Application of a Human Tumor Colony-Forming Assay to New Drug Screening1

[CANCER RESEARCH 45, 2145-2153, May 1985]

Application of a Human Tumor Colony-forming Assay to New Drug Screening1

Robert H. Shoemaker,2 Mary K. Wolpert-DeFilippes, David H. Kern, Michael M. Lieber, Robert W. Makuch, Marmette R. Melnick, William T. Miller, Sydney E. Salmon, Richard M. Simon, John M. Venditti, and Daniel D. Von Hoff

Division of Cancer Treatment,National Cancer Institute, Bethesda, Maryland 20205 [R. H. S., M. K. WO..R. W. M., N. R. M., R. M. S., J. M. V.]; Departmentof Surgery, University of California at Los Angeles, Los Angeles, California 91343 [D. H. K.]; Departmentof Urology, Mayo Clinic, Rochester, Minnesota 55901 [M. M. L.]; V. S. E. Corporation,Alexandria, Virginia22303 [W. T.M.]; Cancer Center,Universityof Arizona, Tucson,Arizona 85724 [S. E. S.]; and Cancer Therapyand ResearchFoundation of South Texas,SanAntonio, Texas78229 [D. D. V.H.J

ABSTRACT INTRODUCTION The applicability of a human tumor colony-forming assay to Screening for new anticancer drugs has primarily been con drug screening was investigated in terms of feasibility, validity, ducted using a variety of tumor systems in vivo (8, 33). The and potential for discovering new antitumor drugs. Feasibility screening system currently in use at the NCI3 is composed of a was addressed in a pilot study during which basic methods, sensitive prescreen, the P388 leukemia model, and a "tumor appropriate assay quality controls, and a standardized protocol panel" composed of 3 transplantable murine cancers, the L1210 for screening were developed. Considerable variability was noted lymphocytic leukemia, the B16 melanoma, and the M5076 sar in the availability and colony growth of different tumor types. The coma; and a human mammary tumor xenograft, the MX-1 (34). majority of the Ã©valuableexperiments utilized breast, colorectal, The demonstration by Hamburger and coworkers (10, 23) and kidney, lung, melanoma, or ovarian tumors. For many tumor others (7) that tumor cells from human solid tumors could be types, little evidence of growth was observed, or only rare selectively grown in primary culture for drug sensitivity assay specimens formed colonies. Colony-forming efficiencies ranged indicated a new and highly relevant system which could poten from 0.05 to 0.11% for the six most useful tumors listed above. tially be used for drug screening. Initial work with this in vitro A set of quality control measures was developed to address system had focused on its use for predicting clinical activity of technical problems inherent in the assay. Testing of standard anticancer drugs on an individual patient basis. Results from a agents in the pilot study established that most of these agents number of independent laboratories have indicated that the could be detected as active. However, it also identified three assay does have some validity in this application, particularly assay limitations: compounds requiring systemic metabolic acti with respect to identification of drug-resistant tumors (5,14,17, vation are inactive; medium constituents may block the activity 31,35,36). The practical value of the assay for patient sensitivity of certain antimetabolites; and compounds without therapeutic testing has recently been the focus for some debate (1, 3, 20, efficacy may be positive in the assay. The assay categorized 22, 26, 27, 32, 40), but it seems likely that, for certain tumor nontoxic clinically ineffective agents as true negatives with 97% types, i.e., those which tend to grow well in the assay and for accuracy. Of 79 compounds which were negative in the current which an adequate number of effective drugs are available, the National Cancer Institute prescreen (leukemia P388), 14 were assay may prove useful as a tool for the clinical oncologist. However, as for any predictive test, the characteristics of the active in the assay. Several demonstrated outstanding In vitro patient population, and in particular the clinical response rates activity and are structurally unrelated to compounds already in to the available drugs, must be considered in utilizing the assay development or in clinical trials. A subset of these active com pounds were found to lack activity in a P388 in vitro colony- data (16, 39). Usefulness of the assay as a tool in preclinical drug develop forming assay. This indication of differential cytotoxicity to human ment has been indicated in analogue studies (21, 24) as well as tumor cells makes this subset of compounds particularly inter in studies of novel agents which were initially identified in other esting as antitumor drug leads. The demonstrated sensitivity to systems (2, 11, 25, 37, 38). The clinical correlations which had most standard agents, discrimination of nontoxic compounds, been reported suggested that the assay might have potential as reproducibility of survival values within assays and between a new drug screening system not only from the standpoint of laboratories, and evidence of ability to identify active compounds identifying active agents, but also for indicating which tumor which were negative in the in vivo prescreen suggest that the types might be most responsive to a new agent. At this point in human tumor colony-forming assay may be a valuable tool for time, it is not yet clear that targeting of clinical trials based on antitumor drug screening. However, because of technical limita "in vitro Phase II trials" will be possible. For example, Von Hoff tions inherent in the current assay methodology, this must be ef al. (37) reported In vitro sensitivity of human pancreatic tumors confined to selected tumor types and limited to screening on a to bisantrene. Clinical trial data available to date do not indicate activity for bisantrene in this tumor.4 In the autumn of 1980, the moderate scale. NCI initiated a multiinstitutional, contract-based program to eval 1This work was supported by Contracts N01-CMO-7251, N01-CMO-7327,N01- CMO-7419, N01-CMO-7420, N01-CM1-7497, and N01-CM3-7652 from the Na uate the assay and its role in drug development. The goal of the tional Cancer Institute. present study was to evaluate the applicability of the assay to 2To whom requests for reprints should be addressed at the Drug Evaluation Branch, Developmental Therapeutics Program, Division of Cancer Treatment, 3The abbreviations used are: NCI, National Cancer Institute; DMSO, dimethyl National Cancer Institute, Landow Building, Room 5CO3, Bethesda, MD 20205. sulfoxide. Received1/30/84; revised 11/19/84; accepted 2/7/85. 4D. Hoth, personal communication.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. NEW DRUG SCREENING WITH A HUMAN TUMOR COLONY-FORMING ASSAY large scale drug screening in terms of feasibility, validity, and performed using 10-fold dilutions of test compounds. potential for identification of new antitumor agents. Data Collection and Analysis. Patient demographic and experimental The work presented here represents a cooperative effort of data were collected on floppy disks integral to the image analysis system NCI staff and investigators from 4 laboratories (Cancer Therapy and transmitted by telecommunication linkage to the NIH computer facility in Bethesda, MD, for decoding and analysis. Drug effects were and Research Foundation of South Texas, Mayo Foundation, expressed in terms of the percentage of survival, obtained by dividing University of Arizona, University of California at Los Angeles). the mean number of colonies observed in the treated plates by the mean Work has proceeded through 3 phases: a pilot phase, during number observed in the appropriate control plates multiplied by 100. A which basic methods and an assay protocol were developed; a compound was considered "active" if it reduced colony formation to 30% validation phase, during which the assay protocol was evaluated or less of the control value. This criterion has been widely used in other using standard agents; and an implementation phase, during applications of the assay (14,18,23). Initial plate counts were used only which screening was initiated. for establishing assay evaluability and were not subtracted from control and drug-treated group counts prior to calculation of the percentage of survival. MATERIALS AND METHODS RESULTS Drugs and Test Compounds. All compounds were provided by the Division of Cancer Treatment, NCI, and shipped under coded numbers for "blinded" testing in the laboratories. Each compound was weighed Pilot Study Results. In the first phase of this study, a set of 50 compounds was tested "blindly" in each of the 4 laboratories. out on an analytical balance, taking appropriate safety precautions, and solubilized in either water, 0.9% NaCI solution (saline), DMSO, orethanol. Included among these compounds were a variety of established Stock solutions of compounds were stored in small aliquots at -70Â°C anticancer drugs, new compounds currently undergoing clinical until required for testing. Compounds not soluble in any of the vehicles trials, some compounds without therapeutic efficacy, as well as listed above were handled as suspensions for testing in the assay. compounds selected as either positive or negative in the NCI in Tumor Specimens. All specimens were obtained from patients who vivo tumor panel. Each of these compounds was tested in 10 had not received prior chemotherapy. Specimens were collected using different tumor specimens consisting of 3 to 5 different histolog- aseptic techniques and transported in medium containing penicillin and ical types, with at least 4 specimens of one tumor type. Results streptomycin. Specimens which could not be immediately processed were minced into fragments 5 cu mm or smaller prior to transport. from this testing are summarized in Table 1 and are expressed Effusions were collected with 10 units of preservative-free heparin per as in vitro response rates with all tumor types pooled. In this ml of effusion. Specimens were processed to obtain cell suspensions as table, compounds are ranked from high to low, based on their In soon as possible after removal from the patient. While the majority of vitro response rates. Since testing was done at a fixed concen tration (10 Mg/ml, "continuous" exposure) using a heterogeneous specimens were plated within 8 h of removal, because of delays in transport, some were held as long as 80 h prior to plating. Procedures population of human tumors, this effectively ranks compounds for preparation of cell suspensions varied somewhat among the 4 labo according to potency. The failure of many tumors to respond to ratories. One laboratory used mechanical methods exclusively, while the potent agents such as actinomycin D under these high "concen others used a combination of mechanical and enzymatic (12,19) meth tration x time" conditions reflects the natural resistance of the ods. Culture Methods. A 2-layer soft agar culture system was used in tumor cells and, to a lesser extent, the tendency of the assay to these studies. While the basic system was as described by Hamburger yield an indication of drug resistance when adequate quality and Salmon (10), some modifications, particularly in medium formulation, controls are not applied. This tendency relates to several tech were used in the individual laboratories as have been previously de nical problems which are discussed below. Extreme levels of scribed (12,14,23,35). Cell counts were performed on a hemacytometer drug resistance including resistance to multiple agents of differing after lysis of erythrocytes in acetic acid. A total of 500,000 nucleated mechanisms of action (pleiotropic drug resistance) have been cells were plated in a volume of 1 ml (0.3% agar) over 1-ml base layers documented extensively in Chinese hamster cell populations (6, (0.5% agar) in 35-mm Retri dishes. Cultures were incubated in humidified 15) and also observed in human tumor cell populations (4, 28). 37Â°Cincubators with an atmosphere of 5% CO2 in air, and control plates The sporadic activity by compounds such as DMSO indicated were monitored for growth using an inverted microscope. At the time of the need for additional "blinded" testing, including noncytotoxic maximum colony formation (7 to 21 days in culture), final colony counts materials. Additional testing of known compounds was per were obtained with a Bausch and Lomb Omnicon (FAS II) image analysis system (13). Objects presenting a circular profile in 2 dimensions with a formed after implementation of quality control measures which minimum diameter of 60 um were scored as colonies by the system. For were developed to address the technical problems described quality control purposes, control plates were also counted on the day of below. plating (Day 0) or the following day (Day 1). Technical Problems Associated with the Assay. Two im Drug Treatment. Cultures were plated in triplicate with the test portant technical problems were identified in the pilot study, (a) compound incorporated into the top (cellular) layer at the time of plating. Preparation of single cell suspensions from human solid tumors Initial screening of all compounds was performed at a concentration of is extremely difficult, and completely monodispersed suspen 10 f/g/ml. In each experiment, 6 untreated or vehicle-treated cultures sions are not achieved in the majority of experiments, (b) The were plated for determination of control growth. Statistical considerations variability of the estimate of control growth varies from assay to prompted the use of 6 control plates per assay, since each set of 3 drug- assay and in some instances interferes with measurement of treated plates was to be compared to the same set of control plates. Because of spotty laboratory contamination or laboratory accidents, drug effects. With the use of the automated image analysis system, it was possible to quantitate the quality of cell suspen individual cultures were occasionally lost to analysis. For an assay to be sions obtained by performing "colony" counts on cultures im considered complete, at least 3 control and 2 drug-treated plates had to be available for counting at the termination of the experiment. For detailed mediately following plating. In this counting procedure, cell ag studies (beyond the initial screening step), dose-response assays were gregates as well as pieces of tissue debris or other artifacts of

CANCER RESEARCH VOL. 45 MAY 1985 2146

1Colony-forming Table compoundsCompound8Actmomycinassay response rates for pilot study blinded

response30/6421/5121/5321/5525/7419/5817/5316/57

DSpirogermaniumDihydroxyanthracenedioneSangivamyoinHomoharringtonineBleomycinAT-125Sodium21-C2 18'* 15-tffl 12â€¢fp 9-6- azide AZQC 13/50 26 Aphidicolin 13/50 26 3-III Maytansinec/s-PlatinumL-Alanosine 16/6417/7013/54 252424 I0 610162025303540*80866066707680869096100-100AVERAGE CULTURESChart INITIAL PLATE COUNT FOR CONTROL 268562CBDCA2404191 14/5912/5111/4812/5513/6113/6117/8112/6110/518/4110/529/489/309/539/538/516/446/55C.IÃ‚-J3/*t242423222121212020201919181717161411-t countsfor 1. Frequency distribution of average initial (Day 0 or Day 1) plate whichthecontrol cultures. The columns represent the percentage of experiments in -/3-D-ArabinofuranosylcytosineVinWastine average initial counts fell into the indicated categories. Data from 1274 sequen sulfateBCNU5-FluorouracilVincristine tialchart.15!ZQÅ’ experiments are presented in this

sulfateAcer â€¢j negundoEllipticineCotehicineDON295453Gallium

101u_OHUJli.Ou0-I nitrateHenkel compoundPALAO.P'-DDDHadacidinD~M6TC3ptopunri6

1\ 15/497/704/414/445/564/46 t101010999 Hi1 TaxolMelphalanIButyric !Rjfl ?| 1 SoSÃ¯lali IF MB fi M ol ifl B H Ã•[I/ X Moo Ju IA llB/ 1Â»1Ã•VuaJU TiPi, Aplf^/Q acidStreptozotocinCCNU Jp376J sb/^oisWli IMI JJJAÃ•Â°lÂ¿tÂ°Â°o g \j}*â„¢ Â°a

386 396 406476EXPERIMENT 416 426 436 446 456 466 HydroxyureaProcarbazineDMSO 4/475/594/48 988 NUMBERChart 2. Plot of control group coefficients of variation for a representative series Cyclophosphamide 5/61 8 of 100 experiments. The horizontal reference line at 50% indicates the upper limit 276374 4/50 8 setexperiment.served for an Ã©valuable DTICara-AII265595Hexamethylmelamme 5/654/564/573/433/48877765

ofexperiments.among replicate control plates in a typical series Coefficients of variation (30) are seen to range from W-Methyl formamide 2/38 MethotrexateTumor 2/48%4741403834333228430-27-- approximately 5% to greater than 50% in a few instances. The * Compounds identified by 6 digit numbers (NCI accession or "NSC" number) represent compounds recently evaluated in the NCI in vivo screening system. which a mean control colony count of at least 70 was observed Compounds identified by Roman numerals were obtained under commercial dis (1099 experiments) was 29%. creet agreements. 6 All compunds were tested at 10 tÂ¡g/mtby "continuous" exposure. Tumors Assay Quality Control. During the initial phase of the pilot showing reduction of colony formation to 30% or less of the control value were considered to "respond" in vitro. project, the only provision for assay quality control was micro c AZQ, (2,5-bis(1 -aziridinyl)-3,6-dioxo-1,4-cydohexadiene-1,4-diyl)bis-cart>amic scopic inspection of control plates immediately after plating. acid, diethyl ester; CBDCA, Diammine (1,1-cydobutanedicarboxylato(2-)-O,O')- Experiments showing large numbers of input aggregates were platinum; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; DON, 6-diazo-5-oxo-L-nor- teucine; PALA, N-(phospnonacetyl)-L-aspartic acid; O.P'-DDD, 1-chloro-2-(2,2-di- discarded. To be considered as an Ã©valuableassay, at least 70 chloro-2-(4-chlorophenyl)ethyl)benzene; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-ni- colonies per plate were required at the termination of the assay. ttosourea: DTIC, 5-(dimethyltriazeno)imidazole-4-carboxamide; ara-A, 9-,f-n-arabi- Seventy was selected as corresponding to a count of 30 in the nofuranosyladenine. actual area (slightly less than 50% of the total plate area) scanned by the image analysis system. This value has been widely used size, shape, and density comparable to formed colonies are for defining adequate growth in the assay (14,18, 23). All colony recognized by the image analysis system as "colonies." Chart 1 counts presented or discussed in the remainder of this paper shows a frequency distribution of initial mean colony counts in a represent equivalent whole-plate counts. In subsequent work, a series of 1274 experiments. While the majority (>70%) of counts comprehensive program for assay quality control utilizing 4 pa are observed to fall in the range of 0 to 20, a substantial number rameters was implemented. Experiments were qualified for eval- of experiments were performed where higher counts were ob uability based on the number of objects scored as colonies by served, and in some instances, the initial mean colony count the image analysis system immediately after plating (Day 0 or exceeded 100. Chart 2 illustrates the amount of variation ob- Day 1 colony counts). An upper limit for the mean initial colony

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. NEW DRUG SCREENING WITH A HUMAN TUMOR COLONY-FORMING ASSAY number of 47 was established. This upper limit was selected on Table 2 the basis of the pilot study data on initial plate counts (Chart 1). Summary of experience with the human tumor colony-forming assay The limit was selected empirically with the goal of retaining as large a fraction of experiments as possible, while excluding study trolled proto exper ience*666 assays in which very large initial plate counts were observed. As ResultsSuccessful protocol388 col278 (25)" seen in Chart 1, this value excludes approximately 13% of experiment (23) (24) experiments. Providing an upper limit in this manner increases Inadequate growth 776 (50) 318(26) 1094(40) the uniformity of screening assays, while taking into considera Unevaluable assay 186(12) 511 (43) 697 (25) SpecimencontaminationTotal 202(13)15524-Parameterquality-con93(8)1200Overall 295(11)2752e7859" tion the intrinsic variability of tumor specimens and their response to the procedures required to obtain cell suspensions. Control experimentsTotalno. of colony formation in the assay was established as the net increase drugassaysPilotno. of Ã©valuable (mean evaluation day count minus mean initial colony count) in colony number obtained over the course of the incubation period * Results presented are for the cumulative project experience. During the pilot (7 to 21 days). A mean net increase of at least 70 colonies was study, only 3 quality control parameters were used to qualify Ã©valuableassays (no positive control compound was included). During this period, 25% of experiments required for an assay to be considered Ã©valuable.Limitation of were successful. Following implementation of the fourth quality control parameter, the maximum variability of the control growth estimate was the successful assay rate fell to 17%. In the most recent testing, where breast, accomplished by setting 50% as the upper limit for the coefficient colorectal, kidney, lung, melanoma, and ovarian tumors were selected for use, the successful assay rate was 32%. of variation for the mean colony count of the control group. This Numbers in parentheses, percentage. value was also selected empirically and was designed to exclude c For 140 specimens, assays were aborted because of ineligibility (previous the rare, highly variable (see Chart 2) assays from the Ã©valuable treatment) or other technical reasons (inadequate size of specimen, etc.). An average of 11.8 compounds was tested in each of the Ã©valuableassays. data base. Taken together, these 3 quality control parameters provide standardization of assay conditions and ensure that real proliferation occurs in each assay evaluated for drug effects. To remote surgical units (with the associated differences in transport ensure that each inidividual assay would have sensitivity for and storage times), preselection of specimens for use in screen measurement of drug effects, an internal positive control com ing versus utilization of all specimens regardless of quality, etc. pound was incorporated into every experiment. For this purpose, Results of Blinded Testing. To evaluate the false positive sodium azide was applied to the plates as a liquid overlay rate of the assay, a series of nontoxic sugars, salts, and amino immediately after they had hardened to achieve a final concen acids were subjected to blinded testing in each of the 4 labora tration of 600 /ig/ml. Treatment was done in this fashion to avoid tories. As shown in Table 4, results of this testing indicated the induction of cellular aggregates which occurs when cells are activity in 3 of 112 assays for a false positive rate of 3%. exposed to this concentration of sodium azide in liquid medium. Because of the changes in assay protocol which were made Positive control plates were counted on the experiment evalua based on observations in the pilot phase of this project, an tion day, and tumors which failed to show reduction of colony additional set of 20 blinded compounds was tested under the formation to 30% or less of the control value were considered fully developed protocol. This set of compounds included 10 unevaluable. Recently, sodium azide was replaced by mercuric established clinical agents, 5 highly toxic compounds without chloride (100 /Â¿g/ml)as the positive control compound because known therapeutic efficacy, and 5 nontoxic clinically ineffective of concern over the instability of the compound and because compounds. Inasmuch as compounds requiring metabolic acti more consistent results were obtained with mercuric chloride. vation and certain antimetabolites had been shown to be inactive Additional details regarding the rationale for these quality control in the pilot project, further testing of such compounds was felt measures and, in particular, their value for identifying and ex to be unnecessary. Using a 10% in vitro response rate as a cluding artifactually drug-resistant assays have been published minimal criterion for activity, 10 of 10 established agents were elsewhere (29). Table 2 summarizes our experience with assay active, 5 of 5 toxic compounds were active, and all 5 nontoxic evaluability. As indicated in the table, quality control parameters compounds were inactive in this testing (Table 5). Using a 20% were implemented in stepwise fashion. The largest factor inter in vitro response rate as activity criterion, 9 of 10 established fering with assay evaluability was inadequate growth of colonies. agents were active, and the results in the other categories were Culturability of Tumors. Data from 2509 e/periments with the same as using the 10% activity criterion. In order to minimize the potential "false positive" yield of the assay, the 20% response respect to assay evaluability rates for 38 different tumor types are summarized in Table 3. A wide range of colony-forming rate criterion was selected for use in screening of unknown efficiencies and assay evaluability rates were observed. Because compounds. of the limited number of specimens of any one tumor type Evaluation of Assay Reproducibility. Interlaboratory assay obtained, meaningful evaluation of the relative culturability of reproducibility was evaluated in a control experiment using a histolÃ³gica! subtypes, e.g., lobular versus ductal carcinoma of human lung tumor cell line. Cryopreserved aliquots of cells were the breast, was not possible. Based on performance in the assay shipped to each laboratory for testing with a panel of 12 coded and specimen availability, breast, colorectal, kidney, lung, mela compounds. This panel was composed of triplicate samples of nomas, and ovarian tumors were selected as preferred tumor 3 established anticancer drugs (Adriamycin, actinomycin D, and types for use in drug-screening trials. Some variation in the vinblastine) and a nontoxic compound (glucose). Using our stand relative culturability of these 6 tumor types was observed be ard activity criterion, I.e., reduction of colony formation to 30% tween participating laboratories. This was largely attributed to or less of the control value at 10 /Â¿g/ml,the 4 laboratories were specimen acquisition procedures and variables unique to each in agreement, with each of the triplicate coded samples of laboratory, e.g., receipt of tumor specimens from local versus Adriamycin, actinomycin D, and vinblastine being active, while all

CANCER RESEARCH VOL. 45 MAY 1985 2148

Table 3 Tables Assay performance by tumor type Colony-formingassay response rates for known compounds no. of CompoundChromomycin rate42/48 colonies A3* formed in ActinomycinD6Anguidine* 48/6938/55 of Ã©valu Ã©valuable k TumorDigestive assays315343160111680143452991904237403285102313116608415923253961171373192332509%able676037383631292927242221212221212017151413131099887600000000024Av.assays57626257136444147143017425019314235937759434235811934512725612997468116107126377224114 MitomycinCrAdriamycin0 45/6643/66 6965 systemMesotheliomaOvaryMelanomaThyroidUverUterusOther, c/s-PlatjnumMercuric 30/5634/6517/5113/4119/6120/6912/5216/70545233323129232321 chloride*Maytansine*BteomycHTVinblastine

sulfate"Sodium azide"BCNU"'CS-Ruorouradl" sitesCotorectalPancreasAdrenalKidneyAdenocarcinoma,ill-defined Melphalan" 14/67 ChtorambucÃ¼" 5/48 10 L-Glutamine"Sodium 3/613/721/580/680/63%88707054200 chloride"Water"D-Glucose"Marmitol"Response primarysiteStomachLungBreastEsophagusProstateSkinSarcomaBenignSmallunknown

* Cytotoxic compound without proven clinicalactivity. Establishedclinicalantitumor drug. c BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea. Noncytotoxic compound. bowelBladderHead/neckBrainTestisCarcinoma, Table6 Interlaboratoryreprooucibillty This experiment was performed using a cryopreserved human lung tumor eel line. Experimentalconditions were as described in 'Materials and Methods" except that the number of cells plated was reduced to 5.0 Â»10'. siteLymphornaHematopoietic/reticuloendothelialsystemEndocrineGallbladderunknown primary laboratoryB0.50.30.2NT*0.30.00.10.00.2104.5112.9NTC1.21.20.71.51.10.02.20.86.3NT118.5124.3D0.50.21.00.00.30.31.93.01.1108.5103.284.3survival by CompoundActlnomycm ductsGenitaland DActinomycin femaleGenitalsystem, DActinomycin maleLeukemiaMyelomaNervoussystem, DAdriamycinAdriamycinAdriamycinVinblastineVinblastineVinblastineGlucoseGlucoseGlucoseSampleB2B3B4B2B3B4B3B4B5B2B3B4Av.A0.10.00.20.10.10.30.30.10.1102.6104.2115.6% systemNeuroblastomaThymusTotalNo.

Table 4 NT, not tested because of contamination or technical error. Colony-formingassay response rates for negativecompounds CompoundValineD-GalactoseNaCIPhenylalanineD-GlucoseMgCfeD-MannitolL-GlutammeTotalTumorresponserate1/181/211/210/170/170/150/20/13/112%655000003with the tumor cell line may not generalize precisely to the situation obtained with fresh surgical specimens utilized for drug screening, they do provide some assurance that the basic assay can be reproduced in different laboratories. In order to more fully evaluate intralaboratory reproducibility, several established agents were tested blindly in the same tumor (fresh tumor specimens) under 2 different coded numbers. As shown in Table 7, a total of 50 such comparisons were made. Correlation analysis of the percentage of survival values obtained in this fashion yielded an overall correlation coefficient of 0.80. A samples of glucose were inactive (Table 6). A tumor cell line was scattergram of these data is shown in Chart 3, where each point selected for these interÃaboratoryreproducibility studies because represents the percentage of control growth obtained under the of the logistic problems associated with obtaining a suitable fresh 2 different coded numbers. or frozen surgical specimen. These include: the fact that a very Initial Screening Results. To evaluate the potential of the large number of cells is required for this experimental design; human tumor colony-forming assay for identifying compounds fresh specimens are sometimes delayed in transit between lab which might be missed by the conventional in vivo screening oratories; frozen specimens are not always reproducibly re program, a group of 100 compounds which had been negative covered for subsequent growth in soft agar; and not all speci in the in vivo P388 leukemia prescreen was selected for screen mens give rise to Ã©valuableassays under the best conditions ing in the colony-forming assay. While this screening is still in (rapid local processing). While the reproducibility results obtained progress, early results seem to warrant presentation at this time.

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In order to obtain a high probability (83%) of identifying an 340307, responses were noted for 2 of 3 colorectal tumors, one active agent with a true in vitro response rate of 20% or greater, of 3 lung tumors, but no responses in any of 3 other tumor types each compound was designated for testing in 15 different tumors tested. In the case of NSC 338600, responses were observed at a concentration of 10 /Â¿g/ml.Table 8 summarizes initial test in 2 of 3 lung tumors and one of 2 melanomas, but none in the data for the first 79 of these compounds tested. While the 3 other tumor types tested. majority (82%) of these in vivo prescreen-negative compounds Complete evaluation of active compounds will include 5 dose- were also negative in the colony-forming assay, significant activ response experiments in each of the 6 tumor types considered ity (activity in 2 or more tumors) was observed with 14 com feasible for use in drug screening (breast, colorectal, lung, kidney, pounds. Detailed human tumor colony-forming assay response melanoma, and ovary). The purpose of this testing is to confirm rate data for compounds active in 2 or more tumors are shown the activity observed at 10 Mg/ml, to define the spectrum of in in Table 9. While testing of these compounds has been taken to vitro antitumor activity, and to estimate the minimum effective various stages of completion, a substantial range of in vitro concentration. Testing is scheduled at each of the 4 participating response rates is apparent for these compounds. Those yielding laboratories by means of an interactive computer program resi high in vitro response rates tend to show broad spectrum activity. dent in the central NIH computer facility which gives priority for For 2 compounds, NSC 340307 and NSC 338600, some indi further testing to compounds showing the highest in vitro re cation of selective antitumor activity was observed. For NSC sponse rates in the initial testing. While this testing has only recently been initiated, data are available for some of the most Table 7 active compounds. For example, NSC 338698 (Chart 4) has Reproducibility ot drug effects within tumors shown the highest overall in vitro response rate at 10 /Â¿g/ml (Table 9). Results of 3 dose-response experiments with this of experi coefficient*0.80 Compound5-Fluorouracil ments17 compound are illustrated in Chart 5. In all 3 experiments, signif icant activity was observed at doses as low as 1.0 mg/ml. c/s-Platinum 19 0.78 Heterogeneity in tumor response was seen at 0.1 pg/ml, with 2 L-PhenylalaninemustardNo. 14Correlation 0.86 tumors showing reduction of colony formation to less than 60% 0.80o Total 50 of control and one tumor showing no inhibition of colony forma * Correlation analysis of the percentage of survival values obtained when the tion. As for virtually all new compounds acquired by NCI, no same compound was tested twice using the same fresh human tumor specimen under different coded numbers. The Pearson product-moment correlation coeffi information is yet available regarding plasma concentrations cients are given. In all instances, these are significant (P < 0.001). which can be achieved with this compound. If no in vivo model 0 Overallcorrelation coefficient. can be found in which this compound is active, detailed phar- macokinetic studies will be required before this compound is taken to clinical trial. In a series of experiments utilizing human tumor xenografts in nude mice and the same tumors in a colony- forming assay, Friedman ef al. (9) have recently shown that in _12Hzooo vivo therapeutic effects are observed only when plasma drug concentrations are obtained which are associated with significant inhibition of colony formation in vitro. A number of possibilities could explain the human tumor QA O A Â°â€¢D O colony-forming assay activity of a compound that is inactive zUJutcÂ£225200175150125100-'0V .â€¢' 7550250-o.ooaO against mouse leukemia P388 in vivo. To examine target cell Â°0 "D aspects of this phenomenon, a P388 colony-forming assay was 0aÂ» F 0 Â° devised using the same double-layer agar configuration, medium *Â°*2 o"

1__ 1 1 1 1 , L Table 9 25 50 75 100 125 150 175 Colony-formingassay responserate PERCENT OF CONTROL A tumor was considered to respond if colony formation was reduced to 30% or less of the control value. Charts. Scanergram of intraassay reproducibility data. Three established agents were tested twice, under different blinded code numbers, in a series of Tumor type experiments. Reduction in colony formation, expressed as percentage of control, Compound*338698ABCD343522339675E340307338600FG343556341964Breast1/11/10/10/10/10/20/1Colorectal2/30/40/1Kidney1/10/10/11/10/10/10/10/11/1Lung1/11/21/11/11/32/31/21/2Melanoma2/23/31/21/32/30/21/21/11/51/21/3Ovary6/74/61/22/42/72/40/71/40/80/80/70/40/80/4 drugsis plotted are on represented the horizontal as follows: and vertical 5-fluorouracil(["]):cisplatin axes for the different codes. (A); and Data L-phenylala- for the 3 nine mustard (O).

Table 8 Initial colony-forming assay screening results for P388 prescreen negative compounds Colony-formingassay negatives8 65/79 (82)" Compounds active in 2 or more tumorsc 14/79(18) Compounds active in 3 or more tumors 9/79(11) Compounds active in 4 or more tumors 4/79(5) * Each of the negative compounds was tested in at least 6 tumors. " Numbers In parentheses, percentage. : The number of assays conducted on the active compounds ranged from 5 to * Compunds are identified by NCI accession (NSC) number or, for compounds 18. obtained under commercialdiscreet agreements,by alphabeticalcharacter.

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Table 10 In vitro assay results for in vivo P388 prescreen negative compounds colony-form tumor colony-forming Compound8338698AD343522339675E340307338600FG343556341964P388ing assayactivity^++-+â€”â€”+â€”â€”â€”â€”-Humanassay responseratec12/15(80)"10/13(77)5/15(33)3/12(25)3/13(23)2/9 C' 'N II C, ;,C

(22)3/20(15)3/22(14)2/14(14)2/15(13)2/15(13)2/17(12)

Chart 4. Structure of NSC 338698.

120h " Two compounds were unavailablein sufficient quantity for testing in the P388 100 colony-forming assay. * Activity criteria were the same as used in the human tumor colony-forming o ocI- assay, i.e., reduction of colony formation to 30% or less of the control value. 80 c Pooleddata for all tumor types tested. 8 d Numbers in parentheses,percentage. 60 an additional 25% of experiments were judged as unevaluable because of excessive initial counts, excessive control group o 40 oc variability, and/or lack of activity of the positive control com pound. While this is a rather large fraction of experiments to 20 exclude from the Ã©valuabledata base, we feel that these quality control measures are essential for obtaining reliable data and in 0.1 1.0 10.0 particular for excluding from the data base experiments which artifactually demonstrate an extremely drug-resistant phenotype MICROGRAMS/ML Chart 5. Dose-responsedata for NSC 338698. Results from assays in 3 differ (29). A substantial increase in assay sensitivity was realized as ent tumors are summarized here. Symbols used are: melanoma(D); colon (A); and increasingly stringent quality control measures were imple sarcoma (O). mented in this study. This can be seen in the increase in in vitro response rates for established clinical drugs tested in the pilot formulation, and drug exposure mode as used in the human study (Table 1), during which only 3 quality control parameters tumor colony-forming assay. Because of the high colony-forming were in use, and in the subsequent blinded testing under the efficiency and rapid cell cycle time of the P388 cells, the cell fuliy developed protocol (Table 5). input was reduced to 2.0 x 10" cells, and the incubation period The 38 tumor types evaluated in this project showed a wide was limited to 7 days. Table 10 summarizes results for 12 of the range of culturability. Certain tumor types such as head and 14 active compounds described above for which material was neck tumors, lymphomas, and sarcomas performed very poorly. available for testing in the P388 colony-forming assay. Using the This presumably reflects a requirement for growth factors or same activity criteria as applied to the human tumor colony- nutrients not present in the culture medium. In recognition of forming assay, 8 of 12 compounds were inactive against P388 these problems, we have limited drug-screening studies to a in vitro. In Table 10, it can be seen that the human tumor colony- subset of tumors (breast, colorectal, kidney, lung, melanoma, forming assay response rates have fallen to less than 20% for 6 and ovarian tumors) which are both available in adequate number of the initial 14 active compounds. While this finding reduces our and tend to perform well in the assay. The possibility exists that, level of interest in these compounds, we plan to perform addi in selecting these tumors for use in drug screening and further tional dose-response and in vivo tumor panel testing with these selecting as Ã©valuableassays only those specimens giving rise and any future compounds which meet our initial activity criterion, to formation of 70 or more colonies, we have focused on an i.e., reduction of colony formation to 30% or less of the control atypical subpopulation of tumor cells which merely tend to grow value in 2 or more of the tumors tested in the initial (single dose) rapidly. Alternatively, it could be argued that, in these specimens, phase of screening. a truly relevant "stem cell" population can be measured. In any event, low colony-forming efficiencies are observed for all tumor DISCUSSION types, and the practical demands of drug screening indicate that these 6 tumor types are most useful for routine testing. The data presented here indicate that the human tumor colony- The value of a drug-screening model is ultimately established forming assay can be used for new drug screening against by its ability to identify compounds which are useful in clinical selected tumor types. Lack of adequate growth, observed in treatment. At the preclinical level, the validity of a model may be approximately 40% of the experiments, was the principal reason assessed in terms of its responsiveness to known active and for unsuccessful assays. It should be noted that the criterion for inactive agents and in terms of its reproducibility. The utility of adequate growth was relatively stringent in these studies, re the model may be judged by its ability to discover active agents quiring a net increase of at least 70 colonies in the control plates. which are not found by existing models or which are only found In most other studies, only 20 or 30 control colonies were by more costly models. Human tumor xenograft models have required at the termination of the assay. In the present studies, been utilized for drug screening but are very expensive, largely

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. NEW DRUG SCREENING WITH A HUMAN TUMOR COLONY-FORMING ASSAY because of the requirement to utilize immunodeficient "nude" 338698 have modest, activity in P388. It is of interest that the mice as hosts. The cost of screening one compound in the latter 2 compounds were active in the P388 colony-forming human tumor colony-forming assay (6 tumor types) is only slightly assay. These 2 compounds had been negative in the P388 more than the cost of screening in a single xenograft model. The prescreen and only demonstrated activity when retested in vivo, human tumor colony-forming assay has been shown to detect utilizing DMSO (the in vitro vehicle) to formulate the compound. the majority of established agents. Major exceptions were com It could be argued that testing in the standard NCI tumor panel pounds requiring systemic metabolic activation, such as cyclo- is largely irrelevant for these human tumor colony-forming assay phosphamide, procarbazine, 5-(dimethyltriazeno)imidazole-4- active compounds, since only a single human tumor xenograft is carboxamide, and hexamethylmelamine, and certain antimetab- included in the current panel. At this point, however, we feel that olites, such as methotrexate and hydroxyurea. The relative in any indication of in vivo activity would provide support for further vitro response rates obtained for established clinical agents development of these compounds. Several new human tumor tested at a fixed concentration of 10 ng/m\ reflect the relative xenografts are currently under development which may be more potency of these compounds rather than their therapeutic effi appropriate in vivo models for testing human tumor colony- cacy. The activity observed with a number of highly toxic com forming assay active compounds. The identification of com pounds which have not been shown to have therapeutic efficacy pounds which are inactive in P388 in vivo and in vitro and yet is not surprising and reflects the sensitivity of the assay to show significant activity in the human tumor colony-forming cytotoxic compounds. The observation of a 3% false positive assay is perhaps the most important result of our initial screening rate in testing with noncytotoxic compounds seems acceptable studies. Indeed, this observation suggests that it might be advis for an in vitro drug-screening model. Inter- and intralaboratory able to utilize the P388 colony-forming assay as an in vitro reproducibility also supports the validity of the assay protocol. prescreen and thus reduce the number of compounds which The results of the initial drug-screening trials with the assay would have to be tested in the human tumor colony-forming seem promising. Fourteen compounds which would otherwise assay. have been missed by the NCI screening system have been In this study, we have addressed the feasibility, validity, and identified by the assay. While it is not known at this time whether potential of the human tumor colony-forming assay as a new the colony-forming assay is detecting a different class of agents system for antitumor drug screening. While only a limited number than the in vivo prescreen or is simply more sensitive, it would of tumor types can currently be used efficiently in the assay, and seem that use of the assay for rÃ©Ã©valuationofprescreen negative only a subset of these gives rise to Ã©valuableassays, a sufficient compounds may be beneficial. The observation that 8 of 12 of yield of Ã©valuableassays can be obtained with breast, colorectal, the compounds identified by the human tumor colony-forming kidney, lung, melanomas, and ovarian tumors to make drug assay were not cytotoxic to P388 cells in vitro indicates that the screening feasible on a moderate scale. Sensitivity to the majority assay is not simply identifying cytotoxic compounds that lack in of established agents and reproducibility of drug effects between vivo activity as a result of systemic metabolic inactivation or laboratories and within assays support the validity of the assay other pharmacological reasons. Indeed, these compounds ex for use in screening. The ability of the assay to identify promising hibit differential in vitro cytotoxicity between different human agents which would be missed by the conventional in vivo tumor specimens as well as between the murine and human screening system indicates that the human tumor colony-forming cells. This differential cytotoxicity, if favorably expressed in vivo, assay may be a useful addition to the NCI drug-screening pro could be the basis for selective antitumor activity. Among the gram. agents identified, Compounds D, E, and NSC 339675 would seem to be most promising. These agents show in vitro response ACKNOWLEDGMENTS rates greater than 20%, have relatively high 50% lethal dose values in P388-bearing mice, and have novel structures, i.e., no We would like to acknowledge the valuable contributions of Dr. Michael Alley, Dr. Gary Clark, Dr. John Kovach, Dr. Thomas Moon, Dr. Donald Morton, Dr. Jerry active analogues in the NCI data base. NSC 338698 demon Phillips, Robert Brennan, Mary Ann Campbell, and Rosa Liu to the design of this strated a higher in vitro response rate than any of the established project and to the conduct of the work. In addition, we would like to acknowledge agents tested in the assay. However, the evidence for in vitro the dedicated work of the technical personnel at each of the participating institu tions. cytotoxicity of this compound in P388 cells in the absence of significant in vivo activity or toxicity may indicate that this com pound is metabolically inactivated in the host. NSCs 340307 and REFERENCES

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Robert H. Shoemaker, Mary K. Wolpert-DeFilippes, David H. Kern, et al.

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