Br. J. Cancer (1980) 41, Suppl. IV, 56

IS ANY SINGLE IN SITU ASSAY OF TUMOUR RESPONSEADEQUATE?

J. DENEKAMP

Fromn the Gray Laboratory of the Cancer Research Campaign, Mount Vernon Hospital, Northwood,Middlesex, England

Summary.-The different assays available for measuring the response of undisturbedtumours in situ after therapy are reviewed. These are: animal survival time, regres-sion rate of tumours, regrowth delay, local tumour control and loss of incorporatedradioactivity. The relative advantages and disadvantages of each assay are reviewedin terms of cost-effectiveness and the relevance of the data they yield.For comparisons of different treatment modalities any single assay seems adequate

provided a dose-response relationship can be demonstrated. The assay of choicewill depend upon: the dose-range to be investigated, the amount of prior informationthat is required and the skills and apparatus that are available. No single assay isclearly best, but survival time and regression rate studies probably yield the leastvaluable information.

If the main question is the absolute number of cells surviving a particular treat-ment, or the mechanisms leading to a given response, no single assay will yield asmuch information as a combination of several in situ techniques, together withexcision assays. For clinically oriented questions, however, a single assay may beadequate. The choice of an appropriate tumour model is the most important factorin determining the relevance of the data obtained from mice for man.

THE CHOICE of a tumour system for in-vestigating the response of experimentaltumours to therapy involves 2 aspects;the type of tumour and the type of assay.This paper deals mainly with the choiceof assay, and the relevance of differenttumour models is discussed in this volumeand elsewhere (Scott, 1958, 1961; Hewittet al., 1976; Denekamp, 1979).The techniques available for studying

experimental tumours include all thosethat can be used clinically (i.e. life-span, regression, recurrence and localcontrol) plus several which are eitherunethical or impractical for routine usein patients. The latter include the studieswith tracer isotopes (e.g. 125IUdR whichstably labels DNA and whose loss shouldindicate cell death), the study of tumoursin observation chambers and the excisionassays whereby cell survival is assessedeither in vitro or in another recipientanimal. The excision assays are dealt within many other papers in this volume and

I shall confine my attention to the in situmeasurements on undisturbed tumours inthe treated host, i.e. a situation mostclosely resembling that of a cancer patientundergoing therapy.The experimental oncologist may seek

to study tumour response in order to pre-dict the outcome of a new treatment or tocompare various forms of treatment.Alternatively he may be interested in themechanisms underlying the response ofthe tumour to any particular form oftherapy. For the comparison of treatments,any assay which will give a graded re-sponse with increasing dose of the agent(i.e. a dose-response curve) is useful. Someof the assays are more appropriate forstudying low doses, e.g. 125IUdR loss,whereas others are only useful at high doselevels, e.g. tumour control probability.Each investigator will have developedexperience in using a particular techniqueand in handling the data derived from it.In choosing his system he should have

IS ANY SINGLE IN SITU ASSAY OF TUMOUR RESPONSE ADEQUATE?

considered the statistical accuracy that isdesired, and the possibility of achieving itwith a given number of mice, manpowerand cost. However, even one's own well-tried techniques should be periodicallyreviewed to guard against artefacts arisingas an incidental part of the experiment.For example, the problems resulting fromtumour antigenicity influence the inter-pretation of most experiments, and cannotbe over-emphasized.For assays where the precise surviving

fraction of cells is desired no single in situassay is sufficient. The combination ofseveral different assays may allow sur-viving fraction estimates to be made,which can then be compared with theestimates obtained from excision techni-ques (e.g. Wheldon, McNally, Twentyman,Barendsen, this volume). Such comparisonswill yield valuable insight into the mech-anisms that underlie the gross tumourresponse.

Animal survival timeThis assay, or variants of it, are com-

monly used clinically and are fraught withproblems resulting from deaths due toother causes, e.g. from distant metastasescausing death after localized treatment,or death resulting directly or indirectlyfrom the treatment itself, such as frominfection being fatal in an immune-sup-pressed patient. All these problems arealso inherent in the assessment of animaltumours, together with the problem thatsome tumours have artefactual immuno-genicity, resulting from transplantationinto non-syngeneic mice. In this caseeither the immune response may eradicatea reduced tumour burden after localizedtreatment, or the animal may becomeimmune-suppressed and less able to copewith a tumour after systemic therapy. Ineither case the measured response of thetumour will not be a simple reflection ofthe extent of tumour cell kill caused bythe cytotoxic agent. Thus, unless thecause of death is verified in each animal,and data are collected about the status ofthe primary tumours and about any dis-

seminated disease, survival time is notparticularly informative. It is more com-monly used in experimental chemotherapy,where the systemic effects are important,than in radiobiology where the treatmentis generally localized. Furthermore, in theU.K. the restrictions placed on experi-ments by the Cruelty to Animals regula-tions make this an unpopular endpoint,difficult to justify as ethical when otherassays are available.

Regression of tumoursThe rate of shrinkage of tumours after

radiotherapy and its prognostic signifi-cance in terms of long-term local controlhas long been an area of dispute and con-troversy. Thomlinson & Craddock (1967)showed that a rat fibrosarcoma, RIB5,regressed at approximately the same rateafter large single doses, whether the cellkill was only a few decades or whether thedose was curative. They pointed out thatthe regression rate depends mainly on theability of the host organism to resorbthe killed cells rather than on the preciseextent of cell kill. This view was supportedby the clinical data on head and necktumours, and the experimental single dosestudies of Suit et al. (1965). They couldfind no correlation between shrinkage andprognosis in patients or mice. However,more recently a number of clinical studieshave shown a significant correlation be-tween the degree of shrinkage at the endof therapy, or within a few months there-after, and the ultimate local control (seeDenekamp [1977] for references). Thismay be a classic case ofapples and oranges:single dose treatments in rodents may notbe predictive, whereas multiple fractionsin man are. The apparent paradox couldresult from the more rapidly shrinkingtumours becoming more radiosensitiveduring the course of therapy (e.g. bybetter reoxygenation), rather than theshrinkage being a simple reflection of cellkill. For fractionated treatments to ourC3H mouse tumours, shrinkage duringtherapy did appear to be a prognosticindicator (Denekamp, 1977). The shrink-

57

J. DENEKAMP

age rates of individual tumours duringchemotherapy may similarly reflectchanges in the proliferative status of cells,e.g. due to recruitment of non-cyclingcells as the vasculature improves inshrinking tumours. Thus, regression rateor extent may be useful in certain limitedsituations, but will seldom provide datafor the dose-response curves that arerequired in good experimental work.

Regrowth delayThe more prolonged study of tumours

that regress and then recur after therapyhas been shown to yield well-defined dose-response curves after localized treatment(e.g. Thomlinson & Craddock, 1967; Fieldet al., 1968; Denekamp & Harris, 1975) andhas become widely used as a technique forcomparing different modes of therapy(e.g. X-rays, neutrons, radiosensitizers,hyperthermia). The sort of dose-responsecurves that can be obtained are illustrated

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Fia. 1. Regrowth delay data after irradia-tion of Carcinoma NT under aerobic orclamped conditions + misonidazole (1 mg/g).The upper quadrants can be transformedinto "pseudo" survival curves by convert-ing the delay into numbers of cell doublings.Only volume doublings (TD) give a reason-able estimate of the cell kill. (Redrawnfrom Denekamp & Harris, 1975.)

in Fig. 1 for a CBA mammary carcinoma,used with or without the radiosensitizermisonidazole. The shape of the dose-re-sponse curves gives some insight into therelative sensitivities of the subpopulationswithin a tumour, and can be used to de-duce, for example, the fraction of radio-resistant hypoxic cells. It is possible toderive "pseudo" cell survival curves fromsuch data if the dose that is almostcurative has been determined and theapproximate number of cells in the tumouris known. In the example shown (Fig. 1)interpreting the delay in terms of numbersof volume doubling times yields reason-able estimates of surviving fraction(_ 10-8), whereas the use of cell cycle timeor potential doubling time for the calcula-tion gives highly implausible estimatesof 10-20 or 10-36 (Denekamp & Harris,1975).Not all tumours give such small errors

as those shown in Fig. 1, and are thereforenot suitable for obtaining precise datausing this assay (Denekamp & Harris,1976). The advantages and disadvantagesof regrowth delay as an assay are sum-marized in Table I. It is applicable to mosttypes of tumour, providing they will growas discrete nodules in an accessible site.

TABLE I.-Regrowth delay experimentsAdvantages:

1. Suits most types of tumour.2. Yields good dose-response curves.3. Can be used over a wide dose-range4. Experiment design easy-requires little prior

information.5. Can be compared with normal tissue injury

obtained over similar dose range (low doses).6. Can be compared with clinical studies.7. Dose response curve can distinguish subpopu-

lations of differing sensitivity.8. Pattern of regression and of regrowth yield

insight into parenchymal/stromal components.

Disadvantages:1. Tumours must be accessible for measurement.2. Distant metastases may kill animal before

regrowth of primary (cannot include these inthe analysis).

3. At high doses some tumours will be cured (howto include these in the analysis?).

4. Tumour bed effect, i.e. damage to stroma.5. Cannot assess prolonged fractionated treat-

ments; tumours outgrow treatment and re-growth size.

6. Difficult to apply rigid statistical tests.

58

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DOSE (GRAY) DOSE (GRAY)FIG. 2.-Regrowth delay data for Ca Sq.D treated with X-rays alone or with X-rays plus heat toclamped tumours. Many of the animals treated with the combined therapy died of metastases beforeregrowth of the primary tumour was observed: A1l, A- This prevents any average curve beingdrawn. (Redrawn from Hill & Denekamp, 1978.)

The ability to measure a response over awide dose range makes it particularly suit-able for assessing therapeutic benefits,because the low dose regions correspondto the doses that will produce an accept-able level of normal tissue damage; thetreatments can then be "normalized" to atolerance level, as a simulation of clinicalpractice (Field et al., 1968; Denekamp etal., 1976). Regrowth delay has recentlybeen used clinically, for studies of radio-sensitizers in patients with disseminatedsubcutaneous nodules. It has permittedestimates of enhancement ratios and ofthe fraction of hypoxic cells in humantumours (Thomlinson et al., 1976; Dene-kamp et al., 1977; Ash et al., 1979;Denekamp et al., in prep.).The problems are also listed in Table I.

With non-immunogenic tumours, meta-stases may occur so that the animal diesbefore regrowth of the primary hasoccurred. This makes averaging of the

growth delay for groups of animals im-possible in certain situations and makes itdifficult to compare treatments quanti-tatively, as shown in Fig. 2 (Hill &Denekamp, 1978). At higher doses sometumours will be locally controlled, and aproblem arises in how to handle theseanimals in an averaging process. Fowleret al. (1980) have analysed such data inseveral different ways; by regarding thetumour growth delay as infinite and usingreciprocals to obtain an average, by usingthe lifespan of the mouse, or using thelatest time at which a recurrence has everbeen observed. This latter is the mostlogical approach, since it implies that thisis the time taken for a single cell, or viableunit, to cause a recurrence after a highdose treatment that will almost cure allthe tumours (Hill & Denekamp, 1979).The correlation between regrowth delayand cure data when analysed in this wayis good (Fig. 3).

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59

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J. DENEKAMP

Some emphasis has been placed on theprecise size at which regrowth delay isassessed (Begg, 1980). It may correspondto a doubling in volume, an increase byX mm in diameter, where X may be assmall as 1P5 mm (Denekamp & Harris,1975) for a rapidly shrinking mousetumour, or as large as 16 mm in a rapidlygrowing rat sarcoma (Thomlinson &Craddock, 1967). When larger size incre-ments are used there will be an increasinginfluence from damage to stromal elements,the so-called "tumour bed effect", whichmay result in a second plateau in the re-growth curve (Thomlinson & Craddock,1967) or in a slower growth rate intumours treated to different dose levels.Robinson et al. (1974) actually use theslope of the regrowth line as their measureof effectiveness, rather than delay to reacha certain size. Other papers in this volumeare concerned with the relative contribu-tions of stromal and parenchymal cell

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FIG. 3. Tumour control probability and re-growth delay data for tumours treated withsingle doses of X-rays with or without theradiosensitizer misonidazole. This demon-strates the increasing SER' with increasingradiation dose, but the tumour control dataare clearlv an extension of the regrowthdelay data. Sensitizer enhancement of MTtumour by 1 mg/g misonidazole. (Redrawnby J. F. Fowler from data of P. W. Sheldon,Ph.D. Thesis, 1979.)

damage in the gross response of thetumour (e.g. Begg, 1980).When comparing different types of

tumours, or tumours with a wide spreadof growth rates within a transplant, it maybe more meaningful to use the specificgrowth delay, i.e. the delay divided by thepreirradiation doubling time (e.g. Howlettet al., 1975). Data analysed simply asgrowth delay, or instead as specific growthdelay may lead one to quite different con-clusions.Two major problems with the regrowth

delay assay are the difficulty in applyingstrict statistical tests to the data, and thechoice of which day to consider as dayzero when assessing fractionated treat-ments, because the overall treatment timeis not small compared with the end timeof assay. Unless care is taken in theanalyais of such data the regrowth delayquoted may be shorter than the overalltreatment time, i.e. the tumour may haveoutgrown the size range of interest beforetreatnient has even been completed. Whenplotted as delay from final treatment thiswould appear as a negative delay!

Tumour cure (local control)The logical extension of a regrowth

delay experiment to higher doses and moreeffective treatment leads to a region wherevarious proportions of the tumours havean infinitely long regrowth delay or are"cured". These data can be plotted astumour control probability curves and canbe analysed using conventional statisticaltechniques, e.g. by probit or logit analysis.Fowler et al. (1980) have compared dataobtained for several different forms oftherapy, where both a regrowth delay anda cure assay have been used. In most casesthe enhancement ratio was seen to varywith the radiation dose level and the"cure" data simply represents an exten-sion beyond the dose range that is useablein delay experiments (Fig. 3). No sig-nificant differences have been shown inthe conclusions from these 2 assays. Thetime at which the assessment of localcontrol is made needs to be determined

60

.

IS ANY SINGLE IN SITU ASSAY OF TUMOUR RESPONSE ADEQUATE?.

TABLE II.-Tumour cure experimentsAdvantages:

1. Good resolution (small errors).2. Can apply rigid statistical tests.3. Can use more prolonged schedules.4. Easy to score: yes/no.5. Obviously relevant to clinical practice.

Disadvantages:1. Slow (80-180 days).2. Expensive in animal numbers and space.3. Not suitable for metastasizing tumours.4. Restricted to large single or total doses.5. Not directly comparable with normal tissues

which respond at lower doses.6. Experimental plan requires prior information

about relevant dose range.

for each tumour system, and is generallyshorter in a fast-growing tumour than inone which grows slowly. This time shouldbe determined in the initial experimentsfrom the time at which the latest recur-rence occurs, or at least beyond whichonly a very small percentage of tumourswould recur. The advantages and dis-advantages of this assay are listed inTable II. Whilst it is precise, relativelyunambiguous and can be used more easilywith prolonged treatments, cure experi-ments are slow to yield information andcorrespondingly expensive in animal housespace and costs. It is often a problem thatthe dose region from 0-100% cures isnarrow, and hence a large amount of priorknowledge is needed to design each experi-ment: furthermore this dose range maynot match the range over which the re-sponse of any normal tissue can bemeasured. This was found to be a problemin the comparison of fractionated andsingle dose treatments, with and withoutsensitizer, in the radioresistant anaplasticMT tumour used by Sheldon & Fowler(1978).Loss of incorporated radioactivityThe loss of incorporated 125IUdR has

been used as a tool for measuring cell lossin undisturbed tumours and in tumourafter cytotoxic therapy. The tumours canbe assessed in situ after injection of theisotope into the tumour-bearing mouse,either using collimated scintillation coun-ters, or by excision of the tumour and

counting in a well-scintillation counter.Alternatively the total radioactivity peranimal can be monitored after transfer oflabelled cells into an unlabelled host. Begg(1977) showed that the estimates of cellloss in untreated tumours obtained byexternal counting of IUdR compared wellwith those obtained from traditionaltritiated thymidine and autoradiographictechniques in some tumours, but not all.He and others (e.g. Begg & Fowler, 1974;Hofer, 1969; Porschen & Feinendegen,1973) have also used this technique as arapid assay for treatments that increasethe rate of cell loss.

Whilst the technique allows a rapidassessment of damage (within 10 days)from a small number of animals, the re-sponse generally saturates fairly quicklyand so is limited in usefulness to relativelylow dose levels. It is clear that the per-centage reduction in radioactivity is nota direct reflection of the percentage ofcell kill. Problems with this technique in-clude the choice of normalization time,i.e. when to start counting; the influenceof surrounding normal tissue cells whichare rapidly turning over, and hence alsolabelled; the influx of non-tumour cells,e.g. macrophages; and the possible re-utilization of the labelled compound afterdisintegration of dead cells in the tumourand from elsewhere in the body. In generalit has not been widely adopted as a methodfor assessing response of solid tumours leftin situ, either experimentally or clinically.

Tumours implanted in observation chambersFor several decades tumour prepara-

tions have been made in Algire chambersor in variants of this, such as the elegantsandwich preparations of Reinhold. Thesepreparations allow direct observation ofthe response of individual tumour regionsin relation to their vascular supply. Whilstthis has allowed elegant investigations ofblood flow changes and of the cytotoxicityof treatments in regions of differing oxygentension, pH, etc. it is seldom used as ameans of quantitatively measuring overalltumour response. This technique is dis-

61

62 J. DENEKAMP

cussed in more detail by Reinhold &Berg-Blok (1980).

Other factors of importanceAs has been reviewed previously (Dene-

kamp, 1979) a number of factors, otherthan the precision of the assay, may playan important role in the relevance orotherwise of the data obtained. The sizeof the tumour at treatment should bestandardized. For example, small tumours( < 2 mm) have been shown to containfewer nutritionally deficient, non-dividingand hypoxic cells than larger tumours.This is presumed to relate to a pro-gressive failure of the vascular system asit is called upon to produce new vessels forthe expanding tumour mass. It is stillcontroversial whether large tumours arethe most relevant to the human situation,or whether it is the ratio of tumour massto body mass that is the importantparameter.A variation in the availability of

nutrients is also likely to occur if tumoursare implanted into different regions in thebody. Some sites are naturally less restric-tive than others, e.g. the loose skin of thebody of the rodent as contrasted with theclosely attached skin on the head, tail orfeet (Denekamp, 1979). This has beenshown to grossly influence the response oftumours to therapy (Hill & Denekamp,1978).The tumour origin (Hewitt et al., 1976),

the number of generations it has beentransplanted, its histological type (Dene-kamp, 1972) and the use of anaesthetics(Sheldon & Chu, 1979; Denekamp et al.,1979) can all influence the response that ismeasured to any form of therapy. Theseare factors which may appear to be trivial,but have been shown to exert a more sig-nificant effect on an experimental inter-comparison than the choice of one in situassay rather than another. The differencesbetween in situ assays and excision tech-niques, however, lead to much greaterdiscrepancies as are described elsewherein this volume.

In summary, any single in situ response

may be appropriate for clinically orientedstudies on experimental tumours, pro-viding it yields well defined dose-responsecurves over the dose-range of interest.The assay chosen should depend upon theskills of the investigator, the tumourmodel that he will choose to work with,and his familiarity with any artefacts orproblems inherent in that method ofassessing damage. By contrast, for inter-pretation in terms of surviving clonogeniccells, several independent sets of data areneeded, as discussed by Wheldon (1980).

I am grateful for the critical comments of DrsJ. F. Fowler & A. C. Begg and to S. A. Hill & J. F.Fowler for permission to use the data in Figs 2 & 3.This work was entirely supported by the CancerResearch Campaign.

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6