Radiotherapy for Non Small Cell Lung Cancer ... · There are presently no biomarkers in routine...

Cancer Therapy: Clinical

Radiotherapy for Non–Small Cell Lung CancerInducesDNADamageResponse in Both Irradiatedand Out-of-field Normal TissuesShankar Siva1,2, Pavel Lobachevsky1,3, Michael P. MacManus1,2, Tomas Kron1,4,Andreas M€oller5, Richard J. Lobb5, Jessica Ventura3, Nickala Best3, Jai Smith3,David Ball1,2, and Olga A. Martin1,2,3

Abstract

Purpose: To study the response of irradiated and out-of-fieldnormal tissues during localized curative intent radiotherapy.

Experimental Design: Sixteenpatientswithnon–small cell lungcarcinoma (NSCLC) received 60 Gy in 30 fractions of definitivethoracic radiotherapy with or without concurrent chemotherapy.Peripheral blood lymphocytes (PBL) and eyebrow hairs were sam-pled prior, during, and after radiotherapy. Clinical variables ofradiotherapydose/volume,patientage,anduseofchemoradiother-apy were tested for association with g-H2AX foci, a biomarker ofDNA damage that underlies cellular response to irradiation.

Results: Radiotherapy induced an elevation of g-H2AX foci inPBL, representing normal tissues in the irradiated volume, 1 hourafter fraction one. The changes correlated directly withmean lungdose and inversely with age. g-H2AX foci numbers returned tonear baseline values in 24 hours and were not significantlydifferent from controls at 4 weeks during radiotherapy or 12

weeks after treatment completion. In contrast, unirradiated hairfollicles, a surrogate model for out-of-field normal tissues, exhib-ited delayed "abscopal" DNA damage response. g-H2AX focisignificantly increased at 24hours post-fractionone and remainedelevated during treatment, in a dose-independent manner. Thisobserved abscopal effect was associated with changes in plasmalevels of MDC/CCL22 and MIP-1a/CCL3 cytokines. No concor-dant changes in size and concentration of circulating plasmaexosomes were observed.

Conclusions: Both localized thoracic radiotherapy and che-moradiotherapy induce pronounced systemic DNA damage innormal tissues. Individual assessment of biologic response todose delivered during radiotherapy may allow for therapeuticpersonalization for patients with NSCLC. Clin Cancer Res; 22(19);4817–26. �2016 AACR.

See related commentary by Verma and Lin, p. 4763

IntroductionLung cancer is the leading cause of cancer-related death in both

sexes (1). Non–small cell lung cancer (NSCLC) accounts for themajority of lung cancer cases (�85%). High-dose (or definitive)radiotherapy, alone or in combination with chemotherapy, is thestandard of care for curative intent treatment of patients withlocally advanced NSCLC or inoperable patients with early-stagedisease (2). Overall, more than half of the patients with NSCLCare treated with radiotherapy.

The expected side effect profile of definitive lung radiotherapy isnot inconsequential. Damage to normal pulmonary tissues is arecognized dose-limiting toxicity associated with curative intentirradiation of localized NSCLC (3). Unfortunately, acute lunginjury still occurs despite efforts tominimize dose to normal lungand is a potentially debilitating toxicity. A complex interactionbetween radiation-induced damage to parenchymal cells, sup-porting vasculature and associated fibrotic reactions results inacute and late radiation toxicities (4). In the lung, these changescan manifest in reduced pulmonary function and in a chronicinflammatory cascade known as pneumonitis. A recent meta-analysis suggests that symptomatic pneumonitis still occurs in29.8% of patients and fatal pneumonitis in 1.9% (3). Further-more, definitive lung radiotherapy can manifest generalized toxi-cities such asmalaise, nausea, and anorexia. At an individual level,it is poorly understood as to why some patients succumb tosignificant systemic toxicities of radiotherapy and why others donot. Responses in cells and tissues that are either adjacent to, orshare the environment with the irradiated target, have beendocumented and classified as either radiation-induced bystandereffects (RIBE), or when referring to responses of other organs andtissues distant from the target tissues, as abscopal ("out-of-field")effects (5).

There are presently no biomarkers in routine clinical use tomonitor individual response for either the localized or systemiceffects of radiotherapy. As such, conventional radiotherapy is notpersonalized to individual radiation response but rather is

1Sir Peter MacCallum Department of Oncology, University ofMelbourne, Melbourne, Victoria, Australia. 2Division of RadiationOncology and Cancer Imaging, Peter MacCallum Cancer Centre, EastMelbourne, Victoria, Australia. 3Molecular Radiation Biology Labora-tory, Cancer Cell Biology Program, Peter MacCallum Cancer Centre,EastMelbourne,Victoria, Australia. 4Department of Physical Sciences,Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.5Tumour Microenvironment Laboratory, QIMR Berghofer MedicalResearch Institute, Herston, Queensland, Australia.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Shankar Siva, Radiation Oncologist, Peter MacCallumCancer Centre, St Andrews Place, East Melbourne 3002, Victoria, Australia.Phone: 613-9656-1111; Fax: 613-9656-1424; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0138

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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Published OnlineFirst June 3, 2016; DOI: 10.1158/1078-0432.CCR-16-0138

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delivered as a set course or "recipe" of treatment derived frompopulation-based evidence. It is therefore imperative to establishin vivo biomarkers for early assessment, prediction, and ultimatelyavoidance of delayed organ dysfunction. Our group has previ-ously investigated imaging biomarkers of lung perfusion andventilation injury secondary to radiotherapy that may result infunctional deficits (6). Another potential biomarker of toxicity isradiation-induced DNA damage, in particular, DNA double-strand breaks (DSBs), possibly the most serious molecular con-sequence of radiation treatment (7). In response to irradiation,phosphorylated histone H2AX (g-H2AX) molecules surround aDSB to open the chromatin structure and to serve as a platform forthe accumulation of many factors involved in DNA damageresponse (7). These sites can be marked with anti–g-H2AX anti-bodies with fluorescent "tags." Our group has previously dem-onstrated that the number of foci per cell is proportional to theradiation dose and follows well-studied kinetics in normal tissuesfrom healthy donors (8, 9). This sensitivity allows detection ofradiotherapy-induced DNA damage in situ in human tissues, suchas peripheral blood lymphocytes (PBL) andhair follicles (10). Theapplication of this assay in the case of homogeneous total bodyirradiation is straightforward and relies on the measurement ofthe average number of g-H2AX foci per cell (11, 12). However, arobust relationship between partial body irradiation dose/vol-ume and resultant DNA DSBs are yet to be established. Anapproach has been suggested to apply the g-H2AX assay to assesssubpopulations of PBL with evidence of heavy DNA damage toevaluate the irradiated fraction of the blood volume and the dosereceived by that fraction, and as a result, act as a biodosimeter forpartial body irradiation (13, 14).

Here, we studied a DNA damage response in directly exposedand unexposed normal tissues outside of an irradiated volume tolocalized radiotherapy. We established the kinetics of DNA DSBsand dose/volume effects of partial lung irradiation in patientsreceiving curative intent radiotherapy and chemoradiotherapy forNSCLC. We report that radiation-induced DSBs in lymphocytescirculating through the irradiated thorax correlate to the clinical

radiotherapy dose/volume characteristics of the irradiated lung.Furthermore, for the first time, we document nontargetedresponses to radiotherapy in terms of DSB induction in out-of-irradiated field normal tissues, using eyebrow hair follicles, whichare substantively distant from the irradiated thorax. Finally, in asearch of factors that may mediate the observed abscopal effect,we performed a parallel analysis of a panel of plasma cytokinesand explored patterns of circulating exosomes.

MethodsPatients and therapy

This research was a biologic substudy of a clinical trial inves-tigating ventilation/perfusion positron emission tomographythat received ethics committee approval at the Peter MacCallumCancer Centre (Universal Trials Number U1111-1138-4421). Allpatients provided written consent to participate in this study. Allpatients had biopsy confirmed NSCLC and were treated withcurative intent, with a total dose of 60 Gy in 30 fractions ofradiotherapy delivered over 6 weeks with a 3-dimensional (3D)conformal technique. In total, 16 consecutive patients were con-sented and enrolled. The median age of the patient populationwas 68 years (range, 46–89 years). Patient characteristics areshown in Table 1.

Typically, a 3- to 4-field 6 MV photons radiotherapy techniquewas used with effort made to avoid the contralateral unaffectedlung and spare spinal cord. The planning target volume (PTV)wasa direct isotropic expansion of 1.5 cm on gross disease includingmotion observed on 4-dimensional computed tomography(4DCT). In patients receiving concurrent chemotherapy, this wasdelivered using platinum doublets. This consisted of either 2 � 3weekly cycles of 50 mg/m2 cisplatin days 1 and 8, with 50 mg/m2

etoposide days 1–5, or 6 � weekly cycles of carboplatin (areaunder the curve ¼ 2) day 1 with 45 mg/m2 paclitaxel day 1. Thefirst cycle of concurrent chemotherapy was commenced imme-diately prior to the first fraction of radiotherapy. No patientreceived adjuvant chemotherapy after the concurrent chemother-apy delivery.

Tissue sample processingThe patients underwent serial eyebrow hair collection and

venipuncture for blood collection. The samples were collectedand processed at 5 time points, before therapy (baseline, BL), at

Table 1. Patient characteristics (N ¼ 16)

Characteristics Value (n)

Age, median, (range), y 68 (46–89)TreatmentRadiotherapy 7Chemoradiotherapy 9

StageI 3II 4III 8IV 1

Radiotherapy PTV, median, (range), cc 349.1 (87.2–1,137.7)MLD in Gy, (range) 14.5 (6.4–20.7)Clinical respiratory toxicitya

Y 7N 9

aClinical respiratory toxicity is defined by the Common Terminology Criteria forAdverse Events (CTCAE) v4.0 as a grade 2þ toxicity.

Translational Relevance

Radiotherapy is a localized treatment modality that isdirected at the tumor. It is generally believed that the onlynormal tissues at risk are those that are in the irradiatedvolume. However, nontargeted (systemic/abscopal) effects ofradiotherapy are being increasingly recognized. In this study,patients undergoing curative intent radiotherapy for non–small cell lung cancer had samples collected before, during,and after radiotherapy. We observed dose-dependent radia-tion-induced DNA damage in lymphocytes circulatingthrough the irradiated thorax. Furthermore, for the first time,we document clinical abscopal DNA damage to radiotherapyin out-of-irradiated field normal tissues. In a search of factorsthatmaymediate the observed abscopal effect, we performed aparallel analysis of plasma cytokines and explored patterns ofcirculating exosomes. This study opens an opportunity tomonitor individual real-time biologic effects of ionizing radi-ation in patients with cancer undergoing radiotherapy andmay inform strategies for personalized therapy.

Siva et al.

Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research4818




1 hour after the first fraction of radiotherapy, and 24 hours afterthe first fraction of radiotherapy (before the second fraction). Amid-treatment sample was taken at 4 weeks into the treatmentcourse of radiotherapy (within 2–4 hours after the radiotherapyfraction) and 12 weeks after the completion of radiotherapy.

Approximately 7.5 mL of fresh blood was received per timepoint and processed within 3 hours of collection. The blood wasspun and plasmawas removed and stored at�80�C. The resultingpellet from plasma separation was reconstituted to 15 mL withPBS (made-in-house). This was then layered onto Ficoll-PaquePLUS (GE Healthcare Bio Sciences) before centrifuging for 45minutes at 600 � g. PBL layers were collected, fixed with 4%paraformaldehyde (PFA, Electron Microscopy Sciences), andstored in 70% EtOH at �20�C.

PBL were cytospun onto microscopy slides (Mezel-GlaserLomb), using the Cytospin 4 cytocentrifuge (Thermo-Scientific).The samples were then immunostained with mouse monoclonalanti-g-H2AX antibody (Abcam), followed by secondary antibodystaining with Alexa Fluor-488 goat anti-mouse IgG (Invitrogen).Primary and secondary antibody solutions contained 1% BSA inPBS-TT (PBS containing 0.5 Tween-20 and 0.1% Triton X-100,both from Sigma-Aldrich). Slides were counterstained andmounted using Vectashield mounting medium (Vector) contain-ing propidium iodide (PI).

The hairs with attached hair follicles were fixed with 4% PFAand stored at �20�C in 70% EtOH. They were subsequentlyequilibrated to room temperature for batch processing and anal-ysis. The hairs were immunostained in the same manner asdescribed above.

Microscopy and foci countingImage acquisition of the lymphocyte and hair follicle samples

was performed with a laser-scanning confocal microscope Olym-pus FLUOVIEW FV100 (Olympus). The optical z-sections werescanned incrementally and stacked together to enable detection ofall foci throughout the nucleus. At least 100 cells per experimentalpoint were used for analysis with JCountPro software developedby our research team (15).

Analysis of plasma cytokinesThe cytokine analysis was conducted by an external con-

tractor as previously described (16). In brief, a commercialmultiplexed sandwich ELISA-based flow cytometry array wasused (Quantibody custom array, RayBiotech). Each of thesamples was analyzed simultaneously using a panel of anti-bodies to 22 different cytokines. Each test sample, togetherwith the positive and negative controls, was assayed in qua-druplicate for each cytokine. Concentration levels, expressed inpicograms per milliliter (pg/mL), were calculated against astandard curve set for each biomarker from the positive andnegative controls.

Analysis of circulating exosomesThe methodology used for exosome analysis has been

described previously (17). Briefly, plasma was clarified ofplatelets and large vesicles by sequential centrifugation at1,500 � g and 10,000 � g for 10 and 20 minutes, respectively.Clarified plasma was overlaid on qEV size exclusion columns(Izon Science) followed by elution with PBS. Exosome fractionswere pooled and concentrated in Amicon Ultra-4 10-kDanominal molecular weight centrifugal filter units to a final

volume of 100 mL and subsequently filtered with an Ultrafree0.22-mm centrifugal filter device. The concentration and sizedistribution of exosomes was then analyzed with tunable-resistive pulse sensing (qNano, Izon Science) using a NP100nanopore. The concentration of isolated exosomes was stan-dardized using a multi-pressure calibration with 70-nm car-boxylated polystyrene beads.

Statistical analysisPatients were grouped into those receiving concurrent che-

motherapy and those receiving radiotherapy alone. Two-wayANOVA was used to assess variance in g-H2AX foci numbersbetween the treatment groups and across the sampled timepoints. Paired 2-sided t tests were used to compare the averagenumber g-H2AX foci per cell and the proportion of cells with�6 foci from baseline to the other sampled time points. ThePTV, mean lung dose (MLD), and age were recorded for eachpatient and correlated with the change in g-H2AX levels frombaseline to 1 and 24 hours after the first fraction of radiationusing a Pearson statistic and a linear regression model fit. Thesechanges from baseline levels were measured at an individualpatient level. The average numbers of g-H2AX foci per cellwere grouped by treatment type (chemoradiotherapy vs.radiotherapy alone) and compared using multiple t tests withHolm–Sidak corrections for multiple comparisons. The averagenumbers of g-H2AX foci per cell from baseline to 1 and 24hours after the first fraction were fit with a linear regressionmodel and tested for correlation using a Pearson statistic.Changes in cytokine levels were analyzed and grouped intotreatment type (chemoradiotherapy vs. radiotherapy alone).Two-way ANOVA assuming repeated measures testing for dif-ferent time points was used to assess differences in cytokineconcentrations between chemoradiotherapy and radiotherapygroups and across sampled time points. Corrections for mul-tiple comparisons were performed using Dunnett method. Allstatistical analyses were performed using PRISM v6.0 softwarewith a threshold of significance of 0.05.

ResultsDetection of DNA damage in directly irradiated PBL

Representative images of g-H2AX foci in PBL at each ofthe sampled time points are depicted in Fig. 1A. The average(�SEM) number of g-H2AX foci per cell at baseline in thestudy population was 0.974 � 0.183. This was significantlyelevated at 1 hour post the first fraction of radiotherapyat 1.960 � 0.225, P ¼ 0.029 (Fig. 1B), consistent withpostirradiation g-H2AX formation reported elsewhere(7). The foci numbers were not statistically different frombaseline at the 24-hour, 4-week, or 12-week time points (P ¼0.494–0.872).

At 1 hour after the first fraction of irradiation, a considerableportion of PBL included a subpopulation of heavily irradiatedcells (with �6 g-H2AX per cell), which were apparently tra-versing the irradiated volume, and subjected to a dose of closeto 2 Gy. This proportion (10.7% � 0.1%) was significantlyhigher (P ¼ 0.002) than the average proportion of cells atbaseline in all patients (2.5%� 0.9%; Fig. 1C). However, it wasnot statistically different from baseline at the 24-hour, 4-week,or 12-week time points (P ¼ 0.205–0.460). The average num-ber of g-H2AX foci per cell and the proportion of heavily

DNA Damage in Normal Tissues during Lung Radiotherapy

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irradiated cells varied across the sampled time-points (F¼ 2.75,P ¼ 0.036 and F ¼ 3.85, P ¼ 0.008, respectively) but notby treatment group (radiotherapy alone vs. chemoradiother-apy, P ¼ 0.506 and P ¼ 0.639, respectively). There were nosignificant differences in g-H2AX values in patients receivingradiotherapy alone as compared with chemoradiotherapy,when considering either the average number of foci per cell(P ¼ 0.340–0.722) or the proportion of cells with �6 foci (P ¼0.339–0.640) as shown in Fig. 1D and E.

The median (range) MLD of all patients was 14.5 Gy (6.4–20.7 Gy). MLD correlated with the excess (above the baselinevalue) average number of g-H2AX foci per cell at 1 hour post thefirst fraction of radiotherapy (r ¼ 0.634, P ¼ 0.033, Fig. 2A). TheMLD correlated somewhat better with the excess proportion ofcells with �6 foci per cell at 1 hour post the first fraction ofradiotherapy (r ¼ 0.739, P ¼ 0.009, Fig. 2B). There was a trendfor correlation of PTV for both the excess in average number offoci per cell and the proportion of cells with �6 foci at 1 hour,but this did not reach statistical significance with P values of

0.051 and 0.092, respectively (Supplementary Fig. S1). Neitherthe MLD nor the PTV was correlated to changes in g-H2AX levelsat 24 hours post the first fraction of radiotherapy. Consistentwith reported impeded DNA repair capacity caused by age andcellular senescence in in vitro and mouse models (8, 18), age wasinversely correlated with the change in proportion of cells with�6 g-H2AX foci at 1 hour post the first fraction of therapy (r ¼�0.681, P ¼ 0.023, Fig. 2C), and trended toward correlationwith the change in average number of g-H2AX foci per cell at 1hour (r ¼ �0.601, P ¼ 0.052). None of the candidate variablescorrelated with observed g-H2AX foci 24 hours after the firstfraction of radiotherapy.

Detection of DNA damage in out-of-irradiated field eyebrowhair follicles

To exclude a possible effect from radiation leakage from thetreatment head and from internally scattered radiation that mayhave affected results, thermoluminescent dosimetry measure-ments were taken at the patient eyebrow. The radiation dose

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Figure 1.

g-H2AX foci in directly irradiated PBL. A, representative images of g-H2AX foci in PBL before, during, and after the treatment course. Green, g-H2AX foci;red, propidium iodide (PI). Magnification, 600�. B, average (�SEM) number of g-H2AX foci per cell at each time point within the patient population(n ¼ 16). C, average (�SEM) proportion of cells with �6 g-H2AX foci per cell at each time point within the patient populations. D, comparison of theaverage number of g-H2AX foci per cell at each stratified by concurrent chemotherapy use. E, comparison of the average proportion of cells with �6 g-H2AXfoci per cell at each time point stratified by concurrent chemotherapy use.

Siva et al.





measured at the eyebrow was <0.005 Gy for both left and righteyebrows.

g-H2AX foci increases in unexposed eyebrow hair follicleswere delayed compared with directly irradiated PBL, consistentwith reported kinetics of bystander DNA damage in cell andtissue models (19, 20). Representative images at each of thesampled time points are depicted in Fig. 3A. The averagenumber of g-H2AX foci per cell at baseline in the studypopulation was 0.163 � 0.107. This was significantly elevatedat 24 hours and 4 weeks post the first fraction of irradiation at0.858 � 0.737, P < 0.001 and 0.386 � 0.577, P < 0.001,respectively (Fig. 3B). The number of g-H2AX foci per cell was

not significantly higher from baseline at 1 hour post the firstfraction of irradiation (P ¼ 0.199) or 12 weeks post theradiotherapy course (P ¼ 0.110). There was no significantdifference between the average number of g-H2AX foci per cellbetween chemoradiotherapy and radiotherapy alone at any ofthe measured time points, P values ranging from 0.258 to 0.636(Fig. 3C). Given the reported lack of dose response forbystander DNA damage in a cell model (21), the apparentcorrelation between MLD and foci per cell in Fig. 3D wasunexpected, but this proved to be not statistically significant(P ¼ 0.062).

Analysis of plasma inflammatory cytokinesFor DNA damage to appear in tissues distant from the site of an

irradiated tumor, signaling factors must migrate from the irradi-ated site to these other tissues. Abscopal effects of radiotherapyhavebeenproposed tobemediatedby inflammatory cytokines (4,22). In a subset of 12 patients for whom sufficient plasma sampleremained after the primary DNA damage analysis and clinicaltests, we analyzed dynamic changes during the course of radio-therapy for a panel of 22 cytokines that have been reported to beinvolved in intercellular communication and radiation response.Of the 22 cytokines analyzed (eotaxin, IFNg , IL6, IL10, IL11, IL22,IL3, IL33, IP10, MCP1, MCP3, MDC, MIP1a, MIP1b, MIP3a,MIP3b, TGFb1, TGFb2, TGFb3, TIMP1, TNFa, VEGF), results from12 cytokines (eotaxin, IL33, IL6, MCP1, MDC, MIP1a, VEGF,IP10, MCP3, MIP1b, TIMP1, TNFa) were above the limit ofdetection. We recently reported that early changes in levels of 5of these factors, IP10, MCP1, eotaxin, IL6, and TIMP1, wereassociated with pulmonary toxicity after radiotherapy and che-moradiotherapy treatment (16). This toxicity in normal lungtissuesoccurswithinor in close proximity to the irradiated volumeand secondary to damage from direct or scattered irradiation.

To explore whether cytokines could also be involved insignaling outside of irradiated volume, and as elevated abscopalDNA damage in hair follicles persisted during 4 weeks ofradiotherapy treatment and the effect was independent of thetreatment modality (Fig. 3C), we considered overall variationsof plasma cytokine levels during 4 weeks of radiotherapy treat-ment. The plasma levels of eotaxin, IL6, IP10, MCP1, MCP3,MDC, MIP1a, MIP1b, TIMP1, and VEGF significantly variedduring treatment (Fig. 4). Chemoradiotherapy induced changesin 8 cytokines, and radiotherapy alone induced changes in 4cytokines within this time window. While for several cytokines,similar patterns can be observed in both radiotherapy- andchemoradiotherapy-treated patient cohorts, mostly, thesechanges were not statistically significant in one or anothergroup. A statistically significant decrease similar for both treat-ment modalities was observed for macrophage-derived chemo-kine/C-C motif ligand 22 (MDC/CCL22) at 1 hour post firstfraction for both radiotherapy (P¼ 0.009) and chemoradiother-apy (P ¼ 0.028) groups (Supplementary Fig. S2). Plasmalevels of another chemokine, macrophage inflammatory protein1a/C-C motif ligand 3 (MIP-1a/CCL3), were statistically signif-icantly elevated at 4 weeks of radiotherapy treatment in bothradiotherapy only (P ¼ 0.043) and chemoradiotherapy (P <0.001) groups of patients (Supplementary Fig. S2). The changesin both cytokines did not correlate with the irradiated volumeand dose (16), consistent with dose-independent increaseof DNA damage in hair follicles, and suggesting that thesecytokines can play a role in genotoxic stress response. The

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Figure 2.

Association of MLD with (A) excess (above the baseline) average numberof g-H2AX foci per cell, (B) excess average proportion of cells with�6 g-H2AX foci, and (C) association of age with excess proportion ofcells with �6 g-H2AX foci, at 1 and 24 hours post the first fractionof radiotherapy (RT).






changes did not correlate with radiotherapy-induced pulmonarytoxicity (16).

Analysis of exosomesExosomes have been shown to be newmediators of cell-to-cell

communication (23). Their exchange between cancer and nearbycells induces the "cancer field effect," by influencing the pheno-type of recipient cells toward induction of oncogenic properties(24). Exosomes released from 2 Gy–irradiated cells mediate RIBEin in vitromedia transfer experiments, manifested as induction ofDNA damage and chromosome aberrations in na€�ve bystandercells (25, 26).

We hypothesized that changes in concentration and/or sizedistribution of circulating exosomes during a course of radiother-apy would correlate with corresponding changes in the abscopalDNA damage. After the cytokine study, which together withimperative unscheduled clinical investigations, consumed mostof the patients' plasma samples, we could perform exosomeanalysis on all time sets of samples for only 6 patients. Exosomeshad a consistent size distribution with a median of 68 nm (range,64–82 nm, data not shown), which did not change during acourse of radiotherapy for individual patients. Individual datasetsrevealed that the pattern of dynamic changes in the particleconcentration during a course of radiotherapy varied greatlybetween patients (Supplementary Fig. S3). No pattern could beestablished that correlates with the pattern of g-H2AX dynamicchanges in hair follicle cells. No individual correlations have been

found. Although the analysis of this small cohort prevents definiteconclusions, it seems unlikely that release of exosomes duringradiotherapy treatment is correlated with response of normaltissues distant from the irradiated field.

DiscussionExternal beam radiotherapy is generally considered to be a local

form of therapy, as it is delivered precisely to the tumor. It isgenerally believed that the only normal tissues at risk are thosethat are in an irradiated volume. In this study of patients withNSCLC receiving radiotherapy treatment, evidence for biologiceffects outside the target volume was obtained by monitoringg-H2AX foci in eyebrow hair follicles, across the sampled timepoints before, during and after the course of radiotherapy, pro-viding information on how localized radiation can affect anorganism in whole. On the other hand, monitoring g-H2AX fociin PBL provided insight into the effects of radiotherapy ontargeted cells.

PBL are the most common and easiest cells to obtain forg-H2AX analysis in vivo. The induction of DSBs leads to formationof g-H2AX foci in response to as little as 1.2 mGy (equivalent to0.05 excess foci/cell, at 3minutes postirradiation, in cultured cells;ref. 27). Unirradiated PBL exhibit low g-H2AX levels, typically lessthan one focus per cell (28). Differences in g-H2AX response werestatistically significant for both the average numbers of foci percell and the subpopulations of cells postulated to be directly

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Figure 3.

g-H2AX foci in out-of-irradiated volume eyebrow hair follicles. A, representative images of g-H2AX foci in hair follicles before, during, and after the treatmentcourse. Green, g-H2AX foci; red, propidium iodide (PI). Magnification, 400�. B, average (�SEM) number of g-H2AX foci per cell at each time point withinthe patient population. C, comparison of the average (�SEM) g-H2AX foci number stratified by the use of concurrent chemotherapy. D, association ofMLD with the excess (above the baseline) average number of g-H2AX foci per cell at 1 and 24 hours post the first fraction of therapy.

Siva et al.





exposed to ionizing radiation—those with �6 foci per cell;effectively, an approximate empirical deconvolution of the tar-geted subpopulation of PBL. The most dramatic change occurredfrom before treatment to within 1 hour after the first fraction ofradiotherapy, with the average number of g-H2AX foci per celldoubling from0.974 to 1.960, and the proportionof cellswith�6g-H2AX foci per cell within the circulation quadrupling from2.5% to 10.7%. Both the average number of foci and the sub-population of cells with multiple foci significantly correlated tothe treatment plan MLD. The analysis of distributions of heavilydamaged cells (i.e., the directly irradiated subpopulation of PBL)resulted in stronger correlation with not only the clinical radio-therapy treatment plan MLD but also patient age. g-H2AX focinumbers returned to near the baseline controls 24 hours after thefirst fraction of radiotherapy, consistent with reported kinetics ofradiation-induced foci (7).

The observations of DSB induction and repair observed inthis study are consistent with previous clinical studies involvingradiotherapy. A study in patients with a variety of tumor typestreated with 3D-conformal radiotherapy indicated a linearcorrelation between the numbers of g-H2AX foci per PBL andthe integrated total body radiation dose (29). This allowed for

accurate calculation of the applied integral body dose. Similarlyin a clinical model of patients with localized prostate cancer,skin biopsies from low-dose regions below 1.1 Gy in patientsreceiving radiotherapy demonstrated a linear dose–responserelationship for g-H2AX foci (30). A further study investigateddifferences between intensity-modulated radiotherapy (IMRT)and 3D treatment delivery of radiotherapy in patients withprostate cancer using the g-H2AX assay to compare to thephysical model of total body dose distribution (31). The resultsrevealed a similarity between the g-H2AX levels at 10 minutespost radiotherapy with both techniques, with a mean numberof foci per cell being 0.49 for 3D treatment and 0.47 for IMRT.These levels are significantly lower than the mean of 1.96 fociper cell noted in our study at 1 hour post radiotherapy, and it islikely that this difference is due to the larger volumes of tissueirradiated with our cohort of locally advanced NSCLC, which isconcordant with observations from irradiation of differenttumor sites (22).

Our study provided further information. The g-H2AX focinumbers were not significantly different from controls at 4 weeksduring radiotherapy, even though the samples were collectedshortly after irradiation, suggesting that the protective

Figure 4.

Mean (�SEM) changes in concentration of plasma cytokine levels at baseline, 1 and 24 hours and 4 weeks after commencement of radiotherapy (RT).






mechanisms may have taken place, inhibiting induction of exces-siveDNAdamage. Lowdoses of radiation (either acute of chronic)have frequently been reported in a variety of experimentalmodelsto affect the response of cells to subsequent doses (32, 33).Various mechanisms including efficiency of DNA repair andgenetic background have been suggested to affect expression ofa radioadaptive response (interpatient variability can be seenin Fig. 1). This compensatory mechanism can be considered asnaturally radioprotective, to defend normal tissue from accumu-lating DNA damage and to maintain the homeostasis of theorganism. In addition, the average foci values were not signifi-cantly different from controls at 12 weeks after completion ofradiotherapy. All these changes were not attributable to the use ofchemotherapy, with no significant differences in the cohortsreceiving chemoradiotherapy. The observation that PBL appar-ently become refractory to induction of g-H2AX foci has beenreported previously for in vivo studies (34).

While PBLs are an ideal model to measure in-vivo biodosi-metric effects of direct irradiation, we used the model ofeyebrow hair follicles to investigate the long-range effects ofradiotherapy. Hair follicles contain replicating cells that aremore vulnerable to anticancer therapies and bystander DSBformation (35). Recent studies showed the potential of usingplucked hairs to visualize g-H2AX formation in vivo followingboth genotoxic drugs and radiation in humans and animals(12, 36, 37). In this study, we observed significantly increasedg-H2AX formation in eyebrow hair follicles 24 hours after atopologically distant organ irradiation. Importantly, we mea-sured potential contribution from machine leakage as well asinternal scatter doses at the eyebrow with measured results wellbelow that which is plausible for a 7-fold increase in the averagenumber of g-H2AX foci. Furthermore, the image guidanceprocedures using low-dose thoracic cone beam computedtomography (CBCT) would not contribute doses sufficient toinduce measurable g-H2AX at the eyebrow. Importantly, theobserved effect is not influenced by the concurrent adminis-tration of chemotherapy, suggesting a true abscopal effect. Thedelayed formation of DSBs is in concordance with previousexperiments demonstrating that the increase in bystanderg-H2AX foci did not peak until several hours after radiationexposure of directly hit cells in gap junction involving exper-imental models or after receiving conditioned bystander medi-um (19, 21). In another study performed in 3D human tissuemodels following charged a-particle microbeam irradiation,our group found an increased incidence of g-H2AX foci inbystander cells; the DNA damage response was maximal at 12to 48 hours and gradually decreased over 7 days post irradi-ation (20).

Figure 1B and C indicates that there are patients with elevatedlevels of foci in PBLs at 4 weeks during radiotherapy and 12weeksafter radiotherapy. This may signify the sensitivity of thesepatients to radiation treatment. Hair follicle results suggest thatdistant normal tissues, including bone marrow and spleen, arealso exposed to long-lasting abscopal effects of radiation. Thismay alter the differentiation of precursors into mature lympho-cytes, thereby contributing to the elevated post-therapy g-H2AXlevels. Whether kinetics of g-H2AX induction and repair correlatewith the risk of secondary hematologic or other malignancy inradiotherapy-treated patients remains to be confirmed with lon-ger term follow-up studies. More generally, the observation ofabscopal DNA damage in distant hair follicles, and by analogy

with other preclinical data (38), in other normal tissues with ahigh proliferation index, raises concerns in the context of radio-therapy-related second malignant neoplasms (39).

The underlying mechanisms of the abscopal effect are incom-pletely understood. The term "abscopal" can refer to the sce-nario of regression of distant tumor sites after localized irradi-ation, but although these events are exceedingly rare, they havebeen previously reported in the context of NSCLC (40, 41).Inflammatory mediators, such as chemokines, cytokines, andprostaglandins, as well as reactive oxygen and nitrogen speciesmediate this effect (4, 22). We have previously demonstrated adose/volume dependence of cytokines induced in plasma with-in 1 hour of thoracic irradiation in patients with localizedNSCLC (16). In the present study, we report a statisticallysignificant decrease of plasma MDC levels in both treatmentgroups. This observation is consistent with the fact that lowerdoses of radiation tend to promote anti-inflammatory re-sponses, due to the inherent susceptibility of na€�ve immunecells to radiation (42). MDC is known to be constitutivelysecreted by differentiated dendritic cells and macrophages butalso can be produced by monocytes, granulocytes, and naturalkiller (NK) cells. It is involved in homeostasis and immune cellhoming, as well as in chronic inflammation (43). Interestingly,higher expression of MDC in lung cancer signifies better prog-nosis—longer disease-free survival and lower risk of recurrenceafter treatment (44). MDC is a potent chemoattractant forimmature dendritic cells, NK cells, and T-cell subsets. MDCmay play a role in intercellular communication via initialinduction of misbalance in cellular microenvironment of theirradiated volume and trigger activation of other factors respon-sible for radiation-induced systemic effects. Indeed, a recentreport suggests that downstream factors IL1b and TNFa canmediate radiotherapy-induced systemic effects of localized radi-ation in mice; in this study, TNF-a was proposed as a trigger ofnausea and fatigue (45).

Plasma levels of MIP-1a were statistically significantly elevat-ed at 4 weeks of radiotherapy treatment in both treatmentgroups of patients. This factor is produced by macrophages,dendritic cells, and lymphocytes and is crucial for inflammatoryresponses via activation of human granulocytes and chemotacticmobilization of macrophages, monocytes, and lymphocytesinto inflammatory tissues (46, 47). MIP-1a triggers the synthesisand release of other proinflammatory cytokines such as IL1, IL6,and TNFa from fibroblasts and macrophages, which have beenshown to be involved in bystander and abscopal signaling(discussed in refs. 5, 22). The late elevation of this factor isconsistent with the notion that high single doses (>15 Gy) andaccumulated doses during a course of radiotherapy effectivelycontrol tumors, and irradiated tumor and stromal cells becomea robust source of diverse antigens which induce proinflamma-tory responses (42). This ongoing inflammation can explain theobserved accumulation and persistence of DNA damageobserved in out-of-field radiotherapy eyebrow hair folliclesfollowing local irradiation of lung tumors. While this study hasfocused on the g-H2AX endpoint, we have identified certaincytokines as potential associated markers, which in contrast tothe other marker investigated, exosome release, have potentialfor translation to individualization strategies.

In summary, in this study, we demonstrate dose/volumedependence of g-H2AX foci in PBL after localized thoracic irra-diation in patients with NSCLC. We followed kinetics of DNA

Siva et al.





damage in PBL and found that generally it decreases over time,however, in some patients it does not. Furthermore, for the firsttime, we demonstrate a direct evidence of abscopal DNA damageinduction in patients' normal tissues at 24 hours post localizedexternal beam radiotherapy.Ourfindings should be validated in alarger cohort with clinical correlations of associated radiotherapy-related normal tissue toxicity and secondary malignancies. Alarger cohort would allow inference of an association betweenobserved abscopal DNA damage and clinical systemic toxicitiessuch as nausea, anorexia, and fatigue. Furthermore, a largersample size would allow for a more detailed analysis of otherclinically relevant 3D radiotherapy dosimetric parameters that areused for plan evaluation, such as volumes of lung receiving 5, 20,and 30 Gy. Such studies represent an opportunity to monitorindividual real-time biologic effects of ionizing radiation inNSCLC patients undergoing radiotherapy and may allow fortherapeutic personalization.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Siva, O.A. MartinDevelopment of methodology: S. Siva, P.N. Lobachevsky, T. Kron, A. M€oller,O.A. Martin

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Siva, T. Kron, A. M€oller, R. Lobb, J. Smith,O.A. MartinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Siva, P.N. Lobachevsky, A. M€oller, J. Ventura,O.A. MartinWriting, review, and/or revision of the manuscript: S. Siva, P.N. Lobachevsky,M. MacManus, T. Kron, A. Moller, R. Lobb, J. Ventura, D. Ball, O.A. MartinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. SivaStudy supervision: S. Siva, D. Ball, O.A. MartinOther (participation in regular group meetings): T. KronOther (experimental work): N. Best

AcknowledgmentsThe authors thank Joel Mason for providing technical assistance and Roger

Martin for critical review of this article.

Grant SupportThis study was supported by the Australian National Health and Medical

Research Council (NHMRC) grants 10275598 and 1038399.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 23, 2016; revised April 15, 2016; accepted May 11, 2016;published OnlineFirst June 3, 2016.

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2016;22:4817-4826. Published OnlineFirst June 3, 2016.Clin Cancer Res Shankar Siva, Pavel Lobachevsky, Michael P. MacManus, et al. TissuesDamage Response in Both Irradiated and Out-of-field Normal

Small Cell Lung Cancer Induces DNA−Radiotherapy for Non

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