Reactivity of antibodies with highly glycosylated MUC1 mucins from colon carcinoma cells and bile.
Sikut R. Sikut A. Zhang K. Baeckstrom D. Hansson GC.
Department of Medical Biochemistry, Goteborg University, Gothenburg, Sweden.
The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with core proteins of the heavily glycosylated MUC1 mucins from the colon carcinoma cell line COLO205 and bile. Both these mucins (designated as H-CanAg and SBG2) are highly glycosylated having 15 and 50 sugar residues per oligosaccharide, respectively. Only a few of the antibodies (129, 139, 153 and 162) reacted with both SBG2 and H-CanAg, and not with a control mucin (L-CanAg) having a similar glycosylation as H-CanAg. These antibodies were tested for their ability to catch soluble mucins, and the antibody 162 was found to be good also in this type of assay. The antibodies selected here should be useful for the detection of high glycosylated forms of the MUC1 mucin in tissues and serum.
Carcinoma-associated MUC1 detected by immunoradiometric assays.
Norum LF. Varaas T. Kierulf B. Nustad K.
Central Laboratory, Norwegian Radium Hospital, Oslo, Norway.
Fifty-four anti-MUC1 antibodies submitted to the International Society for Oncodevelopmental Biology and Medicine (ISOBM) Workshop (TD-4) were evaluated in immunoradiometric assays, using sera from carcinoma patients and healthy donors. The carcinoma serum pool contained sera from 30 patients with advanced cancer (10 breast, 10 colon, and 10 ovarian). This serum pool contained 696 kU/l MUC1, 770 micrograms/l CEA, and 3,700 kU/l CA 125. The reference serum pool was obtained from 10 healthy women combined with 20 sera from pregnant women, of which half had elevated CA 125 (range 82-254 kU/l). The reference serum pool contained 13 kU/l MUC1, 2 micrograms/l CEA, and 65 kU/l CA 125. The Workshop antibodies were tested both as solid-phase antibodies and as tracer antibodies with the carcinoma serum pool. Twenty-two tracer antibodies and 38 solid-phase antibodies gave at least one combination with > 10% binding of the tracer antibody for a total of 836 combinations. These were tested further with the reference serum pool. Antibodies used as tracers could be separated into three categories: Group 1 antibodies, MF06, MF11, B27.29, MF30, and Ma552, gave mainly 'carcinoma-specific' assays in combinations with the solid-phase antibodies, i.e. binding ratio between carcinoma MUC1 and reference MUC1 > 10. Group 2 antibodies, DF3, 7540MR, A76-A/C7, BC4N154, M38, 7539MR, B12, GP1.4, 232A1, Mc5, and Ma695 gave both 'specific' and 'nonspecific' binding ratios depending on the solid-phase antibody used. Group 3 antibodies, 214D4, BC4E549, E29, BCP8, BC3, and 3E1.2, gave mainly 'nonspecific' combinations, i.e. ratios < or = 10. All antibodies used to capture MUC1 on the solid phase gave both 'specific' and 'nonspecific' combinations depending on the tracer antibody used. Ten antibodies were clearly more efficient as solid-phase capture antibodies; Ma695, B12, M38, GP1.4, 214D4, MF06, B27.29, A76-A/C7, BC3, and KC4. Our findings indicate that the ability to detect 'carcinoma-specific MUC1' cannot be deduced from epitope specificity alone.
Rapid quantitative PCR determination of relative gene expression in tumor specimens using high-pressure liquid chromatography.
Odin E. Larsson L. Aram M. Gustavsson B. Larsson PA.
Department of Surgery, Goteborg University, Sahlgren's University Hospital, Sweden.
A reverse transcriptase polymerase chain reaction (rt-PCR) for quantification of gene expression has been optimized for analysis of folylpolyglutamate synthase (FPGS) and thymidylate synthase (TS), using beta-actin as an internal standard (house-keeping gene). Total RNA was isolated from tumor tissue, reversely transcribed to cDNA and PCR amplified with primers specific for TS, FPGS and beta-actin in separate vials. PCR products were separated and quantified by high-pressure liquid chromatography (HPLC) without addition of radioactive or fluorescent markers, which minimizes labor and occupational hazards. The day-to-day variation in the HPLC analysis was 2.7% and the within sample variations for rt-PCR/HPLC analysis of TS and FPGS were 18.5% for both assays. This method provides a tool for convenient gene expression analysis in clinical biopsies.
Correlation between DNA content, expression of Ki-67 antigen of tumor cells and immunophenotype of lymphocytes from malignant pleural effusions.
Sikora J. Dworacki G. Trybus M. Batura-Gabryel H. Zeromski J.
Department of Immunopathology, Karol Marcinkowski University of Medical Sciences, Poznan, Poland.
Malignant cells isolated from 26 malignant pleural effusions, collected from patients bearing metastatic carcinoma, were examined using flow cytometry. Expression of Ki-67 antigen, DNA ploidy, S phase fraction, DNA index of tumor cells and the percentage of aneuploid cell populations were determined. Lymphoid cells from the same malignant effusions and from 10 nonmalignant ones were examined using a panel of monoclonal antibodies (mAb) versus several CD antigens. While the percentages of T cells (CD3+, CD4+, CD8+) and B cells (CD19+) did not differ significantly in malignant versus nonmalignant effusions, those of CD56+ lymphocytes were found to be significantly depressed in malignant diploid and malignant aneuploid effusions as compared to benign ones. Expression of IL-2 receptors on lymphocytes, demonstrated by anti-CD25 mAb, was significantly higher in malignant diploid and malignant aneuploid effusions as compared with nonmalignant ones. An increase in the percentage of Ki-67-positive malignant cells was significantly correlated with the decrease of NK cells. S phase fraction, DNA index and the percentage of aneuploid populations did not correlate with the picture of lymphoid cells from pleural effusion. On the other hand, the percentage of Ki-67-positive tumor cells in pleural effusions was inversely proportional to the patients' survival time, although it was not statistically significant. Our data suggest that the poor prognosis of cancer characterized by the presence of an aneuploid cell population of malignant cells and the high percentage of proliferating cells in pleural effusions can be linked to the depression of NK cells.
Analysis of the T cell receptor variability of tumor-infiltrating lymphocytes in colorectal carcinomas.
Baier PK. Wimmenauer S. Hirsch T. von Specht BU. von Kleist S. Keller H. Farthmann EH.
Abteilung fur Allgemeinchirurgie mit Poliklinik, Chirurgische Universitatsklinik, Freiburg, Deutschland.
While there is good clinical and experimental evidence for immunological tumor control in some tumors--malignant melanomas, for instance--the immunogenicity of colorectal carcinoma (CRC) still remains unsettled. We examined surgical specimens from 4 CRC patients for T cell clones among tumor-infiltrating lymphocytes (TIL). The growth of specific lymphocyte clones in a tumor indicates an immunological response in vivo. We used a T cell receptor V beta family-specific semiquantitative PCR with additional sequencing to examine TIL for clonal expansion. In CRC, specific T cell clones could not be demonstrated. However, we observed a predominance of V beta 9 in 3 of 4 tumors.