The reproducibility and accuracy of the Omnicon (Bausch and Lomb Inc., Rochester, NY) automated tumor colony counter for counting tumor colonies growing in double layer soft agar is evaluated and the reproducibility is compared with manual tumor colony counting. Replicate within day runto-run colony counts of the Omnicon show a median correlation coefficient (r) of >0.985, and day-to-day median r of >0.980. In contrast, for manual colony counting, the best intra-observer reproducibility achieved is a r of 0.943 and the best inter-observer reproducibility is a r of 0.831. Analysis of results from individual culture plates counted by the Omnicon on 5 separate days shows a median coefficient of variation of 10% with 77% of the culture dishes showing coefficients of variation of colony counts over 5 days of less than 20%. Counting of culture plates during incubation shows that the Omnicon is counting tumor colonies developing after plating of a single cell suspension.

Soft-agar culture of transitional cell carcinoma colonies from urine, bladder irrigation fluid, and transurethral resection solid tumor specimens demonstrate generally better growth of tumor cell colonies from urine than from irrigation fluid. Growth of tumor colonies from urine was adequate to evaluate presence of tumor and to study growth parameters of the tumor colonies in vitro, although the number of colonies produced from urine was inadequate to evaluate sensitivity or resistance to chemotherapeutic agents. Growth in soft agar of transitional cell carcinoma colonies from urine may offer a simple, noninvasive method of evaluating the effectiveness of therapy and the prognosis for tumor progression.

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

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To evaluate the persistence of cells with clonogenic properties in patients being treated for transitional cell carcinoma, 272 urine samples were collected from 75 patients and cultured in a double layer soft agar "cloning" system. The development of colonies was evaluated with growth curves based on repeated colony counting with an Omnicon automated colony counter at regular time intervals. Forty-eight patients had at least one evaluable culture. Comparing the results of colony development in culture with the clinical evaluation of the patients, 9 patients had a histologically proven recurrence preceded or accompanied by tumor colony growth in urine culture. One patient had tumor recurrence with growth negative urine culture (false negative). Fifteen patients have had growth negative urine culture with a negative follow-up (mean 19.5 months). Twenty-one patients have had growth positive urine cell cultures with no recurrence in their follow-up (mean 18.7 months). Although the follow-up times are at present relatively short, the present study suggests that repeated soft agar urine culture of patients with low-grade, low-stage bladder carcinoma may provide a means for identifying those patients at a higher risk for recurrence/progression of their disease.