Biomedical subjects

B Avner

Publications and source records attributed to B Avner.

7 recordsLinked to original sources

PubMed1997

Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors.

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.

Animals↗

PubMed1993

Application of the adenosine triphosphate-cell viability assay in human breast cancer chemosensitivity testing: a report on the first results.

Chemosensitivity testing in vitro of breast cancer has been difficult because of small tumour volume, an even smaller yield of viable cells after disaggregation, and the low evaluability rate and sensitivity of current assays. We have employed an alternative approach that quantitates intracellular adenosine triphosphate (ATP) as a measure of cell viability. This ATP-cell viability assay (ATP-CVA) determines in vitro tumor cell viability after exposure to chemotherapeutic agents in comparison to untreated controls following 6 days of incubation. Sixty-one fresh breast cancer specimens upon testing yielded an evaluability rate of 95%. Forty-seven of the tumors were untreated primary breast cancers, the remaining 14 were from patients with metastatic disease. Correlations of in vitro drug sensitivity with in vivo response were obtained for 17 treatment regimens in 14 patients with metastatic breast cancer. The level of sensitivity was 90% and the specificity 86%. These preliminary data demonstrated the ATP-CVA to be a practical in vitro approach to breast cancer testing. It will require a larger clinical study for confirmation.

Adenosine Triphosphate↗

PubMed1991

Evaluation and clinical relevance of patient immune responses to intravenous therapy with murine monoclonal antibodies conjugated to adriamycin.

A retrospective study was performed in order to examine the clinical relevance of human anti-murine antibodies (HAMA) to concurrent clinical events in 21 patients receiving intravenous therapy with cocktails of murine monoclonal antibodies conjugated to Adriamycin. In vivo tumor localization of the murine antibodies was also evaluated. Serum levels of HAMA, human-murine immune complexes (HMIC), and murine antibodies were measured using an automated fluorescence immunoassay. Immunohistochemical staining was performed on frozen sections of tumor biopsies from eight of the patients to examine the in vivo binding of the murine antibodies. The patients were divided into low, intermediate, and high antibody dose groups. The incidence of allergic symptoms (80%) and HAMA correlation (75%) were highest in the low dose group. Specific IgM HAMA was the most highly correlated with allergic reactions, being present in 61.5% of the allergic patients. Thirteen of the 21 patients studied (61.9%) developed allergic symptoms after one or more doses of the murine monoclonal antibody conjugates. The percentages of total antibody doses in the patients' sera at varying intervals post-infusion varied widely from patient to patient for any given time point and dose, suggesting complex factors in the distribution and clearance of the murine antibodies. All eight of the patients biopsied during or post-therapy exhibited tumor localization of the murine monoclonal antibodies. Six of the eight had concurrent HAMA in their sera. Thus, the presence of HAMA did not prevent in vivo localization of the murine antibodies in the target tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Phase I trial of mitomycin C immunoconjugates cocktails in human malignancies.

Nineteen patients with refractory solid malignancies received individualized combinations of mitomycin C conjugated murine monoclonal antibodies selected by immunohistochemical and flow cytometric screening of tumor specimens. There were no responses in this Phase I study. Thrombocytopenia precluded escalation above a mitomycin C dose of 60 mg per treatment cycle. Preclinical, clinical, and toxicity experiences with this investigational approach to the treatment of cancer are discussed.

Adult↗

PubMed1989

Therapeutic murine monoclonal antibodies developed for individual cancer patients.

The purpose of this work was to create antibodies that are highly specific to epitopes on the surface of patient tumor cells that, when added together as a "cocktail," bind to greater than 99% of the patient's tumor cells. We describe the rationale and the methods used to develop new murine hybridomas that secrete monoclonal antibodies reactive with surface markers expressed on breast, lung, colon, kidney, islet cell, and miscellaneous carcinomas and melanoma. A rapid immunofluorescence method (cell concentration fluorescence immunoassay) is also described that has been developed to rapidly screen culture supernatants on viable patient tumor cells, tumor cell lines, or peripheral blood cells. We report the development of one breast, five colon, and three melanoma, three nephroma, and two pancreatic islet cell carcinoma antibodies. The breast antibody binds to 75% of the breast tumors tested thus far and to the same percentage of colon tumors as do the five colon antibodies, 77-85%. The melanoma antibodies described react with 90-100% of the melanoma and prostate cancers tested. The total process of creating the hybridomas and screening the antibodies for potential clinical usefulness has taken from 6 to 9 months to complete, including testing normal tissue reactivity by immunohistochemistry and production of gram quantities of monoclonal antibodies from ascites.

Animals↗

PubMed1988

Characterization of a method using viable human target cells as the solid phase in a cell concentration fluorescence immunoassay (CCFIA) for screening of monoclonal antibodies and hybridoma supernatants.

A new method has been characterized for the use of viable target cells as the solid phase for screening of hybridoma supernatants in a cell concentration fluorescence immunoassay (CCFIA). Briefly, the specific target antigen on the cells is bound by the monoclonal antibodies and revealed by use of a fluoresceinated second antibody. Separation of free from bound antibody is accomplished by filtration in the 0.2 micron filter-bottom wells of specialized assay plates. Processing is automated in a Pandex screen machine, resulting in numerical fluorescence values for each well. This method is rapid (under 1 h per 96-well plate), highly sensitive (down to 0.2 ng/ml) and sparing of target cells (0.3-2.5 X 10(4) cells per assay well). It has been applied to 37 different varieties of human solid tumor cells, as well as to human peripheral blood mononuclear cells. The cells used as targets for the characterization of this method were still capable of attachment and growth when recovered post-assay. This method was compared with a viable cell enzyme-linked immunosorbent assay (ELISA) method, showing similar sensitivity and greatly shortened assay time. Comparison of the results from this method with those obtained from flow cytometric analysis performed on viable cells showed close correlation, whereas a lower correlation was seen with immunohistochemical methods using acetone-fixed cells. Development of this method made it possible to rapidly screen many thousands of hybridoma supernatants and successfully select those which were specific for surface antigens on viable cells.

Adenocarcinoma↗

PubMed1988

Adriamycin custom-tailored immunoconjugates in the treatment of human malignancies.

Twenty-three patients with disseminated refractory malignancies each received a tailored combination of adriamycin-conjugated murine monoclonal antibodies. Tumors were typed using a panel of antibodies. Cocktails of up to six antibodies were selected based on binding greater than 80% of the malignant cells as tested by immunoperoxidase and flow cytometry. These monoclonal antibodies were then conjugated to Adriamycin and administered intravenously. Seventeen of 23 patients had reactions to the administration of immunoconjugates, but these were tolerable in all but two patients. Fever, chills, pruritus, and skin rash were by far the most common transitory reactions. All were well controlled with premedication. In several patients there was limited antigenic drift among various biopsies within the same patient over time. This observation confirms the necessity for the use of a cocktail of antibodies if one wishes to cover all tumor cells. Preliminary serologic evidence suggests that the development of an IgM antibody, which is specific against the mouse monoclonal antibody, has the specificity and sensitivity to predict clinical reactions. Selected patients were re-treated. One patient with chronic lymphocytic leukemia had re-treatment on three occasions and demonstrated regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions and two patients with tongue carcinoma had shrinkage of their lesions. In the course of the study free Adriamycin released from the monoclonal antibodies was discovered to be a limiting factor in the amount of antibody that could be administered. Up to 1 g of Adriamycin and up to 5 g of monoclonal antibody were administered. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody that unpredictably caused hemopoietic depression. This study demonstrates the feasibility and reviews technical considerations in preparing immunoconjugate cocktails for patients with refractory malignancies. The major technical hurdle appears to be the selection of an effective conjugation method that can be used to optimally bind Adriamycin to monoclonal antibodies for targeted cancer therapy.

Animals↗