Cancer Letters xxx (2013) xxx–xxx

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Cancer Letters

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Mini-review

Direct and bystander radiation effects: A biophysical model and clinicalperspectives

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.09.006

⇑ Corresponding author at: Instituto de Biopatología y Medicina Regenerativa,Centro de Investigación Biomédica, Universidad de Granada, Avda. Conocimiento 2,18016 Granada, Spain.

E-mail address: jmrdar@ugr.es (J.M. Ruiz de Almodóvar).

Please cite this article in press as: P.C. Lara et al., Direct and bystander radiation effects: A biophysical model and clinical perspectives, Cancer Lett.http://dx.doi.org/10.1016/j.canlet.2013.09.006

Pedro Carlos Lara a, Jesús Joaquín López-Peñalver b, Virgínea de Araújo Farias b, M. Carmen Ruiz-Ruiz b,Francisco Javier Oliver c, José Mariano Ruiz de Almodóvar b,d,⇑a Radiation Oncology Department, Hospital Universitario de Gran Canaria Dr Negrín, Barranco de La Ballena s/n, Las Palmas de Gran Canaria, CP 35010, Spainb Instituto de Biopatología y Medicina Regenerativa, Centro de Investigación Biomédica, Universidad de Granada, Avda. Conocimiento 2, 18016 Granada, Spainc Instituto de Parasitología y Biomedicina López Neyra, CSIC, Avda. Conocimiento 4, 18016 Granada, Spaind Hospital Universitario San Cecilio, Avda. Dr. Olóriz s/n, 18012 Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:Ionizing radiationDNA damageRadiotherapySide-effectsBystander effect

In planning treatment for each new patient, radiation oncologists pay attention to the aspects that theycontrol. Thus their attention is usually focused on volume and dose. The dilemma for the physician is howto protract the treatment in a way that maximizes control of the tumor and minimizes normal tissueinjury. The initial radiation-induced damage to DNA may be a biological indicator of the quantity ofenergy transferred to the DNA. However, until now the biophysical models proposed cannot explaineither the early or the late adverse effects of radiation, and a more general theory appears to be required.The bystander component of tumor cell death after radiotherapy measured in many experimental workshighlights the importance of confirming these observations in a clinical situation.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Effects of radiation exposure on biological systems

Irradiation of any biological organism generates a series of pro-cesses that differ enormously in a time-scale. The first step consistsof interactions between photons, or particles, belonging to theradiation beam and the atoms the organism is made up of. Asthe radiation tries to penetrate the tissues, it will collide withatoms. Occasionally the collision will be so violent, mainly withinnermost electrons, that some of the electrons may be ejectedand become free. Ionization occurs because an ion pair has beencreated. The principal damaging effect of radiation arises from itsability to eject electrons from molecules within the cells, thuscausing damage to all the molecules in the cell. This step, charac-terized by the process of energy transfer leading to ionizing andexcitation of atoms and the breakage of chemical bounds, is cen-tred on the formation of broken molecules and free radicals. Thevast majority of lesions in DNA are successfully repaired. Some rarelesions fail to repair leading to eventual cell death. Some lesionsare more serious than others and the prevalent hypothesis is thatthe mechanism of radiation-induced cell killing identifies DNA asthe most important sub-cellular target at biologically relevantdoses, and its double strand-break (DSB) as the most severe lesion.

Whilst historically the main application of radiation chemistryof relevance to radiation biology has been research into radia-tion-induced DNA damage, and its chemical characterization, it isinteresting to note that radiochemical processes after this energydeposition lead to altered DNA molecules. The variety of DNAlesions induced by radiation is described on a scale of lesion sever-ity. Severity includes not only the physical size of the DNA lesionbut also its reparability [1]. After a repair interval of a few hours,the unrepaired lesions will predominantly be those with the high-est severity. Within this category, we include DSBs that have notbeen rejoined as well as those that have been mis-repaired; a smallproportion of these DSBs eventually lead to cell death [2]. In gen-eral, DSBs are produced in proportion to dose by two mainprocesses:

(A) Direct action: There are DSBs resulting from a single energydeposit that simultaneously produces the lesion in bothDNA strands and also lesions produced by a cluster of eventsin the DNA [3]. Another possible mechanism of direct actionis that one DSB could be the result of two or more differentenergy deposit events, and the accumulative action of sub-lethal damage might lead to local multiple damage sites [4].

(B) Indirect action: There are DSBs that result from a single free-radical attack [5]. Evidence indicates that this process is thedominant mechanism of DSB formation with low LET radia-tion [6,7].

(2013),

2 P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx

There is compelling evidence to suggest that the DNA DSBs arethe most important lesion causing chromosomal aberrations andcell lethality [4]. Direct action is mainly dependent on the physicalfeatures of the interaction process, such as the properties and qual-ity of radiation (lineal transfer energy, LET) and the size of the tar-get (DNA) which is constant for all human cells. Therefore, it seemsreasonable to consider the number of DSBs produced by this path-way is constant. On the other hand, it has been proposed that theprobability of DNA breakage by oxidative stress at a given site ismainly determined by the intrinsic structure of the double helix[8]. It should be remembered that oxygen radical action is a com-petitive process against the capacity of cells to neutralize the toxiceffect of free radicals, i.e., their scavenging potency [9]. Therefore, aprominent feature of radiation biology is that the chemical condi-tion of the DNA environment has an important influence on radio-sensitivity [10–12] whereas the non-scavengeable DSBs could bedue to direct effects [13]. In general non-scavengeable DNA-dam-age can be separated into two major groups: DSBs and non-DSBoxidative clustered DNA lesions [14]. Theoretically, 30–40% ofDNA DSBs generated by radiation are due to direct action mecha-nisms [6]; the remaining 60–70% may be determined by the bio-logical properties of the cells, including their scavenging capacity,chromatin conformational status, proliferative activity, differentia-tion grade and/or cell cycle phase [9,10,15]. While isolated damageis generally repaired efficiently, clustered DNA lesions have beensuggested to be more difficult to repair [16].

The dose-rate effect in mammalian cells is seen as a change inthe extent of cell killing when radiation is given at dose rates overthe range 0.01–1.0 Gy/min. The interpretation of such results hasshown that at a low dose rate repair can occur during irradiation,thus increasing survival. However, such dose-rate sparing is nevercomplete, and this has led to the suggestion that two types of dam-age are inflicted by ionizing radiation, one that is irreparable andanother that is reparable but which may not be reparable if fixedby mis-repair or binary interaction [1,17,18].

Previous contributions of radiation chemistry to radiation biol-ogy are unmistakable, but there remains considerable potential tohelp advance the biological understanding using the knowledgeand techniques of radiation chemistry [16,19].

2. The three steps of radiobiology: transfer, communication andconsequence

The irradiation of any biological system triggers a series of pro-cesses that are very widely separated in time. The Transfer of en-ergy consists of interactions between the radiation and the atomsthat make up the biological system. This step is characterized bythe appearance of ionisations, excitations, bond-breaking and freeradical formations.

In response to damage produced by ionising radiation, cellsactivate a series of biochemical processes, Communication, thatinitiate the chain of signals of damage induced by genotoxicagents. The DNA damage response (DDR) is a highly complex andcoordinated system that determines the cellular outcome of dam-age caused by radiation. Communication enables cells to increasetheir likelihood of survival by maintaining the integrity of the gen-ome and its corresponding activities. This requires activation of thecell defence system, which consists of two parts, (a) the sensors ofDNA damage and (b) the effectors of damage response.

Most of the lesions produced by radiation are efficaciously re-paired. Some lesions cannot be repaired and cause the death ofthe cell, or can be tolerated remaining as residual damage. A timereference is necessary: cells take time to die, and irradiated cellsare even able to divide once, or more times, before finally disap-pearing [20]. Radiotherapy, like any other cytotoxic agent, pro-

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foundly disturbs the balance between cell proliferation and celldeath in both tumor and normal tissues, through the radiation in-duced DNA damage, increasing the likelihood of cell death byapoptosis, necrosis, autophagy or mitotic catastrophe. The DDRdetermines not only the sensitivity of cells to die following radia-tion, but also the type of cell death that occurs, and the timing ofcell death.

Radiotherapy is highly effective in killing cells. The eradicationof all the clonogenic cells from a tumor leads to cure, and this is thetherapeutic end-point. What we have designated the Consequenceis the result of two factors: (i) the probability of tumor control and(ii) the probability of inducing severe complications in healthy tis-sues. As with any other medical procedure, prescription of a courseof radiotherapy must represent a balance between risk and benefit,the relative weight of which will determine the therapeutic gain. Itis our hope that in the near future medical application of biotech-nology will produce reliable assays for measuring these two vari-ables, so we will be able to use them with a high degree ofcertainty.

2.1. Transfer: from the molecular DNA damage to the differences inradio-sensitivity

When cells are irradiated, three molecular end-points havebeen identified which often correlate with cellular radio-sensitivity:

(i) cells may vary in the amount of damage induced by a givendose of radiation [21–24];

(ii) cells show a different capacity, fidelity and rate to repairradiation damage to DNA [23,25–27]; and

(iii) cells differ in the level of residual damage after repair of theinitial damage and recovery of cells [23,28–30].

Evidently, these three mechanisms may be linked through acommon factor such as the modulation of chromatin conforma-tional structure [31–35].

The transfer of energy to the cells takes place for the time re-quired by the radiation to pass through the cell (t < 10�13 s). Theinitial ionizations and excitations are followed by a series of phys-ical–chemical processes at the centre of which is the formation offree radicals. The half-life of a free radical is 10�5 s. The amount ofDNA damage produced in this time is inversely related to the sur-vival of the treated cell measured as intrinsic radio-sensitivityusing a clonogenic survival assay [21,24,36,37].

The number of DBSs induced in DNA from irradiated cells is be-lieved to be related to the fraction of DNA fragmented under a gi-ven threshold size and is thus able to migrate under pulsed fieldgel electrophoresis [38,39]. Based on this fact we devised a methodto calculate the number of DNA DSBs [21,24]. The final equation ofthe mathematical model has been published in another paper [40].

However, in the sequence of processes that follow the initialevents in the energy transfer step and eventually lead to cellularand tissue consequence, there are a large number of poorly under-stood processes [41]. In response to ionizing radiation, cells imme-diately activate the sensors of DNA damage that actively survey thegenome for the presence of damage. These proteins then signal thethree main effector pathways that together determine the outcomefor the cell: DNA repair, damage checkpoints and programmed celldeath.

2.2. Communication: sensing and signaling DNA damage

DNA repair mechanisms in cells are highly efficient and the vastmajority of the lesions are normally repaired satisfactorily. A keyunresolved issue is how a cell determines when DNA damage is

effects: A biophysical model and clinical perspectives, Cancer Lett. (2013),

P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx 3

excessive and how this determination triggers the shift from repairto apoptosis. It seems plausible that proteins that recognize DNADSBs are physically recruited and grouped by the sites where theinitial damage was produced. A total of about 150 human DNA re-pair genes have been described [42]. The compilation includesgenes encoding DNA repair enzymes, some genes associated withcellular responses to DNA damage, and other genes associated withgenetic instability or sensitivity to DNA damaging agents. One ofthe earliest events known to occur in the DDR is the phosphoryla-tion of the histone H2A [43,44] and thus, the balance of H2A.XTyr142 phosphorylation/dephosphorylation may constitute a no-vel switch mechanism to determine cell fate after DNA damage[45]. In response to ionizing radiation (IR), cells delay cell cycleprogression and activate DNA repair. Both processes are vital forgenome integrity. Chromodomain helicase DNA-binding protein 4(HD4) has been identified as a factor that becomes transientlyimmobilized on chromatin after IR and recently CHD4 has beenconsidered as a novel genome caretaker and as a factor that facil-itates both checkpoint signaling and repair events after DNA dam-age [46]. In fact, a subset of DNA repair proteins as the componentof the DNA repair initiation, cell cycle arrest and apoptosis hasbeen described. ATM, ATR and BRCA1, participate in homologousrepair recombination (HRR) [47,48]; DNA-PK, is involved in nom-homologous end joining (NHEJ) [49,50]; p53 in nucleotide excisionrepair [51,52]; PARP1 in base excision repair (BER) [53–56]; andMLH1 and other proteins that are used in mismatched repair[57,58].

Whether a cell undergoes cell cycle arrest, repair, or apoptosisfollowing DNA radiation induced damage depends on a variety offactors, such as the extent of the damage, and the status ofp53,which is the most commonly mutated tumor suppressorwhose function is to regulate gene expression involved in the con-trol of both cell-cycle check points and cell death; the presence andlevels of several other gene products, including Bcl-2 [59] or PARP-1 whose role in apoptosis or autophagy is well documented[54,60–62], is also significant in the cell’s decision to undergo cell-cycle arrest, repair or apoptosis.

2.3. Consequences

In all normal life situations, DNA is subjected to high levels ofdamage, especially from endogenous reactive oxygen species. Theaverage rate is estimated to be from about 104 damages per cellper day to as high as 106 damages per cell per day [63]. For cellsexposed to extrinsic genotoxic agents, the level of reactive oxygenspecies increases and the rate of damage is indeed much higher[25,64]. The initial reaction of the cell to DNA damage is to activateDNA repair processes to remove the lesions. HRR, NER, BER, MMRand NHEJ are the five major repair pathways in human beings, eachone specializing in certain types of DNA damage. When DNA dam-age overwhelms repair capabilities, some damage persists [2]. TheDNA damage that remains unrepaired may alter bases in the DNAtemplate just as when the damage is repaired inaccurately. Toavoid such errors the DNA repair proteins detect high levels ofDNA damage and induce growth arrest (e.g. by activating p53) toallow time for further DNA repair. If this fails, they induce apopto-sis (for example, through the action of p53 increasing Bax orthrough p53-independent pathways). Cells take time to die aftersmall doses of radiation and they may undergo a number of mitoticdivisions before dying.

The repair of radiation-induced lesions in DNA is widely believedto be a major determinant of the degree of radio-sensitivity in mam-malian cells [25,36,65]. Evidence of this largely comes from radio-sensitive mutants in which clear defects in the rejoining of DNADSBs have been detected [33]. DSBs in DNA are associated with cellkilling of mammalian cells because damage to both DNA strands is

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assumed to deprive repair systems of the template required for effi-cient and accurate restoration of the molecule. Studies of the DNADSB rejoining kinetic during post-irradiation incubation were fittedto a mathematical model defined by three different components –fast, slow and very slow – of the kinetic process [28,66,67]. Defectsin the rejoining process have been related to the increased suscep-tibility of cells to accumulate genetic alterations [68], differences inradiosensitivity could be related with fast kinetic rejoining [23,37]and the extreme radio-sensitivity of some cells seen both in vivoand in vitro, may be ascribed to a reduced DNA double-strand breakrepair, resulting from a defect in NHEJ [69–72].

2.4. From the transfer step to cellular consequence

Research in experimental radiobiology deals at a fundamentallevel with the molecular biochemical and the biophysical natureof radiation damage. Descriptive models are a necessary part ofradiobiology because they provide a framework in which to ana-lyze and compare data and then to build theories of radiation ac-tion both in vitro and in vivo. It has been argued [73,74] thattissue radio-sensitivity has a significant genetic component andmay have an unknown epigenetic component [71,75]. If differ-ences in radio-sensitivity exist for one or more cell types (e.g. fibro-blast, lymphocytes or skin cells [40]) and this is due to geneticdifferences, then individuals exhibiting, for example, lymphocytehypersensitivity might be expected to show hypersensitivity inother normal cell types. In order to study the shape of the distribu-tion of normal tissue response to irradiation we have consideredthe amount of initial DNA damage induced by radiation [76], inparticular we have used the results of the study in which theexperimental radio-sensitivity of a set of lymphocyte samples fromwomen with breast cancer is measured in terms of DNA DSBs [77].Using a methodological approach developed previously [21] wecarried out studies on DNA removed from cells immediately afterirradiation which reveal extensive radiation-induced DNA damage,and when the level of DNA DSBs induction was assessed by usingthis mathematical model in different cell lines, we found that thisnumber and the dose value were directly related [24].

In seeking an explanation for radio-sensitivity amongst cells itseems reasonable to consider that ionizing radiation inducedDNA-DSBs is the most important lesion causing cell lethality. Forthis, we considered the number of DSBs produced per Gy and perDNA unit as a molecular parameter related to radio-sensitivity ofcells [24,40] and furthermore, with the tumor and normal tissueresponse.

To test this hypothesis we have quantified the initial number ofDNA DSBs induced by radiation on lymphocytes as an alternativemeasure of sensitivity to predict normal tissue response afterradiotherapy [40,77–80].

2.5. Long term consequences: normal tissue response in radiotherapy

Radiotherapy is the most important non-surgical modality inthe curative treatment of cancer [81]. Its success in eradicating tu-mors depends mainly on the total radiation dose and in attemptinga cure the dose reaches a level that inevitably causes some effectson the normal tissues surrounding the tumor [80,82,83]. Althougha significant proportion of this variation may be attributed to treat-ment-related factors [84] such as dose inhomogeneity, there isincreasing evidence showing that the major factors determiningthese differences are related to intrinsic biological factors [85].Any inter-patient differences in the intrinsic radio-sensitivity ofnormal and tumor tissues would be relevant to radiotherapy as,if they exist, they may be related to tolerance to treatment andto clinical curability [40]. If sufficient data were available[78,79,86,87], it would be possible to establish a mathematical

effects: A biophysical model and clinical perspectives, Cancer Lett. (2013),

Table 1Assays of radio-sensitivity ‘‘in vitro’’ and complication risks after curative radiotherapy.

Method Sample: Cases/control Early/lateeffects

Conclusions Refs.

Clonogenic cell survival assay Four normal individuals and three patients identifiedas having suffered unexpectedly severe reactions toclinical radiotherapy

Early The data are consistent with the hypothesis thatnormal-tissue response is linked to individual cellradio-sensitivity

[92]

DNA DSB induction 25 Breast cancer patients and 8 over-reactors afterbreast cancer radiotherapy

Early These results suggest that the initial number of DSBscould be used as an indicator of the in vivo response toradiation

[40]

Clonogenic cell survival assay 39 Cases with marked normal tissue changes werematched with several variables with 65 controls withno changes attributable to radiotherapy

Early/late

Inter-individual variation in cell radio-sensitivity maynot be the main determinant of complication risks inpatients undergoing radiotherapy for breast cancer

[93]

Clonogenic cell survival assay and theinduction and repair of DNA DSBs

16 Pair-wise matched head and neck cancer patients2–7 years after curative therapy exhibiting differences(grade 1 vs. grade 3) in late normal tissue reactions

Late This study suggests that lymphocytes are morepromising than fibroblasts to predict patient’s normaltissue response after radiotherapy

[94]

DNA DSB induction 226 Samples of lymphocytes from women with breastcancer were quantified. Among them, we detected 15patients who developed severe skin reactions

Early The ROC analysis of results is indicative of a fair/poordiscriminating capacity of the test to identify thepatients with higher risk of developing a severe acutereaction during the radiotherapy treatment

[77]

Radiation-induced apoptosis inperipheral blood lymphocytes

Normal individuals, ataxia telangiectasia (AT)homozygotes and heterozygotes, and breast cancerpatients who had received radiotherapy

Late The assays of lymphocyte apoptosis are unlikely to beof use in predicting normal tissue tolerance toradiotherapy

[95]

Limiting dilution clonogenic cellsurvival assay, using lymphocytes

83 with carcinoma of the cervix underwent radiationtherapy developing both any and Grade 3 morbidity.

Late The treatment was significantly associated with theprobability of developng late complications. There wasa weak significant inverse correlation betweenlymphocyte SF2 and grade of morbidity

[96]

Lymphocytes irradiated with doses ofeither 3 or 6 Gy and scoring thenumber of chromosomaldeletions.

Two prospective studies were performed: study A with51 patients included different tumor sites and study Bincluded 87 breast cancer patients

Early Individual radio-sensitivity determined at 6 Gy seemsto be a good predictor for risk of acute effects aftercurative radiotherapy

[97]

DNA DSB induction 108 breast cancer patients treated with radicalradiotherapy. Acute reactions were assessed on the lasttreatment day. Late morbidity was assessed after7 years of follow-up in some of these patients

Early/late

Radio-sensitivity values obtained using the in vitro testshowed no relation with the acute or late adverse skinreactions observed. There was no evidence of a linkbetween acute and late normal tissue reactionsassessed in the same patients. A positive relationshipwas found between the treatment volume and bothearly and late side effects

[78]

Clonogenic cell survival assays anddsb repair

Acute reaction after radiotherapy for breast cancer. Thestudy was performed with 25 breast cancer patients

Early The data obtained indicate that the sensitivity offibroblasts measured either by colony assay or by DSBrepair capacity is not a major parameter determiningthe extent of acute reaction after radiotherapy

[98]

DNA DSB induction 40 Patients with locally advanced breast cancer treatedwith a hyperfractionated dose-escalation radiotherapyschedule

Late Data obtained showed a correlation between latetoxicity and higher prescribed protocol dose. Thisopens up the possibility of predicting normal tissueresponse to radiation in individual patients, at least inhigh-dose non-conventional radiation therapyregimens

[79]

DSB quantified by cH2AX/53BP1 fociand chromosomal aberrations inblood lymphocytes

Seven breast cancer patients with minimal (controls)and seven breast cancer patients with marked lateradiotherapy changes (cases) were retrospectivelyselected

Late Higher levels of exchange type aberrations observedamong radiosensitive breast cancer patients suggest arole for DSB misrepair, in addition to residual damage,as determinants of late normal tissue damage

[99]

Gene expression profiling of thetranscriptional response of humandermal fibroblasts to in vitroradiation

Eight patients who developed marked late radiationchange and 8 matched patients without any changewere selected from women entered in a prospectiverandomised trial of breast radiotherapy fractionation

Late No differences between clinically radiation sensitiveand control patients were detected using this approach

[100]

Gene expression arrays, residualcH2AX, apoptosis, G2chromosomal radio-sensitivityand G0 micronucleus assay

Patients with marked (31 cases) or mild (28 controls)late adverse reaction to adjuvant breast radiotherapy

Late Variation in lymphocyte radio-sensitivity does notnecessarily correlate with normal tissue response toradiotherapy. Gene expression analysis can predictradiation exposure and may in the future help theprediction of normal tissue radio-sensitivity.

[101]

Induction and repair of DNA DSB(Comet assay), induction of cH2AXand whole genome expressionanalyses.

15 clinical radio-hypersensitive tumour patients andcompared to age- and sex-matched non-radio-sensitive patient controls and 15 lymphoblastoid celllines.

Late The group of clinically radio-sensitive patients was notunequivocally distinguishable from normal respondingpatients nor were individual overreacting patients inthe test system unambiguously identified by twodifferent laboratories. However, genome-wideexpression analysis revealed a set of 67 marker geneswhich were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitiveand non-radio-sensitive patients.

[102]

4 P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx

model capable of predicting clinical outcome in detail. This modelcould be used for the individual tailoring of the treatment [76].

The ability to predict the determinants in radiation sensitivityfor tumors and normal tissues would have important implicationsfor cancer treatments [41,83,88]. For example a strategy based on

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testing normal human radio-sensitivity to identify patients with ahigh risk of developing unacceptably severe reactions after radio-therapy might permit the individualization of treatment by dose-escalation in resistant patients without increasing normal tissuecomplications [76].

effects: A biophysical model and clinical perspectives, Cancer Lett. (2013),

P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx 5

Although some of the results of predictive biomarker studiesare promising, none of the markers have yet been adequately val-idated to justify widespread introduction into clinical practice (Ta-ble 1). Studies performed to date have often been underpoweredand are mostly observational or retrospective subgroup analysesfrom larger clinical trials based on available tumor material orblood samples from a heterogeneous population of patients. Sounddata from prospective randomized clinical-trial testing, relevantinterventions and incorporating putative predictive markers forthese interventions are therefore required before implementationof predictive tools into routine clinical use can be achieved; forthat, a collaborative effort of biologists, clinicians and physicistsis essential for successful and unhampered development, valida-tion and utilization of predictive biomarker for individualizedtreatment.[89–91].

However, until now the group of clinically radiosensitive pa-tients has been not unequivocally distinguishable from normallyresponding patients [78,79,103] nor were individual overreactingpatients in the test system are unambiguously identified [104].Genome-wide expression analysis, however, revealed a set of 67marker genes which were differentially induced 6 h after in vitroirradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation inlarger cohorts in order to determine parameters potentially predic-tive for clinical radio-sensitivity [102,105].

3. Why does the measuring of radio-sensitivity fail to predictnormal tissue response?

The cellular consequences of the direct action of radiation interms of lethal and potentially lethal damage to DNA can be ex-plained by linear-quadratic radiation cell survival models [1]. Datain the literature strongly support a causal relationship betweenbetter outcomes and improved radio-therapeutic techniques.Changes in radiotherapy practice over the years include recogni-tion of the importance of fraction size, fraction number, total dose,overall time for both tumor and normal tissue reactions, and theintroduction of conservative therapy which can also be used to de-scribe the relationship between the total isoeffective dose and doseper fraction in fractionated radiation therapy [106]. Based on thesemodels, successful tumor control requires that all clonogenic cellsreceive a lethal dose. Radiotherapy outcomes might be further im-proved by a greater understanding of the individual variations innormal tissue reactions that determine tolerance [83]. When accu-rate genetic-based or cell-survival-based predictive assays areavailable for studying tumor and normal tissue radio-sensitivity[107], radiation therapy will become an exact science [108], allow-ing truly individual optimization and the prediction of adversereactions [105].

Most data published so far aimed at establishing a relationshipbetween cellular radio-sensitivity and the risk of late complica-tions [78] might need to be re-evaluated, because the questionsasked up to now have been inadequate to arrive at a meaningfulanswer [109]. Thus, the in vitro test systems hitherto investigatedaimed at establishing a relationship between cellular radio-sensi-tivity and the risk of late complications seem to be inappropriatefor a general prediction of clinical early and late reactions duringor after radiotherapy due to the experimental variability comparedto the small effect of radiation sensitivity. Variation in lymphocyteradio-sensitivity does not necessarily correlate with normal tissueresponse to radiotherapy [78] although some positive results havebeen described [27,110] particularly when total radiation doseswere high [79,110]. Gene expression analysis might be a predictionof radiation exposure and may in the future help predict normaltissue radio-sensitivity [101]. The results obtained so far serve to

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demonstrate that the associations between different measures ofradio-sensitivity are not well understood and the assays are con-fusing due to uncontrolled experimental variables.

4. How have we understood radiation-induced non-targetedaction so far?

The prime goal of therapy is to destroy tumor cells, but antican-cer therapy might have an additional decisive effect: the sub-leth-ally hit tumor cells’ response to damage might be required for thetherapy to be successful [111] as communication with irradiatedcells reduces their survival rate. The tumor suppressor proteinp53 plays a key role in whether the cell cycle is arrested or apop-tosis ensues after a genotoxic attack and Parp-1 participates in thep53 response following DNA damage [54,61,112], but the cascadeof events, including intra- and inter-cellular signaling involvingfree radicals, reactive oxygen species, cytokines or epigeneticchanges, has still to be clarified and the results can vary consider-ably according to the type of radiation and the genotype, DNA re-pair capacity and physiological state of the cells and tissuesinvolved [113].

A short time after the end of radiation treatment, cells withinthe irradiated volume can act in one of three ways: they can growand divide, the basis for the healing of acute injuries; they cannotgrow but they remain alive; or they may survive for a long timewith important immunological changes, disappearing by apoptosisor apoptosis-like cell death very slowly and becoming a chronic fo-cus of immunological system stimulation that could produce thelate side-effects, bystander and abscopal effects observed. There-fore, in our opinion non-targeted action could be considered asthe whole immunological response of the tumor [114,115] andnormal tissues [78,116] to the stress induced by radiation in thetarget volume. As far as this action is concerned, the dose andthe volume of tissue irradiated may be a multiplicity constant ofthe frequency and severity of the late side effects [79]. The individ-ualization of radiotherapy prescriptions and a consequentimprovement in outcomes will only be possible once we haveachieved a greater understanding of individual variations in tumorand normal tissue response.

The observation that the targeted cells release cell damage-in-duced substances into the media was seen when conditioned med-ia on irradiated cultures were shown to induce various types ofdamage in un-irradiated cultures. This phenomenon was namedthe radiation-induced bystander effect [117]. Since the bystandereffect was first described, the biological meaning and extensionof this effect remains open to debate [118], and it can be clearlyseen that the damage induced by the bystander effect is not neces-sarily the same as that responsible for the traditional effects ofradiation [119]. All these effects seem to be substantially more pro-nounced at low-to-moderate doses, with little or no further in-crease at higher doses [116,117].

The central cornerstone in the bystander effect is that the appli-cation of ionizing radiation to a target volume that includes the tu-mor elicits effects that exceed radiation-induced cell deathreflecting intra-cellular communication [117] that results in celldeath [120,121], increased levels of double strand-breaks [115],chromosomal aberrations [122], and/or changes in transcript levelsand gene expression [123–126]. It is proposed that some, as yet tobe identified, secreted factor can be produced by irradiated cellsthat may stimulate effects in non-irradiated cells [127,128]. Afterirradiation, the levels of most of the cytokines increased markedlyin a dose dependent manner [129] presumably acting as part of asystemic response [130]. Recently the bystander effect has beendocumented in response to non-ionizing radiation [131], chemo-therapy [132] and in response to the medium where aging cells

effects: A biophysical model and clinical perspectives, Cancer Lett. (2013),

6 P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx

have been cultured [133] suggesting a generalized response mech-anism to general stresses.

On the other hand, the phenomena of hyper-radiosensitivity(HRS) have a very specific meaning in radiobiology. This phenom-enon has been described by Joiner in an important group of papersin which the authors describe a deviation of the curve of a surviv-ing fraction against a dose of radiation from the linear-quadraticequation in the low-dose region (D < 1 Gy). Doses bigger than2 Gy fit the linear quadratic model well [111]. Until now, the pre-cise operational and activational mechanism of the HRS process isstill unclear. For us, it was attractive to suggest that HRS in this lowdose-region is due to the bystander factor [121]. We have de-scribed an empirical model of cell radiation response that incorpo-rates both local and non-local effects. A mathematical frameworkfor separating the direct and bystander components of cell-radia-tion dose–response has recently been put forward [134]. Evidencenow shows that, as well as these direct DNA damage dependent ef-fects, irradiated tumor cells also send signals to their neighbors;here we consider that clonogenic cell survival (S) after radiationtreatment depends on both the effects of pure radiation interaction(lineal-quadratic model) and bystander interaction (death recep-tor–ligand interaction) [121]. The bystander effect is a manifesta-tion of radiation-induced signals that travel from irradiated cellsto their neighbors, and of molecular mediators that are constitu-tively expressed or induced in target cells [23,50–54], which on ini-tiating a specific or nonspecific action, may produce DNA damagein the non-irradiated neighbor cells. Within this context diffusibleoxygen reactive species and secreted factors have been identifiedas possible mediators in the indirect bystander effect. A key char-acteristic of bystander responses, as opposed to direct irradiationeffects, is the dose–response relationship. Instead of an increasedresponse concomitant with an increase in radiation dose, the by-stander response becomes saturated at relatively low doses[117,121].

Assuming that the radiation and bystander effects on cell sur-vival are independent, the mathematical equation for the biophys-ical model proposed [56] is:

S ¼ e�ðaDþbD2Þ � 1� vmax �d

KBy þ d

� �

where S is surviving fraction, D dose, a and b the coefficients of lin-eal-quadratic model, v represents cell death produced by bystandereffect, vmax is maximum cell death, d the dose at which the bystan-der effect was measured and KBy the dose administered to get a by-stander effect for which the cell-death rate is half maximum. We

Fig. 1. Schematic representation of the biophysical m

Please cite this article in press as: P.C. Lara et al., Direct and bystander radiationhttp://dx.doi.org/10.1016/j.canlet.2013.09.006

took this value to be a measure (Gy-equivalent) of how sensitivethe cell model with a low KBy value (corresponding to the most sen-sitive cells) is.

Thus, we have proposed (Fig. 1) that when radiation therapy isapplied to a tumor-cell population cells can be classified into fourdivisions:

(A) Dead cells: cells that have undergone irreparable lesions,(this is the lethal-lesion compartment in Curtis’s model);

(B) Committed cells: cells affected by potentially lethal lesions,the eventual fate of which depends on competing processesof repair and mis-repair, (these are the potentially lethallesions in the lineal-quadratic equation). Arrows in the dia-gram indicate the possible flow to compartments C or Athrough repair or binary mis-repair;

(C) Activated cells: cells that are either minimally damaged orhave been able to repair their lesions to a level of residualdamage compatible with survival. These cells might becomean active source of cytokines, reactive oxygen species (ROS)and reactive nitrogen species, thus affecting the total burdenof tumor cells via diffusion and systemic circulation, (this isthe bystander component of radiation induced cell death);

(D) Undamaged cells: surviving cells after each fraction of dose,which need to be controlled with successive irradiationtreatments. Clearly, the tumor control probability is depen-dent, moreover on the size of this compartment, on the doseper fraction, total dose, time, oxic conditions and cellularradio-sensitivity.

The cell population (C) is involved in short-range bystander ef-fects [135,136] by means of direct gap-junction including intra-and inter-cellular communication involving free radicals, and alsoin long-range bystander effects, which occur at a distance fromthe irradiated tumor [117]. The long-range bystander effect pro-duced by cytokines released into the lymphatic or vascular circula-tion might materially modify conventional expectation inradiotherapy by producing systemic or abscopal positive effects[137].

5. The long-range bystander effects and the abscopal effect ofradiotherapy

The abscopal effect is an antitumor radiation response observedin metastatic disease distant from the irradiated tissue. Almostfrom the early days of the clinical use of radiotherapy the anti-tu-

odel for direct and bystander radiation effects.

effects: A biophysical model and clinical perspectives, Cancer Lett. (2013),

P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx 7

mor consequences outside the radiation field were recognized[138–148]. Regression of distant metastasis after local radiother-apy has been described very recently in melanoma [149–151]and other tumor locations [152,153]. It was more than 15 yearsago that Mothersill and Seymour [120] demonstrated that mediumfrom irradiated cells caused similar levels of cell death as directirradiation. These clastogenic factors seemed to be involved instress responses and cell–cell signaling. Several studies haveshown clear evidence for a key role for some cytokines [154–159]. Responses to radiation-induced DNA damage include cell cy-cle arrest, repair, chromosomal maintenance and apoptosis if suchdamage is not repaired. Ataxia–telangiectasia mutated (ATM) andataxia–telangiectasia and Rad3-related (ATR) transducer genesregulate other genes called effector genes able to produce such re-sponses. Studies of DNA damage response and repair processesoccurring in bystander cells [122,160], have shown that theremay be differential DNA damage responses in direct and bystandercells. In fact, inhibition of these genes increases radio-sensitivity indirectly irradiated cells but such inhibition of ATR or ATM preventsthe killing of bystander cells [124]. The role of one of these effectorgenes, p53 has been studied by Camphausen et al. [139]. They irra-diated the non-tumor-bearing legs of wild-type p53 and p53 nullmice and observed significant growth delays of distally implantedlung carcinoma and fibrosarcoma cells. The authors implicated p53as a key mediator of the radiation-induced abscopal effect. But thisp53 dependent effect seemed to be modulated by serotonin [161].In fact, adding serotonin to culture medium from irradiated cellsincreased micronuclei numbers in bystander MCF-7 cells andHCT116 p53(-/-) cells recipient of irradiated cell conditioned mediafrom 0.5 Gy a-particle irradiated cells. Data on serotonin modula-tion of bystander effect have been compared in different labs[162]. The general conclusion is that high serotonin concentrationcorrelated with a low cloning efficiency in cultures receiving med-ium derived from irradiated cells. Recent studies confirmed thatserotonin-dependent mechanisms appear to be involved in bystan-der signal production and response to radiation and it has beendemonstrated that serotonin inhibitors produced a dose-depen-dent propagation of the bystander effect in recipient cultures[163].

Recent data suggest that radiation-induced bystander effectmay be epigenetically mediated by microRNAs, which are smallregulatory molecules that target messenger RNA transcripts fortranslational inhibition. In the study of Kovalchuck et al. [164] ion-ising radiation led to a deregulation of miRNA expression in by-stander tissues. In fact, upregulation of the miR-17 family, miR-29 family and miR-16 family were related to modifications in ma-jor bystander end points, including cell cycle deregulation, DNAhypomethylation and apoptosis, respectively. The bystander end-point effect of reduced apoptosis has been studied by Vinnikovet al. [165] showing that irradiated blood plasma did not cause aradiation-induced bystander cell-killing effect in primary humanperipheral blood mononuclear cells (PBM). On the contrary, areduction of apoptosis in irradiated PBM cultured with irradiatedblood plasma would have implications for late consequences asnormal tissue damage or oncogenic risk from mutated cells surviv-ing after high dose radiotherapy.

No relation has been found between intrinsic radio-sensitivityand the bystander effect in both normal and colon cancer patients,although no relation with radiation induced toxicity has been eval-uated [166]. Whilst most of the efforts to optimize radiotherapyhave aimed at improving treatment from the physical and techno-logical standpoint such as precision in dose delivery and optimiza-tion of treatment plans, less progress has been made to includepatients’ individual biological characteristics into treatment deci-sions [90]. Possible therapeutic implications of the bystander effecthave been alluded to, but not discussed in any detail. A decade ago

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Mothersill et al. [136], proposed considering bystander biology inareas of major importance or interest in radiotherapy. Moreover,the existence of abscopal effects has been well known since 1953and the abscopal effect was described as an action at a distancefrom the irradiated volume but within the same organism [167].

6. How does radiotherapy render cancer cells visible to theimmune system?

Radiotherapy is an alternative to surgery for the local treatmentfor oligometastasic disease. Treatment includes high precisiondelivery of radiation (stereotactic body radiotherapy (SBRT/SABR))and very high doses given in a single fraction (stereotatic radiosur-gery (SRS)). This new approach has been increasingly used duringthe last decade, achieving unexpectedly (90%) high rates of localcontrol for metastasis from different tumor primary locations[168–170]. High-dose ablative treatments results in the releaseof detritus of tumor-cell containing molecules that may be immu-nogenic. In fact patients on radiation therapy developed tumorantigen-specific immune responses undetectable before treatment[171]. Therefore, ablative radiotherapy could replicate the effect ofvaccination, as an alternative procedure to present tumor antigens[172] rendering cancer cells more susceptible to T cell-mediatedcytotoxicity [172]. This effect may be related firstly with the great-er than expected effect of single doses by standard models[173,174] allowing for such excellent local control rates and sec-ondly to the unexpected abscopal effect. In fact, surgically-pro-vided local control of metastases is not always related tocorresponding increases in survival due to the appearance of newdisease in other distant organs. Therefore some systemic effectmust be related to the high-dose SABR.

Some facts seem to support the role of radiation in the immune/inflammatory response. Radiation increases the expression of themajor histocompatibility complex (MHC) and class I-restricted tu-mor antigen-specific cells elicited by radiation can upregulate IFN-c in the tumor. The combination of this radiation-induced localinflammation and tumor-specific effector T cells may provide anadditional mechanism for tumor control by modification of the tu-mor vasculature [175]. Dendritic cell (DC) surface antigen presen-tation to T cells requires both MHC and some costimulatorymolecules (B7 and OX40L). Anti-cytotoxic T-lymphocyte antigen4 (Anti-CTLA-4) therapy enhanced effectiveness of radiotherapyin murine [176] and human models [151]. Cytokines released byimmune cells lead to recruitment and activation of leukocytes fromperipheral blood and extravasation into tumor parenchyma [177].Cell adhesion molecules are upregulated on endothelial cells dur-ing inflammation resulting in a critical point for leukocyte move-ment through the endothelium. These upregulation processesafter focal RT (SBRT/SABR) may actually promote changes in the lo-cal tumor microenvironment that enhance infiltration and activa-tion of multiple immune cell types [178]. SABR induces vasculardamage through apoptosis on the endothelial cells with doses over10 Gy [179] and this effect is regulated by the ceramide pathwayorchestrated by acid sphingomyelinase (ASMase) operating as arheostat that regulates the balance between endothelial survivaland death and thus tumor response [180].

Adequate presentation of the antigen by professional antigen-presenting cells (APCs) is needed to achieve effective initiation ofadaptive immune responses to cell-associated antigen. For thiseffective antigen presentation to naive T cells, DCs are criticaland seem to mature only after being loaded with antigens fromnon-apoptotic cells [181]. In a recent paper, Frey et al. suggestedthat necrotic tumor cells may result from continued exposure todeath stimuli and/or an impaired phosphatidylserine (PS) depen-dent clearance of the dying tumor cells. In such circumstances,

effects: A biophysical model and clinical perspectives, Cancer Lett. (2013),

8 P.C. Lara et al. / Cancer Letters xxx (2013) xxx–xxx

mature dendritic cells take up the tumor antigen and mediate theinduction of adaptive and innate anti-tumor immunity [182]. Butthese antigens would also be released after other types of celldeath, such as apoptosis. In fact apoptosis has recently been dem-onstrated to be immunogenic when it is induced by ionizing radi-ation [183] resulting controversially from its original considerationas non-immunogenic and non-inflammatory cell death. In fact, lowdose radiotherapy induced both intrinsic and extrinsic apoptosispathways with an upregulation of macrophage activation-relatedgenes, indicating that macrophage activation was probably in-duced by signals from apoptotic cells [184]. This mechanism canbe blocked by the PS-binding protein Annexin A5 [185].

Active dendritic cells are needed for an efficient presentation oftumor antigens [186]. Combined treatment with radiation and aDC growth factor (Flt3-L), resulted in prolonged survival of treatedmice, compared with treatment alone [187]. Activation of CD8 + Tcell immunity on grafted tumors after radiation, reduced tumorvolume and distant metastases [188]. Critical negative issues forsuch tumor inhibitory immune/inflammatory response would bethat classical anti-cancer treatments such as chemotherapy or tra-ditional fractionated radiation are immunosuppressive either inthe systemic or local setting, severely diminishing tumor anti-gen-specific T cell populations through constant site-specific cyto-toxicity [189].

7. Active clinical trials concerning the abscopal effect

As extensively described above, tumor antigens are taken up lo-cally and systemically by the dendritic cells (DC), which stimulatecellular or humoral specific immune responses. Nevertheless,induction of such a response is, however, regulated by mechanismsable to inhibit immune responses to tumor antigens. Treatmentwith anti-CTLA-4 antibodies would result in an activation of theanti-tumor responses to the antigens released by SABR, throughsuppression of such inhibiting regulatory mechanisms [176]. Cur-rently this approach has been successfully used by three differentgroups in the last year:

(a) Hiniker et al. [149] who treated a metastatic melanomapatient with two cycles of ipilimumab (anti-CTLA-4 anti-body), followed by stereotactic radiotherapy to two of eighthepatic metastases and two additional cycles of ipilimumab.Positron-emission tomography–computed tomographyshowed that all metastases, including the non-irradiatedliver lesions and a non-irradiated axillary lesion, had a com-plete response.

(b) Postow et al. [151] reported a case of the abscopal effect in apatient with melanoma treated with ipilimumab and radio-therapy. There was a temporal association of tumor shrink-age with antibody responses to the cancer-testis antigenNY-ESO-1, changes in peripheral-blood immune cells, andincreases in antibody responses to other antigens afterradiotherapy. A similar report was published this year byStamell et al. [150]: A patient with metastatic melanomareceived palliative radiation to his primary tumor with sub-sequent clearance of all his non-irradiated in-transit metas-tases. A brain recurrence was then treated with acombination of stereotactic radiosurgery and ipilimumab.The patient experienced a complete remission that includedresolution of nodal metastases. In both treatment periods aconcomitant increase in antibody titer against MAGEA3occurred.

(c) A new clinical trial combining ipilimumab and radiotherapy inpatients with metastatic melanoma is currently enrolling atStanford University (ClinicalTrials.gov number,

Please cite this article in press as: P.C. Lara et al., Direct and bystander radiationhttp://dx.doi.org/10.1016/j.canlet.2013.09.006

NCT01449279). This approach for immunotherapy com-bined with radiation as a non-invasive in vivo tumor vaccinestrategy is a novel approach, with possible applicability toother cancers; therefore the possibility of improving theclinical outcome of the treatment in metastatic solid tumorsby combining stereotactic ablative body radiotherapy withsequential immunotherapeutic treatments including vacci-nation strategies, adoptive cell therapy, cytokine therapy,or anti-CTLA-4 therapy [190] has been suggested.

8. Conclusions

Results, hypothesis and perspectives summarized in this reviewallow us to state as a priority the study of:

(1) The bystander short-range effect together with the hyper-radiosensitivity at low doses and the effect observed at thesame dose as a combination that might be exploited clini-cally [111,121] if it is practicable to deliver radiotherapy atdoses of less than 1.6 Gy per fraction, twice a day, 8–12 hapart, to escalate the total dose compared to conventionalfractionation.

(2) The bystander long-range effect through the combination ofnew immunological drugs together with advanced radio-therapy modalities, as this offers the key to improving theradiotherapy outcome in cancer patients.

Clinicians should be aware of these aspects of radiotherapy, andwe feel the need to point out that radiotherapy may not only be asuccessful local and regional treatment but also a novel systemiccancer therapy.

Conflict of Interest statement

None.

Acknowledgements

This work was supported by grants from the Ministry of Scienceand Innovation, CICYT: SAF2009-13281-CO2-02, and MINECOSAF2012-40011-CO2 to J.M.R. de A and MINECO SAF2012-40011-CO2-01 to F.J.O. The authors thank A.L. Tate for revising their Eng-lish text.

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