Translational Cancer Mechanisms and Therapy

The Combination of the PARP Inhibitor Olapariband the WEE1 Inhibitor AZD1775 as a NewTherapeutic Option for Small Cell Lung CancerAlice Lallo1, Kristopher K. Frese1, Christopher J. Morrow1, Robert Sloane1, Sakshi Gulati1,Maximillian W. Schenk1, Francesca Trapani1, Nicole Simms1, Melanie Galvin1,Stewart Brown1, Cassandra L. Hodgkinson1, Lynsey Priest1, Adina Hughes2, Zhongwu Lai3,Elaine Cadogan2, Garima Khandelwal4, Kathryn L. Simpson1, Crispin Miller4,Fiona Blackhall5, Mark J. O'Connor2, and Caroline Dive1

Abstract

Purpose: Introduced in 1987, platinum-based chemo-therapy remains standard of care for small cell lungcancer (SCLC), a most aggressive, recalcitrant tumor.Prominent barriers to progress are paucity of tumor tissueto identify drug targets and patient-relevant models tointerrogate novel therapies. Following our developmentof circulating tumor cell patient–derived explants (CDX)as models that faithfully mirror patient disease, here weexploit CDX to examine new therapeutic options forSCLC.

Experimental Design: We investigated the efficacy of thePARP inhibitor olaparib alone or in combination with theWEE1 kinase inhibitor AZD1775 in 10 phenotypically distinctSCLC CDX in vivo and/or ex vivo. These CDX represent che-mosensitive and chemorefractory disease including the firstreported paired CDX generated longitudinally before treat-ment and upon disease progression.

Results: There was a heterogeneous depth and duration ofresponse to olaparib/AZD1775 that diminished whentested at disease progression. However, efficacy of thiscombination consistently exceeded that of cisplatin/etopo-side, with cures in one CDX model. Genomic and proteinanalyses revealed defects in homologous recombinationrepair genes and oncogenes that induce replication stress(such as MYC family members), predisposed CDX to com-bined olaparib/AZD1775 sensitivity, although universalpredictors of response were not noted.

Conclusions: These preclinical data provide a strong rationaleto trial this combination in the clinic informed by prevalent,readily accessed circulating tumor cell–based biomarkers. Newtherapies will be evaluated in SCLC patients after first-linechemotherapy, and our data suggest that the combination ofolaparib/AZD1775 should be used as early as possible andbefore disease relapse.ClinCancerRes; 24(20);5153–64.�2018AACR.

IntroductionSmall cell lung cancer (SCLC) is an aggressive and recalcitrant

neuroendocrine tumor which metastasizes early (1). The 5-yearsurvival rate for SCLC has been less than 5% for over 30 years,despitemultiple phase I clinical trials with a variety ofmolecularlytargetedagents.Althoughplatinum-based therapies yieldobjectiveresponse rates in the majority of patients, this benefit is usuallytransient with relapse frequently occurring in less than 1 year frominitial diagnosis (2). Recent large-scale genome sequencing effortshave failed to identify recurrent actionablemutations in SCLC, andthe failure to improve survival rates has largely been attributed tothe fact that few patients undergo surgery, and thus the amount oftissue available for research is limited, and tumor biopsies atdisease relapse after chemotherapy are rarely obtained (1).

We recently reported the generation of circulating tumor cell(CTC)-derived explant (CDX) models in which CTCs fromextensive stage SCLC patients are grown subcutaneously inimmunocompromised mice (3). CDX models provide addi-tional material to examine the biology of SCLC and serve as arobust and relevant preclinical pharmacology platform to

1Clinical and Experimental Pharmacology Group, Cancer Research UK Manche-ster Institute, University ofManchester,Manchester, UnitedKingdom. 2OncologyInnovative Medicines and Early Development Biotech Unit, AstraZeneca, Cam-bridge, United Kingdom. 3Oncology Innovative Medicines and Early Develop-ment Biotech Unit, AstraZeneca,Waltham,Massachusetts. 4RNABiologyGroup,Cancer Research UK Manchester Institute, University of Manchester, Manche-ster, United Kingdom. 5Institute of Cancer Sciences, University of Manchester,and Christie NHS Foundation Trust, Manchester, United Kingdom.

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

A. Lallo and K.K. Frese contributed equally to this article.

Corresponding Authors: Caroline Dive, Cancer Research UK Manchester Insti-tute, Alderley Road, Alderley Edge, Macclesfield SK10 4TG, United Kingdom.Phone: 44-161-446-3036; Fax: 44-161-446-3019; E-mail:caroline.dive@cruk.manchester.ac.uk; and Mark J. O'Connor, DNA DamageResponse Biology, Oncology Innovative Medicines and Early Clinical Develop-ment, AstraZeneca, Hodgkin Building (B900), Chesterford Research Park, Cam-bridge CB10 1XL, United Kingdom. E-mail: mark.j.oconnor@astrazeneca.com

doi: 10.1158/1078-0432.CCR-17-2805

�2018 American Association for Cancer Research.

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examine the efficacy of new potential therapies. Importantly,the in vivo response to standard-of-care chemotherapy in CDXmodels closely resembles that of the corresponding donorpatient, suggesting that preclinical results for experimentaltherapies may be more robust for translation to the clinic thanconventional cell line–based xenografts.

Proteomic profiling in lung cancer cell lines revealed elevatedlevels of PARP specifically in SCLC (4). PARP is recruited to DNAsingle-strand breaks (SSB) where it PARylates multiple substratesincluding histones to facilitate the relaxation of chromatin, aswellas itself. The automodification of PARP is required for its disso-ciation from DNA and subsequent repair. The mechanism ofaction for PARP inhibitors with single-agent activity has beenproposed to involve the trapping of PARP onto the DNA SSBspreventing their repair and generating a potential block forcellular DNA replication (5, 6). An important consequence ofthis and the proposed basis of monotherapy activity is thegeneration of DNA double-strand breaks (DSB) that would nor-mally be repaired by the homologous recombination repair(HRR) pathway. In cancers with HRR deficiency, PARP trappingwill result in significant genomic instability until it is no longersustainable and tumor cell death results (7). Althoughdeficienciesin the breast and ovarian cancer–associated genes BRCA1 andBRCA2 are the most investigated examples of defective compo-nents of HRR that lead to increased PARP inhibitor activity,mutations in other HRR pathways genes, including ATM, ATR,and PALB2, can also convey sensitivity to PARP inhibitors (8).

WEE1 is a kinase that regulates both S-phase and G2–Mprogression by phosphorylating and inhibiting the cell-cycle–dependent kinases CDK2 and CDK1, respectively. WEE1 controlof CDK2 activity in S-phase is an important component of thereplication stress response, whereas the control of CDK1 and theG2–Mcheckpoint is critical for ensuring proper DNA repair beforeinitiating cell division (9). Initially, it was hypothesized that cellsdeficient for the tumor suppressor p53 were hyper-reliant onWEE1 function and the G2–M checkpoint, and that efficacy ofthe WEE1 inhibitors could be linked to TP53 status (10). Subse-quent experiments revealed that this correlation was not univer-sally true, althoughWEE1 inhibitor activity is still likely linked toG1–S checkpoint deficiencies in one form or another (11). Recentreports suggest that cells overexpressing potent oncogenes, such asMYC or CCNE1, that induce replication stress are also more

sensitive to WEE1 inhibition, but again does not appear to bea universal prerequisite for response (9).

Given the potential for PARP inhibitors to induce S-phasedamage and a subsequent dependency on Wee1 S-phase andG2–M checkpoint activities, coupled with the high mutagenicburden and proliferative rate of SCLC, we examined the efficacyofthePARPinhibitorolaparibandtheWEE1inhibitorAZD1775inour SCLCCDXmodels. Themost important advantageof theCDXapproach is the ability to assess biology and therapy responses notonly in patient-derived models generated at baseline prior to anytreatment,butalso inCDXderivedfromthesamepatientatdiseaseprogression after treatment. Here, we examine the novel combi-nationofolaparibandAZD1775 for thefirst time in"paired"CDX,providing a clinically relevant test-bed for the introduction of newtherapeutic options following first-line chemotherapy.

Our studies reveal that SCLC CDX tumors exhibit a range ofsensitivities to drugs targeting PARP and WEE1 as single agents,but with clear indications of enhanced efficacy when given incombination. Moreover, these studies have revealed the presenceof previously unappreciated DNA damage response deficienciesin a subset of SCLC tumors. These results therefore provide astrong rationale for the clinical development of AZD1775 incombination with olaparib or other DNA-damaging agents, aswell as candidate biomarkers to explore in tandem toward aprecision medicine approach for this disease.

Materials and MethodsStudy design

The primary goal of this study was to assess the efficacy ofolaparib and AZD1775 monotherapies, as well as the combina-tion, in mouse models of SCLC. Sample sizes (n¼ 7–11mice percohort) were determined to detect statistically a 2-fold differencein tumor response between the various treatments. Once tumorsreached enrollment size, animals were allocated to differentcohorts via stratified randomization inwhich attempts weremadeto evenly distribute initial tumor volume sizes. Data collectionwas stopped at ethical endpoints, including deterioration inhealth orwhen tumors reached�4 times the initial tumor volumeat enrollment. If animals became ill within 3 days of enrollment,they were excluded from the experiment, regardless of whichtreatment they received. No other data were excluded.

CDX establishmentCDX models were generated as previously described (3).

Briefly, 10 mL of peripheral blood was collected from SCLCpatients into EDTA tubes in accordance with the ethicallyapproved CHEMORES protocol (07/H1014/96), processed viaRosetteSep CTC Enrichment Cocktail (15167; Stemcell Tech-nologies), and resuspended in 100 mL of a 1:1 solution ofHITES medium and Matrigel (354234, BD Biosciences). Cellswere then subcutaneously implanted into flanks of 8- to16-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG)mice (Charles River). All procedures were carried out in accor-dance with Home Office Regulations (UK) and the UK Coor-dinating Committee on Cancer Research guidelines and byapproved protocols (Home Office Project license nos.40–3306/70-8252 and Cancer Research UK ManchesterInstitute Animal Welfare and Ethical Review Advisory Board).In a parallel patient blood sample, CTC burden was quantifiedvia CellSearch according to the manufacturer's instructions.

Translational Relevance

The clinical management of small cell lung cancer (SCLC)has remained largely unchanged for over 30 years due in partto the lack of model systems that predict clinical outcomes.SCLC prognosis remains dismal. Our recent introduction ofSCLC circulating tumor cell–derived explant models (CDX)that can be generated from a simple patient blood draw beforetreatment and again after treatment upondisease relapse offersresearch opportunities to test new therapies and explore pre-dictive biomarkers. Here, we report promising data inmultipleCDX models that support a combination of PARP and WEE1inhibitors in SCLC that is superior in efficacy to standard-of-care chemotherapy.We also identify a "super-responder" CDXmodel that provides molecular insights into strategies forpatient selection for this drug combination.

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In vivo pharmacology studiesTherapeutic studies were carried out essentially as previously

described (3). A total of 100,000 viable CDX cells in 100 mL 1:1RPMI:matrigel were injected subcutaneously into the right flankof procedure/treatment-na€�ve, 8- to 10-week-old (20–25 g)female C.B-17/lcrHsd-PrkdcscidLystbg-J mice (Envigo). Whentumors reached 150 to 250mm3, they were allocated via stratifiedrandomization into cohorts to be treated each morning withvehicle(s), olaparib (50 mg kg�1 unless indicated otherwise),AZD1775 (120 mg kg�1), the combination, or topotecan usingtolerated dosing regimens previously shown to optimally achievetarget inhibition. Olaparib (AstraZeneca) was formulated at 10mgmL�1 in 10%DMSO/30%KLEPTOSEHPbCD, and AZD1775(AstraZeneca) was formulated at 12 mg mL�1 in 0.5% methyl-cellulose. Compounds were orally dosed at 5 mL kg�1 daily(olaparib) or 10 mL kg�1 on a 5-on, 2-off (AZD1775) schedulefor 21 days. On days when both drugs were administered, ola-parib was administered 1 hour after AZD1775. Topotecan (TheChristie NHS Foundation Trust) was administered at 3 mg kg�1

daily for 3 days via intraperitoneal injection. Cisplatin and etopo-side (The Christie NHS Foundation Trust) were administered aspreviously described (3), and these studies were performed sep-arately from studies on olaparib and AZD1775. Mice wereobserved for a period of time after dose to ensure no adverseeffects were seen. Tumors were measured twice a week by caliperuntil they reached 4 times initial tumor volume (4 � ITV), asdetermined by the formulaV¼ (L�W2)/2, or until animal healthdeteriorated. For pharmacokinetic and pharmacodynamic stud-ies, mice were sacrificed 2 or 24 hours after a single dose ofindicated drugs was administered and relevant tissues were col-lected. Statistical assessment of tumor response was performed asdescribed (12). Tumor growth delay (TGD) measures the nor-malized time to quadrupling of initial tumor growth, and there-fore a higher TGDvalue is indicative ofmore robust tumor growthinhibition.

Pharmacokinetic analysisNote that 25 mL plasma was prepared using an appropriate

dilution factor, and compared against an 11-point standardcalibration curve (1–10,000 nmol/L) prepared in DMSO andspiked into blank plasma. Acetonitrile (100 mL) was added withthe internal standard, followed by centrifugation, and superna-tants were then diluted 1:7 in water and analyzed via UPLC-MS/MS using a Xevo TQ-Smass spectrometer (Waters) and the systemand optimization parameters listed (Supplementary Tables S1and S2).

Ex vivo studiesCDX tumors were dissociated as previously described (13), and

cells were grown in HITES medium supplemented with 5 mmol/LY-27632 (Selleckchem) and 2.5% FBS. CDX-derived cells wereseeded into 96-well plates, incubated for 48 hours, and treatedwith increasing concentrations of the compound(s) of interest.Seven days after treatment, cell viability was assessed via Cell-TiterGlo 3D assay (Promega). Single dose response curves were com-puted with the open-source CRUK software Combenefit (14).Combinatorial activity was assessed using the Loewe additivitymodel (15) and computed using the R package synergyfinder(16). For radiosensitization assays, cells were irradiated withindicated doses using a Gulmay Bipolar 320 kV X-ray source.

After irradiation, cells were plated into fresh media, and viabilitywas measured after 5 days.

RAD51 focus assayCDX-derived cells were irradiatedwith 10Gy and incubated for

the indicated times. After incubation, cells were cytospun at aconcentration of 150,000 cells per slides, fixed in 4% PFA for 20minutes at room temperature, permeabilized with 0.2% triton X-100 (Sigma), and washed with 10% FBS 0.2% Tween20 in PBS.Samples were then blocked with avidin/biotin-blocking solution(VECTOR laboratories, SP-2001) and 10% goat serum (Dako).Sections were then incubated with primary antibodies (Supple-mentary Table S3) overnight at 4�C overnight, washed, andincubated with secondary antibodies for 1 hour at room temper-ature. Slides were mounted in ProLong DAPI (Life Technologies)and left dry in the dark. Images were acquired with a Leica gatedStimulated EmissionDepletionmicroscope, and cells were scoredas HR-proficient if there were �5 foci per nucleus.

ImmunohistochemistryFormalin-fixed paraffin-embedded tumor sections (4mm)were

stained for the indicated antigens using the BondMax autostainerand Bond polymer refine detection system (Leica Biosystems),Venatana Discovery Ultra, or I6000 BioGenex autostainer andDako EnVision systemHRP (Dako; Supplementary Table S3). Forthese antigens, digital images of whole tissue sections wereacquired using a Leica SCN400 histology scanner (Leica Micro-systems). Images were analyzed using Definiens Developer XDand the Tissue Studio Portal version 4.4 (Definiens AG).

Immunoblot analysesFlash-frozen CDX tumors were homogenized in Fastprep tubes

with matrix A using a TissueLyser LT (Qiagen) in ice-cold lysisbuffer (10 mmol/L HEPES, pH 7.1, 50 mmol/L NaCl, 0.3 mol/Lsucrose, 0.1mmol/L EDTA, 0.5%Triton X-100, 1mmol/LDTT; or20 mmol/L Tris, 137 mmol/L NaCl, 10% Glycerol, 50 mmol/Lsodium fluoride, 1 mmol/L activated sodium orthovanadate, 1%SDS, 1% NP40 substitute) in the presence of Protease InhibitorCocktail and Phosphatase Inhibitor Cocktail I and III (Sigma-Aldrich). Protein concentration was determined by BCA proteinassay reagent kit (Thermo Scientific). Protein extracts were resus-pended in NuPAGE LDS sample buffer 4x (Invitrogen) and 10xNuPAGE sample reducing agent (Invitrogen). Total protein wasresolved on aNuPAGE 4%–12%Bis-Tris 1.0mmgel (Invitrogen).After transfer onto PVDF or nitrocellulose membranes, blots wereincubated with the appropriate primary antibody (Supplemen-tary Table S3) and the corresponding horseradish peroxidase–coupled secondary IgG. Detection was obtained with the Super-signal West Dura extended duration substrate (ThermoFisher) ona ChemiGenius Imaging System (Syngene) or film.

PAR ELISAFlash-frozen CDX tumor tissue was homogenized for 3 � 30

seconds at speed 6.0 in Fastprep tubeswithmatrix Ausing ice-coldlysis buffer (20mmol/L Tris, 137mmol/LNaCl, 10%Glycerol, 50mmol/L sodium, 1mmol/L activated sodium orthovanadate, 1%SDS, 1% NP40 substitute) in the presence of protease inhibitorcocktail (Roche) and phosphatase inhibitor cocktails 2 and 3(Sigma-Aldrich). Lysates were sonicated, incubated on ice for 30minutes, centrifuged, and supernatants were boiled for 5minutes.They were then cooled on ice for 1 minute, centrifuged, and

PARP and WEE1 Inhibition in Patient-Derived Models of SCLC

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protein concentration was estimated using a Pierce BCA assay.Lysate (2mg)was used to quantify total PAR levels according to themanufacturer's recommendations (Trevigen). Samples werequantified on a Tecan Safire II microplate reader (Tecan), andabsolute values were determined via interpolation from a stan-dard curve.

RNA-seqTotal RNA was extracted from three independent RNAlater

(Qiagen)-treated tumors from each model with RNeasy mini kits(Qiagen) and quantified by Qubit (Thermo Fisher). RNA with anRNA integrity number > 8 (Agilent 2100 Bioanalyzer) was used togenerate libraries with SureSelect poly A samples (Agilent) andsequenced on the NextSeq 500 using 75-bp paired-end reads.RNA-seq data were aligned to Human GRCh38 and MouseGRCm38 assembly using Mapsplice (version 2.1.6). Xenograftdata were filtered via a novel algorithm to distinguish human andmouse reads (17). The filtered reads are then used to generatecounts data using Rsubread package (version 1.16.1) withEnsembl 77 GTF file in R. The counts were converted into RPKMvalues using edgeR (version 3.10.5).

Whole-exome sequencingGenomic DNAwas extracted from a representative flash-frozen

CDX tumor from each model with DNeasy Blood and Tissue kits(Qiagen) and quantified by Qubit. Note that 150 bp paired-endreads were obtained from samples assessed on a NextSeq 500High run (Illumina). The whole-exome sequencing (WES) datawere aligned to Human GRCh37 and Mouse GRCm37 assemblywith bwa-mem (version 0.7.12). Deduplication, realignment,and recalibrationwere performedon the aligned data using Picard(version1.96) andGATK tools (version 3.3), as stated in theGATKbest practices. The reads aligning tomouse genomewere removedusing an in-house developed algorithm (17). Somatic mutationcallingwas doneon thefiltered reads usingMutect (version1.1.7),and the mutation calls were annotated using Ensembl VariantEffect Predictor.

Statistical analysisAll statistical analyses were performed on Prism v7.04 (Graph-

Pad) using one-wayANOVAor Student t test formultiple or singlepairwise comparisons, respectively. Single and double asterisksdenote P < 0.05 and P < 0.005, respectively. The absence of anasterisk means that there is no statistically significant differencebetween groups.

ResultsTo examine the efficacy of olaparib and AZD1775 in SCLC, we

first treated two previously characterized CDX models represen-tative of chemosensitive and chemorefractory patient donors (3).The chemosensitive CDX3 and chemoresistant CDX4 modelswere generated from baseline blood samples taken from a patientthat lived for 9.7 months and 0.9 months, respectively, followingtreatment with platinum-based therapies. Treatment of micebearing CDX3 tumors with either olaparib or AZD1775 mono-therapy led to complete ormodest regression,with tumors rapidlyrecurring after treatments ended (Fig. 1; Supplementary Fig. S1A).Remarkably, combination therapy in CDX3 promoted completeand durable regressionswith 5 of 7mice remaining tumor-free forover 1 year. Conversely, CDX4, a model completely resistant to

cisplatin/etoposide, showed no response to olaparib, whereastreatmentwith either AZD1775monotherapy or the combinationled to disease stabilization during the treatment period (Fig. 1;Supplementary Fig. S1B).

Topotecan, a DNA-damaging agent that is the only approvedsecond-line therapy for SCLC, was more effective than cisplatin/etoposide against CDX4, eliciting amedian 66% tumor regression(Supplementary Fig. S1E). Furthermore, combination of topote-can with AZD1775 yielded impressive tumor regression in thismodel, with median 83% tumor regression. These data indicatethat AZD1775 can cooperate with an effective DNA-damagingagent even in a platinum-resistant model.

The majority of SCLC patients present with chemosensitivedisease followed by rapid acquisition of chemoresistanceand disease progression. To examine the efficacy of the ola-parib/AZD1775 combination in patients with acquired

Figure 1.

Combination treatment with olaparib/AZD1775 is superior to cisplatin/etoposide. A, The cohort average best response (�SD) is shown for cisplatin/etoposide (white), AZD1775 (red), olaparib (green), and AZD1775/olaparib(blue). B, The cohort average benefit (�SD) of each treatment over vehicle isshown for each treatment. Cohort sizes are �7. � , P < 0.05; ��, P < 0.005 vs.cisplatin/etoposide treatment.

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chemoresistance, we utilized a pair of CDX models, CDX8 andCDX8p, which were derived from blood samples from thesame patient taken at baseline and upon disease progressionafter platinum treatment (Supplementary Fig. S2A). Treatmentof CDX8 tumors with cisplatin/etoposide induced a partial

response (median 47% regression) and rapid relapse (medianTGD ¼ 1.9), whereas mice bearing CDX8p received littlebenefit from cisplatin/etoposide treatment (median 15%growth and TGD ¼ 1.3), consistent with the outcome ofchemotherapy in the donor patient (Fig. 1; SupplementaryFig. S2B and S2C). When these models were treated witholaparib or AZD1775, CDX8 exhibited relatively stable diseaseduring the treatment period, whereas CDX8p tumors grew atthe same rate as the vehicle control cohort (Fig. 1; Supple-mentary Fig. S1C and S1D). Similar to that seen in CDX3, thecombination exhibited synergistic activity against CDX8 withmany complete regressions. Although CDX8 tumors recurredmore quickly than CDX3 tumors (Fig. 1B), olaparib/AZD1775yielded a more durable response than cisplatin/etoposide(median TGD ¼ 1.9; Fig. 1). Consistent with reports ofreduced olaparib efficacy against platinum-resistant ovariancancer (18), treatment with olaparib alone or in combinationwith AZD1775 achieved only TGD rather than regression inCDX8p (Fig. 1; Supplementary Fig. S1D). Taken together,these results indicate that although the efficacy of olaparib/AZD1775 was model dependent, the activity of the combi-nation is consistently superior to that achieved with conven-tional cisplatin and etoposide in chemosensitive models.Furthermore, the combination of AZD1775 and an activeDNA-damaging agent has activity in a chemoresistant model.

As platinum-based regimens are initially quite effective intreating SCLC, it is unlikely that olaparib/AZD1775 or any othernew agent will be used clinically in treatment-na€�ve patients. Wetherefore sought to examine the efficacy of olaparib/AZD1775 in asecond-line setting following initial treatment with cisplatin/etoposide. Toward this end, we treated mice bearing CDX3tumors with a single cycle of cisplatin/etoposide to induce aninitial tumor response. After a 3-week drug holiday during whichtumors began to regrow,micewere randomized to receive vehicle,olaparib, AZD1775, or the combination. In this setting, only 1 of12 combination-treatedmice remained tumor-free, and themedi-an time to initial tumor volume was 101 days following com-pletion of dosing (Supplementary Fig. S3), suggesting that thiscombination is still active after initial treatment with cisplatin/etoposide but may prove most beneficial as a first-line option ifthe proper patient cohort could be identified.

In vivo pharmacology experiments are both time consumingand expensive, so we sought to use an in vitro platform to expeditedrug screening as previously reported for colorectal and breastcancers (19, 20). Using an ex vivo SCLC CDX culture system thatfaithfully recapitulates in vivo drug response in CDXmodels (21),we investigated the in vitro efficacy of olaparib, AZD1775, and the

Figure 2.

AZD1775 and olaparib are synergistic in vitro. CDX3 (A), CDX4 (B), CDX8 (C), orCDX8p (D) ex vivo cultures were treated with AZD1775 and olaparib for 1 week,and relative cell viabilitywas assessed.One representative experiment of at leastthree is shown. For the combinatorial index, red indicates synergy, and greenindicates antagonism between drugs. E, GI50 values for various CDX ex vivocultures treatedwith cisplatin, etoposide, AZD1775, or olaparib are shown after 1week of drug treatment. For the combination, we have shown the GI50 values forolaparib at 150 nmol/L AZD1775.

Table 1. Response to standard-of-care for each patient and derived CDX, with the corresponding CTC count at baseline

Model Pt treatment Tumor responsea Chemosensitivity Pt survival (days)a CellSearch countb

2 Carbo/etop PR Refractory 108 1,6253 Carbo/etop PR Sensitive 295 5073p Relapse 295 4634 Carboplatin PD Refractory 27 1,3758 Carboplatin PR Refractory 181 3888p Relapse 181 1,79610 Carbo/etop PR Sensitive 317 16014p Relapse 227 36215p Relapse 556 36115p2 Relapse 556 165aSurvival is defined as the time from diagnosis until death.bThe number of EpCAMþ/CKþ CTCs in a parallel 7.5 mL sample taken from the same patient blood that yielded the corresponding CDX.

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combination. Both monotherapies as well as the combinationcorrelatedwith in vivo response, with evidence of synergy in CDX3and CDX8, but not CDX4 or CDX8p (Fig. 2A–D; SupplementaryFig. S4). We therefore decided to screen additional CDX modelswith varying sensitivity to platinum, derived from baseline andrelapse patients (Table 1). Examination of ex vivo cultures derivedfrom CDX2, CDX3p, CDX10, CDX14p, CDX15p, and CDX15p2revealed a range of sensitivity to cisplatin and olaparib mono-therapies, whereas most models were relatively sensitive toAZD1775 with submicromolar GI50s. Similarly, the majority ofmodels were sensitive to olaparib/AZD1775, although CDX4 andCDX8p were among the most resistant in vitro (Fig. 2E). Theseresults from a broader panel of CDXmodels tested in vitro supportour findings that olaparib/AZD1775 is an effective combinationand may represent a promising treatment for SCLC.

It has previously been reported that olaparib is a CYP3Asubstrate and that AZD1775 is aCYP3A4 inhibitor (22); therefore,it was possible that the combination effect seen in some CDXmodels could be attributed to a drug–drug interaction in whichelevated levels of olaparib result from inhibition of CYP3A byAZD1775. To examine this possibility, we administered 50 mgkg�1 olaparib (olaparib low) both alone and in combinationwithAZD1775 as previously described, or 100mg kg�1 olaparib alone(olaparib high) to CDX3-bearing mice. Increasing the dose ofolaparib to 100 mg kg�1 had no significant effect on antitumorefficacy against CDX3 (Fig. 3A), and pharmacokinetic analysis ofolaparib and AZD1775 indicates that mice dosed with the com-bination are not exposed to plasma drug concentrations signif-icantly different tomice receivingmonotherapies (Fig. 3B and C).Therefore, the efficacy of the olaparib/AZD1775 combination isunlikely to be due to altered olaparib exposure.

To examine the molecular mechanism of olaparib andAZD1775 efficacy, we treated parallel cohorts of mice with asingle dose of drug and examined intratumor signal transductionpathways and cellular behavior. WEE1 kinase regulates G2–Mprogression by directly phosphorylating and inhibiting the cell-cycle kinase CDK1, and this has been used both preclinically andclinically as a pharmacodynamic biomarker of WEE1 inhibition(9). Consistent with WEE1 inhibition, phospho-CDK1 levelswere reduced, and the number of mitotic cells was increased incohorts treated with AZD1775 either alone or in combinationwith olaparib (Fig. 4A and C; Supplementary Fig. S5). Similarly,global PAR levels were decreased in all cohorts treated witholaparib (Fig. 4B). Together, these data indicate that the differentlevels of efficacy in different CDX models cannot be attributed todifferential target inhibition. Examination of cleaved caspase 3levels revealed that increased levels of apoptosis correlated withantitumor efficacy (Fig. 4D; Supplementary Fig. S5). Becausetreatment with both olaparib and AZD1775 promoted increasedDNA damage, we investigated several pathways associated withthe DNA damage response.

CHK1 phosphorylation by ATR is induced by the formation ofregions of single-strand DNA (ssDNA) either resulting from theprocessing of DNADSBs or stalled DNA polymerase during DNAsynthesis due to replicative stress (9). The extended ssDNA iscoated by replication protein A (RPA) which binds ATR andassociated proteins that initiate the replication stress responsethat includes the phosphorylation and activation of CHK1. Theinability to resolve replication stress–induced fork stalling canlead to replication fork collapse, the generation of a single-endedDSBs, andATM-inducedH2AXphosphorylation. Consistent with

Figure 3.

Pharmacokinetic analyses. A, Mice were treated with 100 mg kg�1 (dark green)or 50 mg kg�1 (dashed green) olaparib, or with a combination of low-doseolaparib and AZD1775 (blue) for 21 days, and tumor volumes were measuredbiweekly. Individual relative tumor volumes are depicted. Tumor lysates fromcohorts of mice bearing different CDX models treated with AZD1775 (red),olaparib (green), or the combination (blue) were analyzed and quantified forAZD1775 (B) or olaparib (C) at 2 hours after a single dose. Dotted lines representthe reliable limits of detection. Cohorts of �4 tumors were used.

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previous reports, treatment with AZD1775 promoted potentCHK1 phosphorylation, indicative ofWEE1 inhibition increasingreplication stress (23). However, this effect was similar in allmodels examined and did not correlate with efficacy (Fig. 4E;Supplementary Fig. S1). Conversely, induction of gH2AX moreclosely correlatedwith in vivo efficacy, and highest levels were seenwhenAZD1775was combinedwith olaparib inCDX3 andCDX8,the two most sensitive models (Fig. 4F; Supplementary Fig. S1).

The BRCA1 and BRCA2 gene products are key DDR proteinsthat play different but pivotal roles in DNA DSB repair (DSBR).Both proteins have been shown to play key roles in the replicationstress response as well through replication fork stabilization andrestart, and deficiencies in either lead to PARP inhibitor sensitivity(24). Given this association, wewere interested to know theDSBRstatus in our CDX models. BRCA and broader HRR deficiencieshave been identified via a variety of mechanisms includingtranscriptional or mutational impairment of genes that regulateHRR, the accumulation of "genomic scars," or functional assayssuch as DNA damage–induced RAD51 focus formation (24).Analysis of RNA-seq data on CDX models did not reveal an

obvious HRR mutation signature that correlated with sensitivity,nor did WES reveal an elevated mutation burden associated withimpaired DSBR (Supplementary Fig. S6A and S6B). Despite theabsence of an HRR mutation signature, CDX3 is exquisitelyradiosensitive compared with CDX4, indicating that CDX3 ispotentially deficient in DSBR (Fig. 5A). Functional assessmentof HRR status via examination of ionizing radiation–inducedRAD51 focus formation revealed that CDX3 displayed signs ofHRRdeficiency, suggesting thismodelmayharbor amutation in agene that mediates HRR (Fig. 5B). PALB2 is a BRCA2-associatedDDR protein required for effective HRR (9). WES of CDX3revealed a somatic mutation in PALB2 that resulted in a stop-gain at E178 leading to premature truncation of the protein (Fig.5CandD). Analysis of RNA-seqdata indicates that allPALB2 readsharbor this mutation, suggesting a complete loss of the wild-typeallele (Supplementary Fig. S6C). Therefore, it is highly likely thatthe exquisite sensitivity of CDX3 to both platinum and olaparibcan be attributed to the PALB2 mutation.

Several additional mechanisms of acquired resistance to ola-parib have been reported, including activation of PI3K or MET

Figure 4.

Pharmacodynamic analyses. Tumorlysates from cohorts of mice bearingdifferent CDX models treated with asingle dose of vehicle (white), AZD1775(red), olaparib (green), or thecombination (blue) were analyzedand quantified. Cohorts of � 4 tumorswere used. A, pCDK1 levels weremeasured by immunoblotting 2 hoursafter a single dose and normalized toboth total CDK1 and vinculin in eachsample. B, Total PAR levels weremeasured by ELISA 2 hours after asingle dose. The dashed line indicatesthe limit of detection (2 pg/mL).Proof-of-concept and proof-of-mechanism biomarkers were measuredby quantitative immunohistochemistryfor pHH3 at 2 hours (C), CC3 at24 hours (D), pCHK1 at 2 hours (E), andgH2AX at 24 hours (F) after dosing.� , P < 0.05 and �� , P < 0.005.

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signaling (25, 26), decreased expressionofPARP1or SLFN11 (27–30), and restoration of DNA end resection (31). We did not findany consistent differences in MET or PI3K signaling, suggestingthat activation of these kinases is not a recurrent mechanism ofolaparib resistance in SCLC (Supplementary Fig. S7A). PARP-trapping is now recognized as the primary mechanism of PARPinhibitor–mediated cytotoxicity, and loss of expression of PARP1is thus associated with resistance. However, expression analysisdid not reveal any alteration in PARP1 expression in either CDX4or CDX8p (Supplementary Fig. S7B). SLFN11 binds to RPA, and ithas been postulated that high SLFN11 levels inhibit HRR bypromoting the destabilization of the interaction between RPAand ssDNA (32). Although CDX4, the most olaparib-resistantmodel, exhibits the lowest SLFN11 levels, this correlation doesnot extend to CDX8p (Supplementary Fig. S7C). End resection isthe process of creating stretches of ssDNA necessary for RAD51-induced strand invasion and subsequent repair via homolo-gous recombination. In the context of functionally relevantBRCA1 mutations, decreased expression of the nonhomolo-gous end joining proteins 53BP1 and REV7 has been reportedto result in restoration of end resection activation of HRR andconsequently resistance to PARP inhibitors (33, 34). However,

neither CDX4 nor CDX8p has decreased expression ofeither protein compared with CDX3 or CDX8 (SupplementaryFig. S7C). Although CDX8 harbors a C342F mutation inFANCD2, a key player in the Fanconi anemia pathway thatincludes BRCA, this mutation is still present in CDX8p and, inthe absence of any TP53BP1-associated mutation, is unlikely toexplain the acquired chemoresistance manifest in CDX8p.Neither WES nor RNA-seq has identified likely candidates ofresistance, and we are actively pursuing additional strategies todiscover the mechanism of acquired drug resistance in CDX8p.

One proposed predictive biomarker of AZD1775 sensitivity isthe absence of a G1–S checkpoint (35). It has been hypothesizedthat cells with a compromised G1–S checkpoint are more depen-dent on the G2–M checkpoint, and therefore sensitive to WEE1inhibition. Given the role for p53 in regulating the G1–S check-point, multiple studies have investigated whether p53 mutationsare stand-alone biomarkers for AZD1775 sensitivity (10, 36).Although all SCLC models tested harbor deleterious mutationsin TP53, chemoresistant models tend to retain relatively highlevels of the cyclin-dependent kinase inhibitor p21CIP1, a primarymediator of p53-dependent cell-cycle arrest (Fig. 6A and B).Although treatment does not induce expression of p21CIP1

Figure 5.

Deficient DSB repair in CDX3. A, CDX3,CDX4, CDX8, and CDX8p ex vivo cultureswere irradiated at indicated doses intriplicate, and relative cell viability wasassessed 5 days later. One of threerepresentative experiments is shown. B,At indicated times following irradiation,CDX3 and CDX4 cultures were assessedfor RAD51 focus formation. Geminin andDAPI staining was used to identify cells inG2, and these resultswere quantified by asemiautomated computer algorithm. Thedotted line indicates a threshold abovewhich cells are considered positive fordamage-induced focus formation. ��, P <0.005. C, Sanger sequencing identifiedthe E178� PALB2 mutation in CDX3, butnot CDX4. D, Schematic of PALB2 withprotein–protein interaction domainsshown in gray and the E178� mutationshown in red.

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(Supplementary Fig. S8), p53-independent activation of p21CIP1

may partially mediate resistance to AZD1775 by maintaining theG1–S checkpoint in p53-deficient cells. Themajority of SCLC cellsalso possessmutations inRB1, which regulates entry into S-phase.We therefore investigated whether RB1 status correlates withAZD1775 sensitivity. WES and RNA-seq revealed RB1 mutationsin CDX3 (splice mutation at 50 of exon 17, c.1499-1G>T), CDX4(87 bp deletion in exon 1 and intron 1, c.99_137þ48del), andCDX8/8p (G509�), and none of the models used in this studyexpress RB1 as determined by Western blotting (Fig. 6A). More-over, there is no consistent correlation between cyclin/CDKexpression and sensitivity to AZD1775. This does not rule outthe potential importance of deregulation of the G1–S checkpointbut does suggest that, at least in SCLC, regulators of G1–S CDKsalone do not appear to be predictive biomarkers of AZD1775sensitivity.

Low expression of PKMYT1, a cell-cycle kinase that is function-ally redundant to WEE1, can predispose cancer cells to AZD1775sensitivity by lowering the threshold of kinase inhibition (37).However, although the most sensitive models do have the lowestlevels of PKMYT1 RNA, there is no correlation between PKMYT1levels andCDK1phosphorylation, suggesting this is unlikely tobea major factor governing AZD1775 sensitivity (Fig. 4A; Supple-mentary Fig. S9A).

Several studies have reported a link between sensitivity toWEE1inhibition and replication stress (38). Replication stress is ageneral term used to describe situations that result in stalled

replication fork progression and the generation of extendedregions of ssDNA (39). Replication stress can be induced byoverexpression of oncogenes such as MYC family members orcyclin E1, which can both deplete nucleotides as a result ofderegulated origin firing and promote collisions between thetranscription and replication machinery (40). WEE1 plays a keyrole in the replication stress response by regulating CDK2, andmarkers of replication stress such as pRPA have been shown toincrease upon treatment with a WEE1 inhibitor (41). Immuno-blot analysis revealed that CDX3, themodel that is most sensitiveto AZD1775, expressed high levels of MYCL, cyclin E1, andphospho-RPA (Fig. 6C). Consistent with a recent report thattumors deficient for the histone methyltransferase SETD2 andribonucleotide reductase subunit RRM2 are synthetic lethal withAZD1775 treatment (42), CDX3 expressed approximately half asmuch SETD2 and RRM2 compared with CDX4, and treatmentwith AZD1775 either alone or in combination with olaparib ledto modest reductions in RRM2 levels (Supplementary Fig. S9Aand S9B) Moreover, AZD1775 treatment further increased pRPAlevels in CDX3 but not CDX4 (Fig. 6D). Together, these datasuggest that both baseline and treatment-induced replicationstress may predict sensitivity to AZD1775.

DiscussionSCLC is a cancer of high unmet need and, despite promising

preclinical (43) and early clinical results (44) utilizing a DLL3-

Figure 6.

Sensitivity to AZD1775 correlates with markers of replication stress. A, Immunoblot analysis of duplicate tumor lysates for indicated proteins. The p53 mutationassociated with a given model is indicated. The positive control for RB is the H1694 cell line. B, Representative images and quantification of p21CIP1 IHC fromCDX3, CDX4, CDX8, and CDX8p. p21CIP1 levels are plotted against tumor regression values for each model. C, Immunoblot analysis of duplicate tumor lysatesfor indicated proteins. Corresponding average RPKM values for MYCL and MYC in each model are indicated below immunoblots. MYCN expression was notinvestigated; however, RPKM values below 5 suggest it is not expressed in the models investigated. D, Immunoblot analysis of duplicate lysates from CDX3 andCDX4 tumors treated with vehicle or AZD1775 for 24 hours. The positive control for MYCL is the H1694 cell line.

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targeted antibody–drug conjugate, there have been no improve-ments in systemic therapy in three decades. Although approxi-mately 100 SCLC trials have been registered on clinicaltrials.govsince 2007, strikingly few involvedmore than 100 patients, manyare single-arm studies, and correlative biomarker assessment israrely obtained largely due to the difficulties in obtaining serialtumor biopsies. The statement in the US Recalcitrant Cancer Act2012 that "more knowledge of the biology at different phases ofSCLC is needed" can partially be attributed to the lack of relevantmodels. Indeed, the recently reported "patient-derived tumorencyclopedia" that contains over 1,000patient-derived xenograftsdoes not contain a single SCLC model (45), and 21 of 26 SCLCpatient-derived xenografts that have been published were derivedprior to treatment of the donor patient with chemotherapy (43,46, 47). Our CDX models and notably those obtained both atbaseline and at progression after chemotherapy and reported herefor the first time offer a new approach to study acquired treatmentresistance and test novel therapies in both first- and second-linesettings. Utilizing these patient-relevant models derived fromliquid biopsy, we demonstrated here that the combination ofolaparib and AZD1775 exerted significant activity against a panelof SCLC CDX models that span the spectrum of responses tostandard-of-care chemotherapy, with some demonstrating a pro-found and extended response to this novel combination.

The PARP inhibitor olaparib is currently being investigated inclinical trials; however, the genetic determinants of drug sensi-tivity remain incompletely defined. Our CDX models offer theopportunity to investigate both predictive biomarkers andmechanisms of drug resistance. We observed the most strikingcombination efficacy in models where both monotherapies hadsome effect.Olaparibwasmost efficacious against CDX3, amodelthat failed to express HRR protein PALB2. Although damagingPALB2mutations are reported to account for outlier responses toPARP inhibitors in other cancer types (48, 49), targeted therapiesthat work in one cancer type do not necessarily work in anothercancer type, even if the genetics are similar (e.g., vemurafenib inBRAF-mutant melanoma vs. colorectal cancer; ref. 50). To ourknowledge, this is the first report correlating a PALB2mutation tocombined PARP/WEE1 inhibition in SCLC. Moreover, SCLCtumors are not typically associated with defects in HRR, a knownpredictive biomarker for PARP inhibitor sensitivity, and our datasuggest further research into this area is warranted.

Multiple studies have shown that olaparib efficacy is signifi-cantly reduced against tumors pretreated with chemotherapy. Wesought to address this by examining the efficacy of olaparib/AZD1775 against a model generated from a patient who pro-gressed on a platinum-based therapy. Consistent with patientprogression, CDX8p was resistant to treatment with platinum/etoposide. Although CDX8p was also relatively insensitive toolaparib/AZD1775, this combination elicited a more durableresponse than platinum/etoposide. Similarly, pretreatment ofCDX3 with platinum/etoposide blunted the response of chemo-naive CDX3 to olaparib/AZD1775. Given the deep, though short-lived response to patient standard-of-care platinum-based che-motherapy, it may be challenging to administer an olaparib/AZD1775 combination to treatment-na€�ve SCLC patients. How-ever, consideration should be given tominimizing the number ofchemotherapy cycles given to debulk the disease prior to testingthis novel combination.

The exquisite sensitivity of CDX3 to combination therapy islikely due to the fact that both olaparib and AZD1775 exhibited

single-agent efficacy against this model. AZD1775 can bothcompromise the ability to restrain mitotic commitment inresponse toDNAdamage andpromote catastrophicDNAdamagein G1–S via inappropriate activation of theMUS81-SLX4 nuclease(51). Thus, combinatorial activity of olaparib/AZD1775 maystem from AZD1775-induced inappropriate cleavage of stalledreplication forks stemming from PARP trapping. However, ourunderstanding of the biology of inappropriate induction of thesepathways in S phase remains in its infancy, and a detailedmechanistic understanding of what role PARP may play in thisprocess remains beyond the scope of this study.

In addition to affecting sensitivity to olaparib, the PALB2mutation could potentially explain the response of CDX3 toAZD1775. BRCA2 and PALB2 function has been linked to repli-cation fork stabilization/restart as part of the replication stressresponse. CDX3, the model that is most sensitive to AZD1775single treatment, already has significantly elevated levels of rep-licative stress as indicated by elevated levels of MYCL, cyclin E1,andpRPA comparedwith the lesser responsivemodels.Moreover,we have demonstrated that following AZD1775 treatment, thereis a further increase in pRPA. Phosphorylated RPA can recruitPALB2 and BRCA2 to the stalled fork, and PALB2 loss has beenassociated with defects in the recovery of the stalled fork andtherefore increased DNA damage (52). In addition, BRCA2 andPALB2 are key regulators of the G2–M cell-cycle checkpoint uponDNA damage (53). Therefore, the absence of PALB2 in combi-nation with the inhibition of WEE1 could increase prematuremitotic entry and therefore genomic instability in CDX3 cells.Together, these data suggest the efficacy of the olaparib/AZD1775combination is likely to involve both an increase in replicationstress response in S-phase and carryover of S-phase–induceddamage into mitosis.

Combination trials using PARP inhibitors are already in earlyphase development, including a second-line study comparingtemozolomide and veliparib with temozolomide and placebowhere the combination reported increased response rates but nosignificant difference in 4-month progression-free survival (54).The ongoing phase I/II trial of olaparib and temozolomide inpatients progressing after chemotherapy also reported a 46%response rate for the combination compared with 14% for temo-zolomide alone (55). Olaparib/AZD1775 has demonstrated pre-clinical activity against AML (56), and a phase I clinical trialutilizing olaparib and AZD1775 has been initiated in patientswith refractory solid tumors (https://ClinicalTrials.gov/show/NCT02511795), including SCLC, and an extended cohort inSCLC is planned.

Conversion of improved clinical response rates to lengthenedprogression-free and overall survival will likely require develop-ment of robust predictive biomarkers. Our preclinical CDX datasuggest that predicting the depth and duration of responses toolaparib and AZD1775 in SCLC patients is unlikely to be straight-forward; the speed with which acquired chemotherapy resistanceoccurs, the genomic instability, and heterogeneity of this devas-tating disease all point to the need for development of broadbiomarker panels for theoptimal use of this combination therapy,where different patients' tumors may acquire resistance via dif-ferent and multiple molecular mechanisms. Further characteri-zation of the DNA damage response status across a wide range ofSCLC tumors, as well as functional validation of potential bio-markers is ongoing.Our results raise thepossibility that a subset ofSCLC may have defects in DNA damage response pathways that

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could be exploited by the combination of PARP and WEE1inhibitors. Moreover, replication stress is often associated withoncogene overexpression and MYC family members, which areknown to be amplified in approximately 20% SCLC, and the roleof MYC as a predictive biomarker of response to olaparib andAZD1775 can now be studied further in our burgeoning panel of45 CDX models (57–59). The CDX3 cellular context represents a"super responder" to the olaparib/AZD1775 combination andhas revealed important molecular insights that we can now takeforward in an assessment in the larger CDX panel.

Surgical specimens and archival biopsies are unlikely to reflectthe disease after first- or second-line treatments. We previouslyreported the prevalence of CTCs in SCLC (60), their prognosticsignificance, and their potential as a readily obtained and repeat-able source of pharmacodynamic and predictive biomarkers.Moreover, mutations in a panel of genes associated with DNAdamage response measured in circulating tumor DNA may alsoassist patient selection. These liquid biopsy approaches shouldnow be fully exploited in upcoming trials of novel therapies inSCLCwhere promising data in patient-relevant preclinicalmodelsare forthcoming.

Disclosure of Potential Conflicts of InterestC. Dive reports receiving commercial research grants from AstraZeneca. C.J.

Morrow is an employee of AstraZeneca. F. Blackhall reports receiving othercommercial research support from and is a consultant/advisory board memberfor AstraZeneca. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception anddesign:K.K. Frese, C.J.Morrow, F. Blackhall,M.J. O'Connor, C.Dive

Development of methodology: A. Lallo, C.J. Morrow, C.L. Hodgkinson,M.J. O'ConnorAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Lallo, R. Sloane, M.W. Schenk, F. Trapani,M. Galvin, S. Brown, C.L. Hodgkinson, L. Priest, A. Hughes, C. MillerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Lallo, K.K. Frese, C.J. Morrow, S. Gulati, F. Trapani,N. Simms, M. Galvin, A. Hughes, Z. Lai, E. Cadogan, G. Khandelwal,K.L. Simpson, C. Miller, F. Blackhall, M.J. O'Connor, C. DiveWriting, review, and/or revision of the manuscript: A. Lallo, K.K. Frese,R. Sloane, F. Trapani, Z. Lai, E. Cadogan, G. Khandelwal, K.L. Simpson,C. Miller, F. Blackhall, M.J. O'Connor, C. DiveAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Galvin, C.L. Hodgkinson, E. CadoganStudy supervision: K.K. Frese, C.J. Morrow, M.J. O'Connor, C. Dive

AcknowledgmentsThe authors thank Matt Carter, Matt Lancashire, the CEP in vivo team,

CRUK Manchester Institute core facilities, and the patients and theirfamilies. They also thank Ian Hagan for his constructive comments onthe article.

C. Dive receives funding from the Cancer Research UK Manchester Institutecore (C5759/A27412), the Cancer Research UK Manchester Centre Award(C5759/A25254), the Cancer Research UK Lung Cancer Centre of ExcellenceAward (C5759/A20465), and the Cancer Research UK/AstraZeneca biomarkersalliance (10001080/AgrID486).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 22, 2017; revised April 5, 2018; accepted June 20, 2018;published first June 25, 2018.

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