Biological Basis for Chemo-Radiotherapy Interactions

European Journal of Cancer 38 (2002) 223–230 www.ejconline.com

Biological basis for chemo-radiotherapy interactions

C. Hennequina,a,*, V. Favaudonbb aaService de Cance´ ´ rologie-Radiothe´ ´ rapie, Ho ˆ ˆ pital Saint-Louis, 1 avenue Claude Vellefeaux, 75010 Paris, France bbUnite´ ´ 350 INSERM, Institut Curie-Recherche, Labs. 110-112, Centre Universitaire, 91405 Orsay Cedex, France

Received 29 June 2001; accepted 29 August 2001

Abstract

For over 10 years, chemo-radiotherapeutic combinations have been used to treat locally advanced epithelial tumours. The rationale for these combinations relies on spatial cooperation or interaction between modalities. Interactions may take place (i) at the molecular level, with altered DNA repair or modification of the lesions induced by drugs or radiation, (ii) at the cellular level, notably through cytokinetic cooperation arising from differential sensitivity of the various compartments of the cell cycle to the drug or radiation, and (iii) at the tissue level, including reoxygenation, increased drug uptake or inhibition of repopulation or angiogenesis. Some mechanisms underlying interaction of radiation with ciscis-diammino-platinum (II) (ciscis-Pt), 5-fluoro-200 -deoxyuridine (5-FU), taxanes and gemcitabine are described. It is shown how various mechanisms including cell synchronisation and reoxygenation concur to paclitaxel-induced radiosensitisation. In the future, specific targeting of tumours, for example, with the epidermal growth factor receptor (EGFR) or angiogenesis inhibitors, should be achieved in order to increase the therapeutic index. ## 2002 Published by Elsevier Science Ltd.

Keywords: Chemo-radiotherapy; Taxane; Gemcitabine

In In 1979, Steel and his coworkers proposed a theo-o- Where the drug carrieries protection againsinst radiatiatioionn retical frame for the prediction of the outcome of com- damage to normal healthy tissues, this would theoretically bined tretreatments with cytotoxic chemotherapy and allow the radiation dose to the tumour to be increased. In radiotherapy [1,2], including: general, however, to obtain spatial cooperation or additivity, it is necessary to use chemo- and radiotherapy each .. Spatial cooperation. This concept was devised to at full doses or, at least, drugs at cytotoxic levels, yet there mean that chemotherapy and radiotiotherapy are may be a few exceptions. For example, one European primarily designed to target metastatic disease and Organiznizatiation for Research and Treatment of Cancerr the primitive tumour site, respectively. (EORTC) trial in lung cancer [3] where ciscisplatin was .. Additivity, where it is inintetendnded ththat boboth mod-d- used at a sub-cytotoxic concentration with conventional alalititieies ininteteraract in a pupurerely adaddidititive mode wiwithth radiotherapy, gave evidence of a substantial improvement regard to sterilisation of the target tumour. in local control of the tumour. On the other hand, supra- .. Infra-additivity (or protection), where the chemo- additive interaction usually relies on molecular interac- therapeutic agent inhibits tumour regression (or tion between the drug and radiation. Numerous mechan- protects normal tissue) by radiation. isms of interactions have already been described and will .. Supra-additivity, or enenhahancnced tutumour rerespspononsese,, b e be summarised below. This notwithstanding, in most where the ususe of cocombininatatioions enends in a morere instances the benefit of chemo-radiotherapy combinations potent effect than expected from the summation of in the clinic results from spatial cooperation or additivity. the effects for each agent applied alone. The term ‘radiosensitisation’ is not recommended unless the drug is devoid of any cytotoxic potential. 1 . 1. Quantification of interactions between the drugs and radiations * * Corresponding author. Tel.: +33-1-4249-9024; fax: +33-1-4249 4467. E-mail address: [email protected] If the cytotoxic response to the drug or radiation does (C. Hennequin). not folfollow an exponentintial dose dependence, as is the

0959-8049/02/$ - see front matter ## 2002 Published by Elsevier Science Ltd. PII: S0959-8049(01)00360-4 224224 C. Hennequin, V. Favaudon/ European Journal of Cancer 38 (2002) 223–2300 case in most instances, the determination of the addi- 2.1. Additional damage or modification of radio-induced tivity status of the radiation–drug interaction cannot be DNA damagee reached directly from the inter-comparison of the survival curves. Moreover, data from combined treatment Ionising radiation induces a wide range of lesions in consist of three variables, namely the doses of the two the DNA of target cells, including base damage, alkali- agents and the resresultulting outcome. To overcome thithiss labilile sitsites, sinsinglegle-st-strand breaks (SSB) and double-- problem, Steel and Peckham [1,4] proposed a method strand breaks (DSB). It has long been shown that these based on the isoeffect concept and relying on the con- lesions are rapidly repaired, with the noticeable excep- struction of isobolograms for any given isoeffect. Actu- tion of DSB for which the tt1/21/2 for rejoining extends over ally, when the effect is fixed at a given value, e.g. at 10% 55 mimin or more in repairair-proficient celcells. Unrepaireiredd survival, the system is reduced to two variables so that DSB arare coconsnsisistetentntly reregagardrded as leleththal lelesisionons [6[6].]. isisobobolologograrams fofor ththe chchososen isisoeoeffect may be cocon-n- However, the contritribution to induced celcell kilkill of the strstrucucted by plplotottiting ththe dodoses of ththe twtwo agagenents onon oxidative stress associated with low-low energy transfer separate axes. radiation (LET) and implying poly(ADP-ribose) poly- The central concept in the isobologram method is the merase for its repair, should not be underestimated [7]. determination of the envelope of additivity delineated Many chchemothererapapeueutitic drugs alalso tatargrget DNA,A, by ‘mode I’ and ‘mode II’ frontiers and within which all creating adducts, SSB or DSB. ciscis-diammino-platinum responses are deemed to be purely additiitive. Briefly,, (II) (ciscis-Pt), for example, exert its cytoxic effect throughgh mode I and mode II curves are calculated by the addi- the chelation of guanine residues, yielding monofunc- tiotion of responses to each agent applielied alone, using tiotional adducts and intintrastratrand or intinterserstratrand cross-ss- different regions of the experimental dose–effect rela- links. The bulk adducts are repaired through the exci- tionships. These curves are assumed to represent com- siosion repair pathway [8][8], but mimismatch repair is alsalsoo plplete inindedepependndence of trtreaeatmtmenents (m(modode I) or exactct involved in the processing of cis-Pt adducts by the cell complementation of the effect of one treatment by the [9]. The possibility exists that the presence in close vici- other (mode II). The data points falling below (above) nity in DNA, of both a cis-Pt adduct and a radiation- the envelope of additivity indicate supraadditive (infra- ininduduceced SSSSB may reresusult in a mututual imimpapairmenent of of additive) interaction. Details of the calculations have proper repair (Fig. 1). This model is supported by cal- been described elsewhere [5]. culations made to estimate the probability of interaction For a sufficient degree of significance, the construc- between cis-Pt adducts and radiation-induced SSB [10] tiotion of isoisobolograms requiruires quantintitattative measure-- and by experimental data as well [11]. mentnts. ThThis is onone of ththe lilimimits of ththe meththodod. FoForr EtEtopopososidide is a totopopoisisomererasase IIIIaa popoisisonon. It acactsts example, healthlthy tistissue resresponse may not be readildilyy through the stastabilbilisaisatiotion of abortirtive clecleavable com-- inincocorprpororatated in an isisobobolologograram. Morereovoverer, isiso-o- plexes and creates DNA DSB [12–14], notably in telo- bologram analysis requires a careful, precise determina- meres [15], and elicits maximum cytotoxicity in the S- tion of the dose–effect relationship for each agent. This phase of the cell cycle. A supra-additive interaction with is because drug and ionising radiation produce dissim- radiation was demonstrated in concomitant exposure [5] ilar dose–response effects, i.e. the shapes of the survival and in the radiation-induced G2-block [16]. It has been curves are characteristically different. For the same rea- prpropopososed ththat ththis susuprpra-a-adaddidititivivity reresusultlts frfrom son, the additiitivitvity stastatus may vary wiwith the isoisoeffectct enhanced DNA damage in relation to chromatin con- considered, in such a way that supra-additive interac- formational changes associated with active DNA repair tion, when it occurs, is usually more pronounced at a [16]. low degree of survival [5][5]. However, most precliclinicalal additivity studies have come from in vitro experiments, 2.2. Inhibition or alteration of radiation damage repair and some caution should be taken in the tratransfer of of these concepts to the clinic. Radiatiation recovery is currently considsidered as the main determinant of the fate of late responding tissue. ThTherefofore, ththe ususe of popotetent ininhihibibitotors of rerepapair inin 2. 2. Molecular mechanisms of interaction combination with radiation, is likely to result in late, unmanageable toxicities and should be studied in great Antitumour drugs may provide various mechanisms detail for each drug to prevent the occurrence of dele- of of intinteractiction wiwith radiatiation including DNA repairair terious combinations. inhibition, cell-cycle redistribution, or altered cytokin- The body of data on radiation DNA repair mechan- esesis or apapopoptotosisis. ThThe rerelalatitive imimpoportrtanance of ththesesee isms has grown considerably over the last 15 years. DSB mechanisms has seldom been evaluated, even for in vitro are repaired through two main pathways, namely, non- studies. For each mechanisms, we shall provide exam- homologous end-jo-joiniining (NHEJ) and homologouss ples with current antitumour drugs. recombination. Yet it is intrinsically error-prone, NHEJ C. Hennequin, V. Favaudon/ European Journal of Cancer 38 (2002) 223–2300 225225 largely prevails in somatic mammalian cells. The main suggesting that 5-FU-induced radiosensitisation reliess enzyme complex in NHEJ is the DNA-dependent ser- on the 5-FU fraction which is incorporated into DNA ine/threonine protein kinase (DNA-PK). DNA-PK isis [23,24]. As a matter of fact, radiosensitisation correlates formed from three subunits known as Ku70, Ku86 and wiwith a dedecrcreaease in ththe rarate anand exextetent of rerepapair of of DNA-PKcs. Deletion or mutation in any of these genes radiation-induced DSB and the addition of thymidine conferfers extreme sensitsitiviivity to radiatiation [17,18]. Likee to the culture medium reversesses, in part, the suscept-t- many other enzymes involved in DNA damage signalling ibility to radiation. Leucovorin, however, likely due to or repair (ataxia-telangiectasia mutated (ATM), ataxia the enhanced trapping of fluoro-dUMP by thymidylate telangiectasis Rad3-related (ATR), FRAP, TRRAP), synthase, also acts as an enhancer of radiosensitisation DNA-PKcs has a catalytic domain with high homologygy by 5-FU, suggesting that an imbalanced deoxynucleo- to to phosphatidylinositol 3-kinase (PI3-K). DNA-PKcs siside trtripiphohospsphahate popool may be a majojor papaththwaway of of may therefore be inhibited to near completion by PI3-K altered DNA repair. However, cell cycle redistribution inhibiibitors, most notably wortmannin [19].]. In vitvitroro,, following 5-FU exposure may also explain the enhanced wortmannin is not toxic per se, but it increases drama- radiation susceptibility [25]. ticticallally the cytotoxiciicity of radiatiation [20,21]. Unfortu-- Gemcitcitabine is a pyrimidine analogue with a widee nately, PI3-K inhibitors are highly toxic in vivo.. range of activity against solid tumours. It acts to deplete DNA synthesis and DNA repair often share common the deoxynucleoside triphosphate pool and is incorpo- pathways. This should provide a rationale for investi- rated into DNA, in the same way as 5-FU. A clear cor- gating the potential of DNA synthesis inhibiibitors inin rerelalatition bebetwtweeeen inincocorprpororatatioion ininto DNA anandd combinatiation wiwith radiatiation. In fact, the drugs thatt cytotoxicxicityity, with inhibiibitiotion of DNA synthesis and affect nucleoside and nucleotide metabolism are among probably DNA repairair, has been demonstratrated [26].]. the most effective and most widely used agents to sensitise Gemcitcitabine has been found to exert a major radio-io- tumour cells to radiation treatment, including fluoropyr- sensitising effect in colon, pancreatic and squamous cell imidines (5-fluoro-200 -deoxyuridine (5-FU), 9-bb-D-arabino- carcinoma cell lines in relation to the S-phase cell con- furanosyl-2-fluoroadenine monophosphate (fludarabine),), tent [27–29]. The effect was observed for relatively low 2200 ,2,200-difluoro-200 -deoxycytosine (gemcitabine)), thymid-d- drug concentrations, reached a maximum of 24 h after ine analogues (5-bromo-200-deoxyuridine (BrdUrd), 5-5- the onset of drug exposure [30] and persisted for more iodo-200 -deoxyuridine (IrdUrd)) and hydroxyurea. than 48 h after contact with drug [31]. Depletion of the 5-FU inhibits thymidylate synthase and depletes the deoxynucleoside triphosphate pool contributes to the pool of nucleotide triphosphates, leading to cell cycle enenhahancnced raradidiatatioion sususcscepeptitibibilility [2[27,7,3030], bubut nono redistribution, DNA fragmentation and cell death [22]. inincrcreaease in ththe inincicidedencnce or rerepapair of DNA strstranandd ThThe inincocorprporatition of 5-5-FU ininto DNA anand RNA asas breaks by gemcitabine could be evidenced in an analysis fluoro-deoxyuridine, contributes to its cytotoxicity. 5-5- of of 55200 kbp fragments by pulsed field gel electrophor- FU is a well known radiosensitiser. There is evidence esis [32,33]. This seems to rule out any direct inhibition

Fig. 1. Model showing response to DNA damage. A cisplatin DNA adduct or a radiation-induced single-strand break is likely to be repairable. In contrast, when both are observed, there may be a mutual impairment of proper repair resulting in the death of the cell. 226226 C. Hennequin, V. Favaudon/ European Journal of Cancer 38 (2002) 223–2300 of DNA repair pathways. In spite of this, major toxicity and metaphase [55,56]. In fact, both drugs act through has been reported during the preliminary clinical trials disdisruptiotion of the centrosome network, thus inducingng of the gemcitabine–radiation combination [34], such that faulty mitosis and cytokinesis [57]. Pioneering studies of it has been recommended that this association should not radiatiation intinteraction with paclitlitaxel indicaicated thatt be used beyond carefully designed clinical trials. increased radiosiosensitiitivitvity occurred at the time of the BrBrdUrd anand IdIdUrUrd rereadadilily susubsbstititutute fofor dedeoxoxy-y- G2-M block [58–62]. However, further studies showed thymimidine and incorporate intinto DNA, thus inducingng that enhanced radiatiation cell kilkill by taxanes does not potent radiosensitisation with enhanced DNA damage work in all cell lines, and it was recognised that prome- and decreased DNA repair [35]. BrdUrd and IdUrd are taphase arrest upon prolonged contact with drugs, if it believed to act both through an enhanced yield of radi- occurs, may not be a sufficient condition for increased cal damage at the C-5 position of the pyrimidine ring raradidiatatioion sesensnsititivivitity [6[63–3–6666]. ThThis wawas coconfinfirmrmeded [36–38]. The magnitude of the effect in terms of survival through isobologram analysis [67]. Moreover, in some consistently correlates with the amount of drug incor- celcell linlines paclitlitaxel and docetaxel at low doses may porated into DNA [39]. However, halopyrimidines can induce protection against radiation-induced cell kill,, hardly be used as adjuvants to radiotherapy in view of presumably through alteration of signal transductionn their general toxicity. pathways [66,68]. It should be mentioned at this stage that paclitaxel and docetaxel elicit different time-dependent interactions with radiation, due to different rates of 3. 3. IntInteractions at the celcellular levlevelel excretion and cell-cycle specificity [69]. In vitro radiosensitisation of cells by fludarabine [70] 3.1. Cytokinetic cooperation and 5-fluoro-200 -deoxyuridine (FdUrd) [25] has also been proposed to proceed from the accumulation of cells in a It has long been known that radiosensitivity changes radiosensitive compartment of the cell cycle. with the progression of cells through the cell cycle. The Whether celcell cycle redististribributiution might be used toto S phase is most radioresistant, and the G2-M phase is increase the tumour response to radiotherapy is open to usually most radiosensitive [40,41]. For this reason, aa discussion [71]. The usefulness of this approach can be large increase in radiation susceptibility is observed as questioned for many reasons. First of all, tumours are proliferating cells are exposed in close temporal prox- heterogeneous, with an uncontrolled amount of cells in imity with radiation, to drugs which specifically kill cells the quiescent (G0) state, and the possibility of induced in S phase. This is the case for the camptothecin and cell synchronisation by antimetabolites or DNA poly- camptothecin analogues acting as topoisomerase I poi- merase inhibitors in humans is extremely limited. Sec- sons [42], and possibly also for gemcitabine. It is best in ond, as normal tissue surrounding the tumour may also these instances to consider that the effect proceeds from be a target for cell synchronisation, such synchronisa- cytokinetic cooperation rather than radiosensitisation, tiotion may not necessessariarily resresult in an increased thera-ra- since the drug is inactive against non-S phase cells and peutix index. Furthermore, conflicting data have been does not affect the radiation response among survivors reported as to whether G2-M cells are more prone to [42–45]. Moreover, it was shown that the camptothecin– apoptosis than cells in other phases of the cell cycle [72]. radiation interaction represents a pure mode II additivity according to the terminology used for isobolograms 3.3. Promotion of apoptosissis [42]. InIn contrast,topoisomerase I-targeting agentsts are ablee to alter the radiation response in confluent-arrested cells, Cells may undergo lysis or lose reproductive ability possibly through interaction with DNA repair [46–48]. through various unscheduled (immediate, mitotic and dedelalayeyed cecell dedeatath) or prprogograrammed (a(apopoptptososis anandd 3.2. Synchronisation senescence) mechanisms. Mitotic cell death, involvingg abortive mitosis and oncosis (improperly referred to as Maximum radiosensitivity is usually observed in the necrosis), is by far the most frequent mode of cell death G2-M phase of the cell cycle. Therefore, synchronisa- in epithelial tumour celcells. Recent developments havee tion of the cells in G2-M, if it occurs, is expected to elicit shshed lilighght on ththe babasisic mechchananisisms of mimitototitic cecellll ththe maxiximum rerespspononse to raradidiotherapypy. FoFor a fefeww dedeatath, susuch as dedefefects in ththe cocontntrorol of cecentntrorososome years, this was proposed as a rationale for the use of replication [73,74]. However, for the last decade inter- paclitaxel in conjunction with radiotherapy. est has turned mainly to apoptosis, simply because it is Paclitaxel and docetaxel bind with a high affinity to most amenable to studies based on molecular biology microtubules and alter their dynamics [49–54]. At high, and genetics. cytotoxic doses, both drugs were shown to inhibit the Lymphocytes, thymocytes, prostate celcells, salsalivaryry formation of the mitotic spindle and consistently block acini, endothelial cells and intestinal crypts, are the most the progressission of celcells in mitosiosis, between prophasese sensitive cells to apoptosis in response to DSB induction, C. Hennequin, V. Favaudon/ European Journal of Cancer 38 (2002) 223–2300 227227 oxidative stress or hypoxia, ceramide, contact with some the tumour, lealeading to reoxygenation and increased cytokines or deprivrivatiation of growth factors. The p53 radiosensitivity and chemosensitivity. This was clearlyy protein, due for the most part to its role in increased demonstrated by Milas and colleagues [79] in a human (decreased) tratranscripriptiotion of the pro-apoptotic (anti-ti- tumour xenograft treated with paclitaxel prior to irra- apoptotic) mitochondrial proteins Bax (Bcl-2), is man- diation. The fraction of hypoxic cells was measured by datory for radiation-induced apoptosis (for a review, see micro-electrodes introduced in the tumour before and

Ref. [75]). Converselsely,y, TP53 mutatiation or deletietionon aftafter drug tretreatment. The median pO22 values in thithiss reportedly promotes apoptosis after paclitaxel treatment experiment were 6.2 Tor in the untreated tumours, and [76]. A kind of a cooperation depending on the p53 status increased to 10.5 and 31.2 Tor at 24 and 48 h after and the cytotoxic agent could thus be proposed, yet this paclitaxel treatment, respectively. Reoxygenation corre- question is still very much a matter of debate [77]. lated with an increased radiation response, irrespective Apoptosis undoubtedly plays a major role in cell kill- of whether cecelllls acaccucumulalateted in G2-M or nonot [7[79]9].. ing by radiation or DNA-nicking drugs applied alone, Similar reoxygenation and radiosensitisation has been e.g. for topoisomerase I- or II-targeting agents. How- reported following gemcitabine treatment [80]. ever, evievidence in favour of increased apoptosis as aa Fractionated irradiation, through a reduction of the general mechanism to account for increased response toto tumour volume, may alsalso increase the tumour bloodod cocombinined trtreaeatmtmenent, is nonot firfirmlmly esestatablblisishehed. FoForr flow and facilitate drug access to the tumour. Indeed, it example, increased apoptosis might account for radio- has been demonstratrated that radiatiation increases the sensitisation by gemcitabine in cell lines that are prone upuptatake of sosome drdrugugs, lilike cacarbrbopoplalatitin anand 5-5-FU to radiation-induced apoptosis [78]; however, a radio- [81,82]. sesensnsititisisining effeffecect of gegemcititababinine is alalso obobseservrved inin apoptosis-resistant cells. 4.2. Inhibition of tumour proliferation

Tumour repopulation is often invoked to account for 4. 4. The search for tumour specificitcityy the failure of radiotherapy [83]. Although the mechanisms involved in tumour regrowth are not completelyy ThThe waway to a more efficicienent anantiticancecer trtreaeatmtment understood, the role of growth factors is likely to be of would be to tartarget tumours with tretreatments elielicitciting major importance [84]. minimal response in surrounding, dose-limiting normal Modulation of tumour proliferation may be achieved tistissue. Radiotiotherapy takes advantage of differentintialal by epidermal growth factor receptor (EGFR) inhibition, sublethal damage repair in tumours versus normal tis- either with a monoclonal antibody directed against the sue. Unfortunately, there is no convincing evidence to receptor (C225 mAb) or through the inactivation of the show that normal tissue sparing is retretained when the tyrosine-kinase activity of EGFR [85,86]. Preclinical chemo-radiotherapy combination is used, and, in fact, ststududieies hahave dedemonsnstrtratated ththe ababililitity of C2C225 toto randomised trials for the appreciation of the late toxi- enhance in vitvitroro radiosensitivity. Several mechanisms city of chemo-radiotherapy in randomised trials, are still have been proposed to explain this observation, includ- lacking. ing inhibiibitiotion of celcell prolifliferatiotion, of DNA damage SuSuch ststududieies shshouould be enencocoururagaged tatakiking inintoto repair [87], of tumour angiogenesis [88] or, in contrast, account some well-known properties of solid tumours promotion of radiation-induced apoptosis. The inhibi- that make them resistant to treatment. In particular, (i) tion of EGFR seems to be a promising way to increase tumours are frequently hypoxic, in relation to defective the cytotoxic effect of radiation. However, due to the angiogenesis, and respond poorly to radiotherapy; (ii)(ii) fact that repopulation of normal, rapidly responding some tumours contain a large proportion of quiescent tissues is the rule after irradiation, acute toxicity may be cells, which are usually resistant to chemotherapy; (iii) feared. Careful phase I studies with EGFR inhibitors in in contrast, target tumours for radiotherapy are often combination with radiation, should therefore be plan- rapidlidly prolifliferatinting, wiwith a high amount of radio-io- ned before any phase II or III studies. resistant S-phase cells; (iv) epithelial tumour cells over- exexprpresess grgroowtwth fafactctoors rerececeptptorors, or mmay ggroroww 4.3. Inhibition of angiogenesis independentlntly of growth factors; (v) tumour celcells are mutated or deleted for genes involved in genome main- Angiogenesis is essessential for tumour growth. Con-- tenance or cell cycle control. sistently, the search for compounds endowed with anti- neneoaoangngioiogegeninic acactitivivity is flofloururisishihingng, ininclclududiningg 4.1. Reoxygenation and tumour shrinkage angiosiostatin, combretastastattatin, flavone derivativtives oror kinase inhibitors. The combination of angiostatin and A reduction in tumour volume after treatment with irradiation was found to have a major antitumoral effect one modality may result in an improved blood supply to in a human tumour xenograft model [89]. This synergy 228228 C. Hennequin, V. Favaudonon // European Journal of Cancer 38 (2002) 223–2300 may arise from a cytotoxic effect on endothelial cells. Acknowledgements Others compounds, such as TNP-470, may also elicit a radiosensitising potential [90]. The authors wish to thank Electricite ´ ´ de France (RB 2001-02) and the Association pour la Recherche sur le 4.4. Specificity for tumour tissueses Cancer (ARC 9746) for financial support.

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