No abstract

To improve clinical interpretation and use of in vitro clonogenic assay results, the authors reviewed their experience to date with chemosensitivity testing of over 1500 solid tumors. All clonogenic assays were performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Significant growth was defined as 230 colonies/control plate. Clinical responses were determined according to standard criteria. Data were analyzed using two different criteria of in vitro sensitivity (250% and 275% inhibition of colony formation) and independently for each histologic type of tumor. Overall, 68% of specimens plated produced significant growth in vitro. Cloning ability varied from 57% to 82% depending on tumor histology. The assay was 57% reliable for predicting in vivo sensitivity, and 92% reliable for in vivo resistance. Predictive accuracy for sensitivity varied from 30% to 86%, depending on the tumor histology. Use of 250% ICF (inhibition of colony formation) as criteria for differentiating sensitivity from resistance proved most reliable, although criteria should be individualized for each tumor type to maximize predictive accuracy.Cancer 53:1240-1245, 1984.OR OVER TWO DECADES, clinical investigators have F pursued the goal of developing an in vitro test that could accurately predict the chemosensitivity of a malignant tumor. A number of assay techniques have evolved, including the double-layer-soft-agar method reported by Hamburger and Salmon in 1977.' This technique, now commonly referred to as the "clonogenic assay," has been the most extensively studied and clinically applied chemosensitivity test. A number of retrospective reports indicate that the clonogenic assay can predict in vivo sensitivity with approximately 50% to 70% accuracy and in vivo resistance with greater than 85% accuracy.'-' Despite the early favorable reports, there is still considerable skepticism, and several recent critical appraisals of the assay have been published.'-'' Current problems in assay methods have been stressed, including low plating

Ethacrynic acid [2,3-dichloro-4-(2-methylene-1-oxobutyl)phenoxyl] acetic acid, is a water-soluble diuretic agent that has been shown to potentiate the in vitro cytotoxicity of chemotherapeutic agents in established cell lines. We used the differential staining cytotoxicity (DiSC) assay to determine whether ethacrynic acid at 1 and 3.3 microM would potentiate the cytotoxicity of nitrogen mustard and/or doxorubicin in primary cultures of hematologic neoplasms from heavily pretreated patients and in normal peripheral blood lymphocytes. At 3.3 microM, ethacrynic acid was toxic to 8 of 24 (33%) tumor specimens studied. In subsequent studies, ethacrynic acid at 1 microM was toxic to only 2 of 54 (4%) tumor specimens. Significant enhancement for doxorubicin or nitrogen mustard was confined to lymphatic malignancies and to normal peripheral blood lymphocytes. Interspecimen variability was observed, with no enhancement in most individual specimens, 2-fold enhancement in some specimens, and 4-fold enhancement in occasional specimens. Clinical trials will be required to determine whether the observed in vitro activity for ethacrynic acid is associated with clinical benefit in unselected or assay-selected patients.

Two fibrosarcomas of similar histological type, induced in C3Hf mice by either methylcholanthrene or 3,4-benz(a)pyrene, were shown to have individually unique tumor-rejection antigens in classical transplantation-type experiments. By contrast, sera of autochthonous mice, which resisted only transplants of the immunizing sarcoma, were found to contain complement-dependent cytotoxic antibodies, specific for both sarcomas, in vitro. The existence of individually unique as well as common tumor-associated antigens in chemically-induced murine sarcomas is suggested. The private "tumor transplantation-type" antigens elicited tumor rejection responses in vivo. The common tumor-associated antigens, although immunogenic in autochthonous hosts, inducing the production of tumor-specific antibodies, failed to induce transplantation cross-resistance in vivo. This study supports the contention that, in carcinogen-induced murine tumors, and perhaps in human neoplasms as well the evaluation of humoral (and cell-mediated) immune responses in vitro may not reflect tumor rejection-type immune responses in vivo.