtained by transformation of E. coli and transfection of COS cells with plasmid vectors expressing ER. The epitopes of both MAbs were in the N-terminal domain (A/B region) of the receptor. In normal human tissues, IC5 and ID5 reacted with cells known to contain large amount of ER, such as cells of the mammary gland and the uterus. Staining was localized predominantly in nuclei with little or no cytoplasmic reactivity. IC5 and ID5 were unreactive with tissues usually considered to be negative for ER. The reactions of these 2 MAbs were further tested on different tumor types, using immunohistochemical (IHC) method on frozen sections. In breast cancer, a good correlation was found between the results obtained on frozen sections and those using the conventional radioligand dextran-coated charcoal (DCC) assay. Immunostaining with IC5 and ID5 MAbs was also assessed on routinely processed paraffin sections using the antigen-retrieval method. Staining was comparable to that obtained on frozen sections in virtually all the breast carcinomas. Negative reactions were consistently obtained with both antibodies on human neoplasms derived from other non-estrogen-dependent organs. IC5 and ID5 MAbs may thus be of value in routine diagnostic histopathology for assessment of the estrogen-receptor content in human carcinomas.

A novel human lymphoma cell line (Deglis) with dual B/T phenotype and gene rearrangements and containing Epstein-Barr virus genomes.

We report a new and apparently unique human lymphoma cell line termed Deglis. The line was established from a polymorphic centroblastic lymphoma. The cell line and its source carry a dual B-cell and T-cell phenotype and Epstein-Barr virus (EBV) genomes. Simultaneous expression of B-cell (CD19+, CD20+, CD23+, CD37+) and T-cell (CD2+, CD3+/-, CD7+, CD43+) antigens, activation antigens (CD30+, CDw70+) as well as CD68+, a macrophage-associated antigen, was observed on the cell line and its source. Genotypic studies of the cell line showed dual gene rearrangements. JH (on both primary tumor and the cell line) and C kappa were rearranged without expression of cytoplasmic or surface immunoglobulins. T-cell receptor-alpha (TCR-alpha) and TCR-beta genes were rearranged, but TCR-delta and TCR-gamma genes were in germline configuration. Apparently, functional transcripts of TCR-alpha and truncated transcripts for TCR-beta and TCR-delta were observed. EBV-encoded proteins (LMP and EBNA2) were expressed by the parent tumor and the cell line. Southern blot analysis showed the same clonal EBV genomes in the primary tumor and the cell line. Karyotypic analysis of the cell line showed several chromosomal abnormalities but normal chromosomal number. The characteristics of this cell line suggest that neoplastic transformation has occurred in a precursor cell broadly committed to lymphoid lineage. Further studies on this cell line may help resolve some issues in the physiopathology of lymphoid tumors.

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen.

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined.

Production of a monoclonal antibody (IND.64) identifying a cell cycle-associated antigen using spleen cells from nude mice bearing Ichikawa tumour.

Using spleen cells from athymic nude mice grafted with Ichikawa tumour, we have generated the monoclonal antibody IND.64, which detects a proliferation-associated nuclear antigen. Immunoblotting analysis with IND.64 showed a double band with apparent molecular weights of 395 and 345 kD. In normal human tissues, the antigen detected by IND.64 was expressed only by the nuclei of proliferating cells, such as germinal centre cells of reactive lymph nodes, cortical thymocytes, the basal layer of the skin, and proliferative compartments of the stomach, small intestine, and colon. IND.64 did not react with cells known to be non-proliferative or to show only a low turnover, such as cells of the kidney, liver, smooth muscle, cardiac muscle, and brain. The expression of this antigen during the cell cycle was determined using two approaches: IND.64 immunostaining of synchronized adult bovine aortic endothelial cells and flow cytometric analysis of double-labelled PHA-stimulated peripheral mononuclear blood leucocytes with a DNA marker and IND.64. The antigen recognized by IND.64 was found to appear in the late G1 phase, and persisted in phases S, G2, and M, but was absent in the G0 and early G1 phases. IND.64 was further investigated in different tumour types to evaluate the correlation between the percentage of IND.64-positive cells (IND.64 index) and the histological grade. In non-Hodgkin's lymphomas, an excellent correlation was found between the percentage of IND.64-positive cells and the cytomorphological grade. In nodular sclerosis and mixed cellularity Hodgkin's disease, a high number of Reed-Sternberg cells were positive with IND.64. The non-lymphoid neoplasms investigated showed a variable percentage of positive cells. IND.64 appears to be a promising tissue marker to complement the evaluation of prognosis in human cancer.