Clonogenic assays currently define colonies as multicellular growth units above an arbitrarily designated cutoff size rather than by the biological function of different-sized growth units. To define the cutoff size between clusters and colonies in terms of the biological function of the cells within the growth units, we directly measured the self-renewal and proliferative capacity of cells from different-sized melanoma colonies. Primary colonies formed from cells of two patients were removed, pooled according to size, and replated, and the frequency and size distribution of the secondary colonies were analyzed. Cells from primary melanoma colonies that resulted from four to eight population doublings had similar extensive proliferative and self-renewal characteristics. The results demonstrated that self-renewal was not limited to cells in large colonies and suggested that the cutoff may be below 16 cells/growth unit. These data support the use of relatively small multicellular growth units to define colonies and measure highly proliferative human melanoma tumor cells. In addition, these methods may allow the determination of the cutoff size for other tumor types in terms of the biological function of cells rather than arbitrarily designating a cutoff size.

The number of cells within tumor colonies has not been determined accurately in prior reports because, in all but small clusters, cells grow too closely and stacked to allow direct counting of cells by inverted microscopy. Therefore, we stained colonies formed in agar from 38 tumor cell samples of diverse histological origin, removed them with a micropipet, and directly counted the number of cells. The number of cells within colonies increased geometrically with colony diameter and inversely with the size of the cells within the colonies. The relationship can be described using linear regression: [In(no. of cells/colony) = 0.87 - 2.80 In (colony cell diameter) + 2.38 In (colony diameter)] which gave an R2 of 0.92. Measurements of colonies in agar showed that they grew as oblate spheroids rather than spheroids. In an average sample, 60-micron-diameter colonies contained eight to 10 cells; the range for the 38 samples was 1.2 to 48.5. These results precisely define colonies in terms of cell number. They allow calculation of the total number of cells formed from clonogenic cells, a more complete estimate of proliferation than conventional cloning efficiencies which only measure initial proliferation. Furthermore, because of the dependence on the size of the colony cells, if colonies are defined only by a specific diameter then they do not contain similar numbers of cells. Calculations which assume spherical colonies, rather than the oblate spheroid shape we found, greatly overestimate the number of cells within colonies. Our data can increase the accuracy of quantitation in the clonogenic system and thus improve the interpretation of the proliferation of clonogenic tumor cells.

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.

The human tumor stem cell assay (HTSCA) has been used to study the in vitro sensitivity rates of anticancer drugs used in the treatment of 115 patients with previously untreated and relapsing ovarian cancer. The data from these studies have identified patterns of cross resistance and residual sensitivity between these agents, and have allowed the prospective selection of single agents possessing in vitro activity for the treatment of 32 patients with relapsing disease. cis-Platinum and vinblastine were the most active agents in vitro against ovarian TCFUs from both previously untreated and relapsing patients. Prior therapy with even one drug was associated with the acquisition of resistance to several classes of compounds (e.g., melphalan resistance was almost always associated with in vitro adriamycin resistance, P less than 0.001). A clinical trial yielding similar data would have required nearly 450 evaluable ovarian cancer patients. In 11 of 32 patients in vitro testing predicted sensitivity to single agents: eight of these had partial remissions for a predictive accuracy of 73%. In 33 instances the HTSCA had 100% accuracy in predicting the lack of clinical response. Thus, the HTSCA for advanced ovarian cancer appears to have a similar predictive accuracy rate to the estrogen receptor assay for predicting the response to hormonal therapy for disseminated breast cancer.

The retinoid skin cancer prevention (SKICAP) trials are a set of double-blind, randomized, placebo-controlled clinical trials. The SKICAP-actinic keratoses (AK) trial tests the hypothesis that daily supplementation of retinol (25,000 IU) for 5 years reduces the incidence of skin cancers in high-risk individuals, those with a history of greater than ten clinically or pathologically diagnosed AK and, at most, one prior pathologically confirmed cutaneous squamous cell carcinoma (SCC) or basal cell carcinoma (BCC). The SKICAP-SCC/BCC (S/B) trial tests the hypothesis that daily supplementation of retinol (25,000 IU) or 13-cis-retinoic acid (5 or 10 mg) for 3 years reduces skin cancer incidence in very high-risk individuals, those with a history of at least four pathologically confirmed SCCs or BCCs. Between 1984 and 1988, 2800 participants were enrolled at two clinics on the SKICAP-AK trial; and between 1985 and 1990, a total of 719 participants were enrolled at four clinics on the SKICAP-S/B trial. The initial recruitment strategy was referral by dermatologists, but low accrual necessitated the use of other strategies to achieve enrollment goals, which included involving additional clinics and using paid trial-specific advertisements in print and electronic media. Thirteen % of the SKICAP-AK participants and 36% of the SKICAP-S/B participants were enrolled through dermatologist referral, whereas paid advertisements resulted in enrollment of 87% of SKICAP-AK and 43% of SKICAP-S/B participants. A population-based skin cancer registry was used to identify and enroll the remaining 21% of the SKICAP-S/B participants.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective methods have been developed to quantitate results of the in vitro human tumor stem cell assay, the degree of the association between the in vitro assay and clinical response as well as the likelihood of response. Methods considered to quantitate in vitro assay data included first-order kinetics of percent survival with drug concentration, minimal percent of tumor colony-forming unit survival at low drug concentrations, and area under the in vitro percent survival-drug concentration curve. Based upon experimental data, the percent tumor colony survival and the area under the curve (i.e., in vitro sensitivity indices) were concluded to better account than other methods for the commonly observed nonlinear shape of the in vitro curves. The two methods also yielded equivalent quantitative descriptions of the in vitro data. A logistic regression model was used for explicit quantitation of the relationship between the in vitro sensitivity index and predicted probability of clinical response. Very high association was observed between the predicted in vivo and actual clinical response for the cytotoxic drugs considered. Incorporation of other pharmacologic and patient prognostic factors into the quantitative methods is discussed and shown to improve their effectiveness.

We measured the effect of 6 standard (Adriamycin, BCNU, DTIC, melphalan, vinblastine, actinomycin D) and 3 Phase II agents (cis-platinum, vindesine, AMSA) on melanoma colony-forming units (CFU) in soft agar from biopsies of 50 patients with metastatic melanoma. Melanoma CFU demonstrated marked heterogeneity in chemosensitivity to these 9 drugs. Reduction in survival of CFU below 38% at one-tenth the pharmacologically achievable 1h concentration (our operational definition of chemosensitivity) was obtained in only 19% of 200 in vitro trials, and was usually the same whether or not patients had been exposed to prior chemotherapy, suggesting that melanoma CFU are inherently resistant to presently available chemotherapeutic drugs. The soft-agar assay was 86% accurate (25/29 cases) in identifying drugs to which the tumour was resistant in vivo, and 63% accurate (12/19 trials) in identifying drugs to which the tumour was clinically sensitive, counting mixed responses as responses. In contrast, if mixed responses were classified as progressive disease, the accuracy of identification of sensitivity fell to 42% (8/19 trials). These investigations furnish a quantitative description of the chemosensitivity of human metastatic melanoma CFU. Additionally, these studies serve as a useful step towards the development of an in vitro chemosensitivity test for human melanoma, and provide an operational quantitative basis for further exploration of in vitro-directed therapy in metastatic neoplasms.