PII S0360-3016(00)01469-3

ICTR 2000 Translational Research in the Clinical Setting

PREDICTIVE ASSAYS: WILL THEY EVER HAVE A ROLE IN THE CLINIC?

LESTER PETERS, M.D., F.R.A.N.Z.C.R., F.R.C.R., F.A.C.R.,AND

MICHAEL MCKAY, M.B.B.S. (HONS)., PH.D., F.R.A.N.Z.C.R.

Peter MacCallum Cancer Institute, Melbourne, Australia

INTRODUCTION

The term “predictive assays” refers to laboratory tests de-signed to predict the response of tumors and/or normaltissues to radiotherapy on the basis of their radiobiologicalcharacteristics. These tests need to be distinguished concep-tually from clinicopathologic prognostic factors that havebeen determined empirically such as tumor site, stage, typeand grade, and performance status in that predictive assaysare mechanistically based and offer the prospect of rationalinterventions to improve the therapeutic ratio.

The reason for seeking reliable predictive assays is thatwithin any clinically defined strata of patients, there issignificant variability in outcome with respect to both tumorcontrol probability and normal tissue damage. It is intu-itively obvious that knowledge of which patients were des-tined to respond favorably to radiotherapy and which werenot would enable the most appropriate treatment to beselected on an individual basis. This expectation is sup-ported by formal mathematical modeling (1). Although des-perately needed, predictive assays must be shown to bereliable, reproducible, and practical to be accepted intoroutine practice. Despite a great deal of research effort overthe past decade, this has not yet been achieved.

In this article, we briefly review the current state ofpredictive assays and some of the hurdles they would needto overcome to enter routine clinical use and focus on somenew molecular approaches to predictive assays.

TUMORS

Largely because of the accessibility of tumors for obtain-ing fresh tissue, most predictive assay research has beendone on head and neck and cervix cancers. The main typesof predictive assays that have been studied to date and asummary of the results are as follows:

Tumor cell radiosensitivityIt is intuitively obvious that tumors whose clonogenic

cells are more inherently radioresistant should be more

difficult to sterilize. This is clearly the case when tumors ofdifferent histologies are compared. Within a given histologythere is also a wide range of cellular radiosensitivity, pro-viding the rationale for testing this parameter as a predictiveassay. When radiosensitivity is quantified as thein vitrotumor cell surviving fraction at 2 Gy (SF2), several groupshave found a trend toward a higher surviving fraction intumors that recur after radiotherapy than in those that arecontrolled. However, in only one series of cervix cancerpatients has the difference been significant and sustained inmultiple analyses (2). In the case of head and neck cancer,results have generally been inconclusive. The most convinc-ing evidence comes from a recently reported prospectiveSwedish study (3) that showed a significant effect of SF2 onlocal control but not survival. Indirect assays of cellularradiosensitivity based on measurements of DNA or chro-mosomal damage are quicker but no more discriminatingthan SF2.

Tumor cell oxygenationThe radioprotective effect of hypoxia has long been sus-

pected as a cause for radioresistance, leading to a largenumber of treatment strategies aimed at countering thepresence of hypoxic but viable tumor cellsin vivo. Thesehave had only modest success, suggesting that hypoxia isnot a universal factor determining treatment outcome. Thusit is logical to think that quantitation of hypoxia in individ-ual tumors might have utility as a predictive assay. Only inrecent years has direct measurement of tissue oxygen ten-sions been possible. Although limited in number and size,studies of tumor cell oxygenation using polarographic probemeasurements have consistently shown an adverse effect onboth local tumor control and survival in tumors with a lowmean level of oxygenation (4). Besides conferring radiore-sistance directly, it has recently been found that hypoxiaselects for cells with defects in apoptosis, such as tumorcells with mutant p53 (5). Interestingly, hypoxia is an ad-verse prognostic factor even in patients treated surgically

Reprint requests to: Lester Peters, Peter MacCallum CancerInstitute, St. Andrews Place, East Melbourne Vic 3002, Australia.Tel: 161 3 9656 1111; Fax:161 3 9656 1424; E-mail: Lpeters@petermac.unimelb.edu.au

Presented at ICTR 2000, Lugano, Switzerland, March 5–8, 2000.Accepted for publication 31 August 2000.

Int. J. Radiation Oncology Biol. Phys., Vol. 49, No. 2, pp. 501–504, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/01/$–see front matter

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(6), implying that factors other than radioresistance areinvolved.

Tumor cell number and proliferation kineticsTumor control probability is closely correlated with tu-

mor volume. A simple explanation is that larger tumorscontain more clonogenic cells that must be sterilized toachieve a cure.

Because tumor control depends on sterilizing all theclonogenic cells that are present at any time up to the end oftreatment, it is obvious that repopulation of cells that sur-vive the early part of a course of treatment may result infailure to achieve tumor control. This is the reason for theimportance of overall time of treatment. Measurements oftumor cell proliferation kinetics have therefore been studiedas a predictive assay.

Preliminary results suggested that tumor cell potentialdoubling time (Tpot) as an estimate of the regenerativecapacity of tumor cells during fractionated radiotherapymight have predictive value. However, more mature datahave failed to show any significant correlation of Tpot withtumor control, although simple labeling index (LI) did showa weak correlation (7).

Summary of tumor predictive assays and inherentunsolved problems

To date, predictive assays for tumor control have hadlimited success, and none has entered routine practice. Al-though tumor hypoxia appears to have clinical potential,probe measurements will need to be replaced by noninva-sive methods to achieve clinical acceptance.

The failure of any one assay to reliably predict for tumorcontrol is not too surprising, since tumor response is almostcertainly multifactorial. It is therefore appealing to considerpredictive assays based on a test dose of radiationin situ asa surrogate. Morphologic assessments of tumor cell radio-sensitivity based on cytological examination of tumor cellsduring a course of radiotherapy have been correlated withtreatment outcome by various authors for more than 50years. Although proponents of cytologic assays haveclaimed high predictive value, the subjectivity of theseassays probably accounts for their lack of clinical accep-tance. More recent approaches include measurement of tu-mor cell chromosome aberrations (8) and nett tumor cellDNA damage using the comet assay (9). No clinical assess-ments of the utility of these assays have yet been published.

Problems in bringing tumor predictive assays to the clinicinclude the relative inaccessibility of the majority of tumortypes for sampling, patient tolerance of invasive samplingprocedures, precise definition of the clonogenic cell popu-lation, and the precision and time-to-results for most assays.

Normal tissuesIn clinical radiotherapy, the therapeutic ratio depends on

the relative radiosensitivity of the tumor with respect todose-limiting normal tissues. Usually, the limitation im-posed by normal tissue tolerance determines total dose

and/or treatment intensity (10). It follows, therefore, thatpredictive assays of response of normal tissues could beeven more important ultimately in optimizing radiotherapythan assays of tumor response. Most predictive assay re-search on normal tissues to date has been based on theradiosensitivity of normal lymphocytes and skin fibroblasts.A promising new focus is on genetic determinants of radi-osensitivity.

Cellular radiosensitivity.Several small series using cul-tured fibroblasts from individual patients have shown pos-itive but weak correlations between fibroblast radiosensitiv-ity and endpoints such as s.c. fibrosis and telangiectasia.Brock et al. (11) systematically examined the effect of doserate and immediate vs. delayed plating on the predictivevalue of fibroblast SF2 and concluded that cellular radio-sensitivity could at best explain only part of the interpatientvariability observed clinically. More recent larger studies(12) have failed to demonstrate any significant utility forthis assay. Part of the problem with fibroblast SF2 assays isthe precision achievable with a single measurement, butperhaps more important is that cellular radiosensitivity isonly one of several interacting factors determining latenormal tissue response. For example cytokines, especiallyTGFb1, which is rapidly induced after cellular exposure toionizing radiation (13), also play an important role. Anotherpotential contributing factor is the induction by radiation ofterminal differentiation or senescence in the fibroblast lin-eage (14).

Only in cases of extreme radiosensitivity syndromes,such as ataxia telangiectasia and related syndromes, arenormal cell types uniformly radiosensitive. In the spectrumof “normality” there is little correlation between the inter-individual radiosensitivity of different cell types. Thiswould imply that for cellular radiosensitivity to be used asa predictive assay, the critical cell population would have tobe identified for each normal tissue of concern—a dauntingtask.

Genetic profiling.Realization that the underlying basisfor differences in radiosensitivity of different individuals ismost likely genetically determined has led to searches forgenetic aberrations that could be used as predictive assays.Two basic approaches are currently under intensive study.The first is an attempt to link mutated candidate genes suchas the ataxia telangiectasia gene ATM (15, 16), BRCA1,BRCA2 (17), and other genes involved in DNA double-strand break repair with the radiosensitive phenotype. Pre-liminary results from this approach have to date beenlargely unsuccessful, as might be expected based on themultitude of interacting genes involved. However, it ispossible that subgroups of radiosensitive patients mightcarry mutations or polymorphisms in specific ionizing ra-diation damage-processing genes. If this turns out to be thecase, screening of the radiotherapy population for the par-ticular gene defect before therapy may allow treatment to beindividualized for those carrying the gene variant.

The second, more ambitious approach is to attempt todefine a large-scale genetic “fingerprint” of radiosensi-

502 I. J. Radiation Oncology● Biology ● Physics Volume 49, Number 2, 2001

tive individuals using microarray (“DNA Chip”) technol-ogy. In this procedure, DNA corresponding to (part of)thousands of gene sequences is coupled to a solid supportand interrogated with a mixture of two different labeledRNA pools, corresponding to two different states ofinterest (e.g., diseased or not). The relative hybridizationof each sample to each DNA spot gives a measure ofactivity (expression) of each individual gene in the twosamples. Thus, a profile of gene activity in each samplecan be derived. Gene expression profiling with microar-rays has already been useful for distinguishing differentdisease states, including the genetic complexity of cancer(18); thus its application to radiation sensitivity has sig-nificant promise. To pursue this line of research, we haveestablished a relational database of patients known tohave experienced abnormally severe radiation reactionsand have collected blood and skin samples from them.The gene expression profile of these patients has beenassessed using a system based on cDNA arraying on glassslides (S. Bassal, M. Chao, and M. McKay, unpublished).We have identified many mRNA expression differences

between radiosensitive patients and controls, includingknown radiation-responsive genes (Fig. 1).

For clinical applications, the analysis of small numbers ofcells would seem essential in investigating the cellular basisof radiosensitivity in different organs. In this regard, arecent complementary advance has been the development ofquantitative methods for close to proportional amplificationof material from very few cells using the polymerase chainreaction. Combining microarray analysis with histologicmicrodissection of different cell types (19) now enables thederivation of cell-type–specific gene expression profiles.Should profiles of the radiosensitive phenotype be detect-able, their combination with fine-needle aspiration wouldhave good potential for their trial in the clinic.

Because of the sensitivity and potential scope of suchtechnology—to examine the entire genetic expression pro-file of an individual simultaneously—the microarray ap-proach seems to be the best prospect yet for predictinghuman radiosensitivity.

In summary, although initial results are emerging (20), it istoo early to assess the ultimate value of this new approach.

Fig. 1. (Top) Image of the same region of two separate 5,000 gene chips hybridized with pairs of labeled RNA froma radiosensitive individual and a nonradiosensitive control cancer patient. The similar signals across both fields indicateexcellent intrachip reproducibility of the hybridizations. (Bottom) Replicates of the 16 genes on a 5K chip that variedgreater than twofold between lymphoblastoid cell lines from a control and a radiosensitive individual after 2 Gy ionizingradiation and 4 h RNA harvest. The fold differences indicate absolute differences between control and test cases. Notesimilarity between values for replicate samples (light and dark bars), demonstrating good intrachip variation (All genesare spotted twice on the one chip). (Lightning) Bone morphogenetic protein 4, a TGFb superfamily member (TGFb isalso a strong early-phase–induced gene in the IR stress response), and placental and bone alkaline phosphatases(arrowheads), both expressed 2.5–5-fold greater in the control, indicating internal consistency in the experiment.

503Predictive assays and the clinic● L. PETERS AND M. MCKAY

Normal tissue predictive assays: Inherent unsolvedproblems

As for tumors, the relative inaccessibility of, and inabilityto clearly define the relevant stem cell population of differ-ent tissues limits the utility of normal tissue assays based oncellular radiosensitivity in the clinic. Another hazard is thecontribution of factors other than cell survival to the patho-genesis of severe radiation reactions in patients. As fortumor predictive assays, the precision, reproducibility andtime-to-results for cellular radiosensitivity assays are alsolimiting factors, although some surrogate assays for clono-genic survival and microarray analysis give results withinthe window of time required for clinical decision-making.

Will predictive assays ever have a role in the clinic?Unfortunately, this is still unclear at present. It is cer-

tainly disappointing that after more than a decade of inten-sive effort, no predictive assay has yet entered clinicalpractice. However, the rapid rate of development of newtechnologies and their integration into predictive assay re-search are reasons for maintaining an optimistic outlook. In

the case of prediction of tumor response, one possibilityis that a combination of imaging and cell-based assayswill enable the four most important radiobiological pre-dictive factors (inherent sensitivity, tumor oxygen status,proliferative capacity, and clonogenic cell number) all tobe assessed in a practical way. These would then becategorized (e.g., favorable, average, unfavorable), todetermine which is most likely to be cure-limiting in agiven patient. Whether tumor-specific gene expressionprofiles will provide recognizable “signatures” for radio-curability is an unanswered question but may rendermore traditional assays obsolete.

Similarly for normal tissue prediction, the best pros-pects seem to lie in the field of genetic profiling; it iscertainly encouraging to note that preliminary resultshave shown consistent patterns of gene expression inoverreacting patients. However the specificity of thesepatterns and any causal relationship to the radiosensitivephenotype have not been established. The next few yearswill be critical in determining just how close we are tothe “Holy Grail.”

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