combined antitumor therapy with metronomic...

Cancer Therapy: Preclinical

Combined Antitumor Therapy with MetronomicTopotecan and Hypoxia-Activated Prodrug,Evofosfamide, in Neuroblastoma andRhabdomyosarcoma Preclinical ModelsLibo Zhang1,2, Paula Marrano2, Bing Wu1,2, Sushil Kumar1,2, Paul Thorner2,4, andSylvain Baruchel1,3,5

Abstract

Purpose: Tumor cells residing in tumor hypoxic zones are amajor cause of drug resistance and tumor relapse. In this study, weinvestigated the efficacy of evofosfamide, a hypoxia-activatedprodrug, and its combination with topotecan in neuroblastomaand rhabdomyosarcoma preclinical models.

Experimental Design: Neuroblastoma and rhabdomyosarco-ma cells were tested in vitro to assess the effect of evofosfamide oncell proliferation, both as a single agent and in combination withtopotecan. In vivo antitumor activity was evaluated in differentxenograft models. Animal survival was studied with the neuro-blastoma metastatic tumor model.

Results: All tested cell lines showed response to evofosfa-mide under normoxic conditions, but when exposed to hyp-oxia overnight, a 2- to 65-fold decrease of IC50 was observed.Adding topotecan to the evofosfamide treatment significantlyincreased cytotoxicity in vitro. In neuroblastoma xenograftmodels, single-agent evofosfamide treatment delayed tumor

growth. Complete tumor regression was observed in thecombined topotecan/evofosfamide treatment group after 2-week treatment. Combined treatment also improved survivalin a neuroblastoma metastatic model, compared to single-agent treatments. In rhabdomyosarcoma xenograft models,combined treatment was more effective than single agents.We also showed that evofosfamide mostly targeted tumor cellswithin hypoxic regions while topotecan was more effective totumor cells in normoxic regions. Combined treatmentinduced tumor cell apoptosis in both normoxic and hypoxicregions.

Conclusions: Evofosfamide shows antitumor effects in neu-roblastoma and rhabdomyosarcoma xenografts. Comparedwith single-agent, evofosfamide/topotecan, combined therapyimproves tumor response, delays tumor relapse, and enhancesanimal survival in preclinical tumor models. Clin Cancer Res;22(11); 2697–708. �2015 AACR.

IntroductionNeuroblastoma is the most common extracranial childhood

malignancy, responsible for 15% of all childhood cancer-relateddeaths (1).Despite intensive treatment protocols includingmega-therapy with hematopoietic stem cell transplantation and immu-notherapy, 3-year disease-free survival is only about 50% formetastatic disease compared with 95% for localized tumors(2). Rhabdomyosarcoma is the most common pediatric soft

tissue sarcoma and the third most common extracranial solidtumor in children, with an annual incidence of 4 to 7 cases permillion children under the age of 16 years (3). Prognosis ofmetastatic rhabdomyosarcoma remains poor despite aggressivetherapies.

There is strong evidence that tumor cells residing in the tumorhypoxic regions cause drug resistance and tumor relapse (4).Tumor hypoxic zones, which occur in tumors due to nonuniformand inefficient vasculature, hinder the accessibility of chemother-apeutics to tumor cells. Hypoxia can be a direct cause of thera-peutic resistance due to limited blood supply and drug distribu-tion (5). Hypoxia also inhibits tumor cell proliferation andinduces cell-cycle arrest, further enhancing chemoresistancethrough regulating drug transporters, antiapoptotic proteins, andproangiogenic factors (6). Our previous studies have shown thattumor hypoxic microenvironment may serve as a niche for thehighly tumorigenic fraction of tumor cells which exhibit thecancer stem cell features in a diverse group of solid tumor celllines, including neuroblastoma, rhabdomyosarcoma, and small-cell lung carcinoma (7). We also found that hypoxia induces drugresistance to etoposide, melphalan, doxorubicin, and cyclophos-phamide in neuroblastoma cells, which is mediated through anexpanded autocrine loop between VEGF/Flt1 and HIF-1a expres-sion (8). There is evidence that some cells in hypoxic regionsremain alive in a dormant state for prolonged duration and

1New Agent and Innovative Therapy Program, The Hospital for SickChildren, Toronto, Ontario, Canada. 2Department of Paediatric Labo-ratory Medicine, The Hospital for Sick Children, Toronto, Ontario,Canada. 3Division of Hematology and Oncology, Department of Pae-diatrics, The Hospital for Sick Children, Toronto, Ontario, Canada.4Department of Laboratory Medicine and Pathobiology, University ofToronto, Toronto, Ontario, Canada. 5Institute of Medical Sciences,University of Toronto, Toronto, Ontario, Canada.

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

Corresponding Author: Sylvain Baruchel, New Agent and Innovative TherapyProgram, TheHospital for SickChildren, 555University Avenue, Toronto, OntarioM5G 1X8, Canada. Phone: 416-813-5977; Fax: 416-813-5327; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-1853

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 2697

on August 28, 2018. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 30, 2015; DOI: 10.1158/1078-0432.CCR-15-1853

http://clincancerres.aacrjournals.org/

become drug resistant as anticancer drugs preferentially targetrapidly proliferating cells (6). Hypoxia-inducible factors alsoinduce stem cell phenotype and suppress differentiation of neu-roblastoma cells (9). In rhabdomyosarcoma, HIF-1a, induced byhypoxia, upregulates antiapoptotic proteins and glycolyticenzymes (10). HIF-1a inactivation by the inhibition of PI3K/Aktpathway is reported to sensitize tumor cells to apoptosis inrhabdomyosarcoma (11). Moreover, HIF-1a stabilization as theresult of hypoxia increases VEGF expression, increases glycolysisand resistance to chemotherapy (12).

Evofosfamide (previously known as TH-302), a hypoxia-activated prodrug (HAP) has been designed to penetrate tohypoxic regions of tumors. Evofosfamide is reduced at thenitroimadazole site of the prodrug by intracellular reductasesand when exposed to hypoxic conditions, leads to the release ofthe alkylating agent bromoisophosphoramide mustard (Br-IPM). Br-IPM can then act as a DNA crosslinking agent at thetumor hypoxic region and may diffuse to adjacent normoxicregions via a bystander effect (13). Consequently, evofosfamidecan target the malignant cells residing in hypoxic zones, whilehaving little or no cytotoxic effect on the normal cells (14, 15).The antitumor efficacy of evofosfamide correlated with hypoxicfractions in tumor xenografts of lung cancer, melanoma, pan-creatic cancer, renal cell carcinoma, and hepatocellular carci-noma. In adult soft tissue sarcoma phase II trial, evofosfamidein combination with doxorubicin demonstrated 2% completeresponse (CR) and 34% partial response (PR; ref. 16). As asingle maintenance therapy, it had limited hematologic toxicityand cardiotoxicity, whereas with doxorubicin, it caused man-ageable hematologic toxicity without aggravating cardiotoxi-city. A phase III study in advanced and metastatic adult softtissue sarcoma is ongoing (NCT01440088). To date, no studiesinvestigating the efficacy of evofosfamide in pediatric cancershave been conducted.

Topotecan, a topoisomerase-I inhibitor has shown activityagainst both neuroblastoma and rhabdomyosarcoma as a sin-gle agent (17). The combination of cyclophosphamide plustopotecan has been active in rhabdomyosarcoma, neuroblas-

toma, and Ewing sarcoma patients with recurrent or refractorydisease who have not received topotecan previously (18–20).The combination of topotecan, cyclophosphamide, and etopo-side is tolerable and effective in relapsed and newly diagnosedneuroblastoma in phase II clinical trials and upfront phase IIIclinical trials (18, 21). Single-agent topotecan has also beenfound to be antiangiogenic in preclinical models of pediatriccancers (22, 23). Despite initial response, tumors eventuallyacquire resistance to topotecan. Mechanisms of resistanceinclude mutations in topoisomerase-I and involvement oftransporters like BCRP, P-glycoprotein, and multidrug resis-tance associated protein-type IV (24–26).

In this study, we selected themetronomic dose of topotecan forin vivo studies. Low metronomic dose (LDM) chemotherapyusually refers to administration of low dose of cytototoxic agentat close intervals without drug free breaks. LDM chemotherapyhas lower acute toxicity due to lower exposure of the cytotoxicagents. It has been shown active in diverse tumor types, includingmetastatic disease, especially when combined with antiangio-genic drugs (27–30). The availability of the oral topotecan, alongwith our previous studies combiningmetronomic topotecanwithpazopanib in neuroblastoma preclinical models (23), suggestthat oral metronomic topotecan may be an ideal candidate forpediatric solid tumors. Considering the limited effects on hypoxictumors with conventional chemotherapy, hypoxia-targeting cyto-toxic agents along with topotecan therapy may achieve greaterantitumor activities. Therefore, in this study, we investigated theeffect of combined therapy with topotecan and evofosfamide inneuroblastoma and rhabdomyosarcoma preclinical tumormodels.

Materials and MethodsDrugs and reagents

Evofosfamide was provided by Threshold Pharmaceuticals Inc.Topotecan HCl was obtained from Selleck (S1231). Stock solu-tions were prepared in DMSO and aliquots were stored at�80�C.They were further diluted with culture media or saline for in vitroand in vivo studies, respectively.

Cells and cell cultureRH4, RH30, and RD rhabdomyosarcoma cells (31) were gifts

from Dr. David Malkin (The Hospital for Sick Children, Toronto,Ontario, Canada). LAN-5 (32), SK-N-BE(2) (33), BE(2)C (34),and SH-SY5Y (34) neuroblastoma cells were kindly provided byDr. Herman Yeger (The Hospital for Sick Children, Toronto,Ontario, Canada). CHLA-15, CHLA-20, and CHLA-90 (35) wereobtained from the Children's Oncology Group (COG) Cell Cul-ture and Xenograft Repository under a signed and approvedMaterial Transfer Agreement. Cell line authentication was per-formed using short tandem repeats (STR) DNA profiling (Pro-mega's GenePrint 10 System; ref. 36) conducted by the GeneticAnalysis Facility at the Centre for Applied Genomics of TheHospital for Sick Children (Toronto, Ontario, Canada). The DNA(STR) profile for all cell lines match to the profile listed in theChildren's Oncology Group (COG) STR Database (http://strdb.cogcell.org). RH4, RH30, and RD rhabdomyosarcoma cells werecultured in DMEM supplemented with 10% FBS. CHLA-15,CHLA-20, and CHLA-90 neuroblastoma cells were cultured inIscove's modified Dulbecco's medium supplemented with 3mmol/L L-glutamine, insulin, and transferrin 5 mg/mL each and

Translational Relevance

Tumor hypoxia is known to correlate with increased malig-nancy, metastatic potential, drug resistance, and poor patientprognosis in a number of tumor types. Targeting tumorhypoxia with hypoxia-activated prodrugs (HAP), such as evo-fosfamide, may improve cancer therapy. So far, the effect ofevofosfamide has been studied in combination with cytotoxicagents in adult cancers,whereas the impact of evofosfamide onpediatric cancers has not been investigated. This study focuseson the preclinical evaluation of efficacy of evofosfamide andits combination with topotecan, a topoisomerase I inhibitorwidely used in recurrent neuroblastoma and rhabdomyosar-coma. We observed that targeting tumor hypoxia with HAPsimproves antitumor effects of topotecan in our tumormodels,which provides a new therapeutic approach for the treatmentof pediatric solid tumors. All data gathered from this studywillhelp us build the rationale for future phase I clinical trials intreating patients with relapsed/refractory neuroblastoma andrhabdomyosarcoma.

Zhang et al.

Clin Cancer Res; 22(11) June 1, 2016 Clinical Cancer Research2698




5 ng/mL selenous acid (ITS Culture Supplement; CollaborativeBiomedical Products) and 20% FBS (complete medium). SK-N-BE(2) and SH-SY5Y neuroblastoma cells were cultured in AMEMwith 10% FBS.

Test animalsNOD/SCIDmice (Jackson Laboratory) were used for xenograft

mouse models at 4 weeks of age. The mice were housed in anisolated sterile containment facility. All animal studies wereapproved by Animal Care Committee at the Hospital for SickChildren (Animal Use Protocol#1000019356). During the study,the mice were observed daily for possible adverse effects due totreatments. Morbidity signs of ill health, such as ruffled/thinningfur, abnormal behaviors, or local erosion from the tumor werenoted and experiments terminated as indicated.

Cell viability assayCell viability was assessed by Alamar Blue assay as previously

described (37). Alamar Blue cell proliferation assay is based ona reducing environment that indicates metabolically activecells. Briefly, exponentially growing tumor cells was seededinto 48-well plates at 1 � 105 cells per well and grownovernight prior to initiating treatment. On the day of the test,cells were exposed to increasing concentrations of evofosfa-mide and topotecan either as single agents or combined treat-ment for 72 hours. Alamar Blue was added to a final concen-tration of 5%, incubated for 4 hours at 37�C and quantitated ona plate reader at 530/590 nm. IC50 was determined by Graph-Pad Prism software.

We also evaluated drug efficacy under the condition ofhypoxia. To simulate tumor hypoxia in vitro, after drug addi-tion, the plates were incubated for 2 hours at 37�C in ananaerobic chamber. The anaerobic chamber was evacuated andgassed with the hypoxic gas mixture (94% N2/5% CO2/1% O2)to create a hypoxic environment. After 2 hours or overnightexposure to hypoxic condition, cells were removed from thehypoxia chamber and further incubated for 3 days in standardtissue culture incubator. Cell viability was assessed by AlamarBlue assay as described above.

Mouse xenograft modelsTwo neuroblastoma cell lines [CHLA-20 and SK-N-BE(2) and

two rhabdomyosarcoma cell lines (RH4 and RD) were used toestablish murine models (38). Briefly, tumor cells were washedthree times with Hank balanced salt solution before injection.Cell suspensions were mixed 1:1 with Matrigel (BD Bios-ciences). Subcutaneous xenografts were developed by injecting1 � 106 tumor cells into the subcutaneous fat pad of NOD/SCID mice. Treatments commenced when the tumor sizesreached about 0.25 cm3.

Animals were randomized into four groups with 10 animals ineach group: control; evofosfamide, topotecan as a single agent,and the combination of evofosfamide and topotecan. The dosesof evofosfamide and topotecan were 50 mg/kg (every day � 5days/week, i.p.) and 1 mg/kg (every day � 5 days/week by oralgavage), respectively. The injection or gavage volume was 200 mL.Control mice received the same volume of the vehicle (saline).Tumor growth was measured three times a week in two dimen-sions, using a digital caliper. Tumor volume was calculated aswidth2� length�0.5.When tumor volume reached1.5 cm3,mice

were sacrificed, and tumors were dissected and weighed. Tumorgrowth curves were plotted with the relative tumor volumes atdifferent time points. The relative tumor volume of each tumor isdefined as the tumor volume divided by its initial volume.Potential drug toxicity was assessed in tumor-bearing mice bymonitoring animal body weight, appetite, diarrhea, signs ofanimal distress, or any marked neurologic symptoms.

Animal survival studySK-N-BE(2) cells were prepared and injected intravenously

into the lateral tail vein (26-gauge needle, 1 � 106 cells in 100mL total volume). Mice were randomized into four groups:control, evofosfamide, topotecan, and the combination ofevofosfamide and topotecan, with eight mice in each group.All the treatments were initiated 14 days after tumor cellinoculation at the the same schedule and dosage as above.Animals were monitored for body weight and any signs ofstress. Tumor-bearing mice were euthanized at the endpointwhen there were signs of distress, including 20% of bodyweight loss, fur ruffling, rapid respiratory rate, hunched pos-ture, reduced activity, and progressive ascites formation.

IHCDouble immunofluorescence staining was performed on

5-mm-thick frozen sections. Tissue sections were fixed for 10minutes in acetone and allow to air dry for 5 minutes. Endog-enous biotin, biotin receptors, and avidin sites were blockedwith the Avidin/Biotin Blocking Kit (SP-2001, Vector Labora-tories). The tissue sections were incubated with rabbit anti-cleaved caspase-3 antibody (1:50; #9661, Cell Signaling Tech-nology) or rabbit anti-Ki67 antibody (1:200; ab16667, Abcam)for 1 hour at room temperature. Detection of the rabbit anti-bodies was performed by incubation with Texas Red goat anti-rabbit IgG antibody (1:200; TI-1000, Vector Laboratories) for30 minutes. After the washing of the tissue sections, the mouseIgG1 anti-pimonidazole mAb clone 4.3.11.3 (1:50; Hypoxyp-robe, Inc.) was added overnight at 4�C. This antibodywas detected with Fluorescein horse anti-mouse IgG Antibody(1:200; #FI-2000, Vector Laboratories) for 30 minutes.The tissue sections were washed and then mounted withVectashield mounting medium with DAPI (H-1200, VectorLaboratories).

Statistical analysisData from different experiments were presented asmean� SD.

For statistical analysis, Student t test for independent means wasused. A P value of <0.05 was considered significant. To comparethe effects of different treatments on tumor growth in vivo, one-way ANOVA with Dunnett multiple comparison test was used.Survival curve comparisons were performed using GraphPadPrism software for Kaplan–Meier survival analysis. Statisticaldifferences between the various groups were compared pair-wiseusing log-rank (Mantel–Cox) analysis.

Combination synergism was analyzed with MacSynergy IIsoftware (kindly provided by M.N. Prichard) with 95% confi-dence limits according to the Bliss independencemodel (39). Theeffect attributed to combination is determined by subtracting theexperimental values from theoretical additive values defined bythe equation: Exy ¼ Ex þ Ey� ExEy, where Exy is the additive effectof drugs x and y as predicted by their individual effects Ex and Ey.

Preclinical Study of Evofosfamide and Topotecan

www.aacrjournals.org Clin Cancer Res; 22(11) June 1, 2016 2699




ResultsImproved antiproliferation effects of evofosfamide underprolonged hypoxic conditions with neuroblastoma andrhabdomyosarcoma cell lines

We used a panel of neuroblastoma and rhabdomyosarcomacell lines that represented heterogeneity and different stages oftherapy, including five neuroblastoma [CHLA-15, CHLA-20,CHLA-90, SK-N-BE(2), and SH-SY5Y] and three rhabdomyosar-coma cell lines (RH4, RH30, and RD), to assess the effects ofevofosfamide on cell proliferation in vitro. SH-SY5Y is MYCNnonamplified. CHLA-15 and CHLA-20 cell lines are MYCN non-amplified cell lines obtained from tumors of treatment-na€�ve andchemotherapy-treated neuroblastoma patients, respectively. BothCHLA-90 (MYCNnonamplified) and SK-N-BE(2) (MYCN-ampli-fied) cell lines are obtained from bone marrow metastases ofpatients who relapsed after chemotherapy with mutant p53 (35).RH4 and RH30 cell lines are alveolar rhabdomyosarcoma celllines, whereas RD is an embryonal rhabdomyosarcoma cell line.

All these cell lines were exposed to increased concentrationsof evofosfamide in vitro for 72 hours. Under normoxic condi-tion, all the tested lines responded to evofosfamide treatmentin a dose-dependent manner, with IC50 values ranging from4.6 to 151 mmol/L. When exposing neuroblastoma and rhab-domyosarcoma cells under hypoxic conditions (1% O2) for2 hours, no significant IC50 change was documented. However,when tumor cells were exposed to hypoxia (1% O2) overnight,there was a 2- to 65-fold decrease of evofosfamide IC50 (Fig. 1Aand B; Table 1) with the IC50 values ranging from 0.07 to21.9 mmol/L.

Improved antiproliferation effects of evofosfamide whencombined with topotecan in vitro

In our previous studies, we showed that maximal plasmaconcentration (Cmax) of topotecan was 19.8 ng/mL with themetronomic oral dose of 1 mg/kg drug administration inNOD/SCID mouse model (23). In patients, Cmax of topotecan

Figure 1.Increased cytotoxicity of evofosfamide under prolonged hypoxic conditionswith neuroblastoma and rhabdomyosarcoma cells. A panel of 5 different neuroblastomacell lines [CHLA-15, CHLA-20, CHLA-90, SK-N-BE(2), and SH-SY5Y;A] and 3 rhabdomyosarcoma cell lines (RH4, RH30, andRD; B)were selected to assess the effectsof evofosfamide on cell proliferation in vitro. Cell viability was measured by Alamar Blue assays after exposing tumor cells to increasing concentrations ofevofosfamide in vitro for 72 hours under both normoxic and two hypoxic conditions, 1% O2 for 2 hours or 1% O2 overnight. Dose–response curves were fit to the dataand IC50 values were calculated by GraphPad Prism software.

Zhang et al.





is 10.6 � 4.4 ng/mL with the oral dose of 2.3 mg/m2/day. As theterminal half-life of oral topotecan (3.9 � 1.0 hours; ref. 40) issignificantly longer than evofosfamide (0.6� 0.1 hours; ref. 41), aconstant concentration of topotecan (20 nmol/L ¼ 8.4 ng/mL)was selected for in vitro combined treatment.

All selected tumor cell lines were treated in vitro withincreasing concentrations of evofosfamide with or without thepresence of topotecan (20 nmol/L). The dose–response curveswere generated by GraphPad software and IC50 values werecompared between IC50 concentrations of evofosfamide whentested alone and in combination with 20 nmol/L topotecan inneuroblastoma and rhabdomyosarcoma cell lines. As shownin Tables 1 and 2, with the presence of topotecan, evofosfa-mide-induced cytotoxicity was significantly enhanced under

overnight hypoxia as indicated by decreased IC50s in mosttested tumor cell lines.

To understand whether the combination of the drugs wasadditive or synergistic, a Bliss analysis was performed with Mac-Synergy II software with 95% confidence limits. The effect of thecombination was additive in all cell lines.

Topotecan and evofosfamide delay tumor growth and enhanceanimal survival in neuroblastoma model

After verifying in vitro activity of evofosfamide and topotecan,the in vivo effectiveness of topotecan/evofosfamide as singleagents or combination therapy was evaluated in CHLA-20 andSK-N-BE(2) xenograft models. As both CHLA-20 and SK-N-BE(2) cell lines are derived from samples of patients who relapsedafter chemotherapy, we use them to develop aggressive tumormodels for rigorous screening of anticancer agents. All treat-ments commenced when the tumor sizes reached 0.25 cm3. Inboth neuroblastoma models, after 2 weeks of treatment, tumorgrowth delay was observed with evofosfamide or topotecan asmonotherapies (Fig. 2A). In SK-N-BE(2) xenograft model, com-bined regimen induced complete tumor regression, while topo-tecan induced partial tumor regression. When we comparedthe tumor sizes between these two arms, the difference isstatistically significant (P < 0.05) at different time points (fromday 14 to day 33). In CHLA-20 xenograft model, both single-agent topotecan and combined treatment induced completetumor regression, but all those tumors relapsed after drugtreatment was stopped. Therefore, we compared tumor recur-rence between single-agent topotecan and combination groupsafter we stopped drug treatment. As shown in Figure 2B, thecombination group had significant longer recurrent-free inter-val compared with single-agent topotecan group (P < 0.05). Fortopotecan-treated mice, median time of recurrence was 30 days,while median time of recurrence for combined therapy waspostponed to 57 days. In addition, when we rechallenged thoserelapsed tumors with the combined evofosfamide/topotecantherapy, those tumors remained responsive with the originaltreatment schedule (Fig. 2A).

Treatment was well tolerated, with weight loss >10%observed in single-agent and combined treatment groups with-in the first 8 days but returned to baseline thereafter. No animaldeath was observed in any of the groups during the experi-mental period (Fig. 2C).

To determine the antitumor activities of evofosfamide andtopotecan on late-stage metastatic disease, we further assessanimal survival in the SK-N-BE(2) intravenous metastatic model.

Table 1. Comparison of IC50 concentrations of evofosfamide when tested aloneand in combination with 20 nmol/L topotecan in neuroblastoma andrhabdomyosarcoma cell lines

IC50 (mmol/L)

EvofosfamideEvofosfamide þTopotecan P

Neuroblastoma cell linesCHLA-15-normoxia 4.6 � 5.8 3 � 3.5 0.39CHLA-15-1% O2; 2 hours 9.5 � 6.3 7.4 � 4.1 0.57CHLA-15-1% O2; O/N 0.07 � 0.03 0.04 � 0.01 <0.01CHLA-20-normoxia 8.9 � 2.7 1.2 � 0.6 <0.01CHLA-20-1% O2; 2 hours 5.6 � 1.1 0.83 � 0.12 <0.01CHLA-20-1% O2; O/N 0.16 � 0.01 0.1 � 0.01 <0.01CHLA-90-normoxia 13.9 � 4.3 4.9 � 2.5 <0.05CHLA-90-1% O2; 2 hours 10.7 � 2.7 3.5 � 1.3 <0.05CHLA-90-1% O2; O/N 0.47 � 0.10 0.24 � 0.03 <0.01SK-N-BE(2)-normoxia 51.9 � 9.3 46.2 � 16.9 0.55SK-N-BE(2)-1% O2; 2 hours 54.9 � 5.8 49 � 15.6 0.47SK-N-BE(2)-1% O2; O/N 2.4 � 0.59 1.7 � 0.47 0.22SH-SY5Y-normoxia 66.1 � 9.2 57.5 � 13.8 <0.05SH-SY5Y- 1% O2; 2 hours 66.1 � 5.9 39.8 � 5.3 <0.01SH-SY5Y- 1% O2; O/N 1.3 � 0.23 0.5 � 0.07 <0.01

Rhabdomyosarcoma cell linesRH4-normoxia 20.9 � 1.4 16.2 � 1.1 <0.01RH4-1% O2; 2 hours 24 � 1.1 13.8 � 1.7 <0.01RH4-1% O2; O/N 3.3 � 0.27 1.7 � 0.22 <0.01RH30-normoxia 151 � 34.7 120 � 6.5 <0.01RH30-1% O2; 2 hours 115 � 3.8 95.5 � 2.7 <0.01RH30-1% O2; O/N 21.9 � 8.2 15.1 � 1.5 <0.01RD-normoxia 19.1 � 4.9 11 � 2.0 <0.01RD-1% O2; 2 hours 18.2 � 4.7 11 � 1.9 <0.01RD-1% O2; O/N 9.55 � 0.33 4.07 � 0.15 <0.01

NOTE: P values represent statistically significant differences using the extrasum-of-squares F test.

Table 2. IC50 of topotecan in neuroblastoma and rhabdomyosarcoma cell lines

IC50 (nmol/L)Normoxia 1% O2, 2 hours 1% O2, O/N Pa

Neuroblastoma cell linesCHLA-15 17.6 � 3.9 15.6 � 0.9 13.7 � 1.4 NSb

CHLA-20 12.9 � 1.2 12.4 � 1.0 14.1 � 0.6 NSCHLA-90 4,170 � 1,030 4,550 � 1,590 5,260 � 724 NSSK-N-BE(2) 96.2 � 11.8 110 � 13.8 95.0 � 14.3 NSSH-SY5Y 19.2 � 7.0 12.4 � 1.1 14.1 � 0.6 NSRhabdomyosarcoma cell linesRH4 132 � 10.0 110 � 21.8 119 � 3.7 NSRH30 2,251 � 685 1,963 � 416 1,621 � 174 NSRD 387 � 55.8 250 � 87.5 317 � 22.9 NSaP value from one-way ANOVA.bNS, not significant, P > 0.05






Figure 2.Antitumor effects of evofosfamide and topotecan in neuroblastoma xenograftmodels. A, a total of 1� 106 cellswere implanted subcutaneously into SCID/SCIDmice.Once tumors were palpable (about 0.25 cm3), mice were randomized into vehicle control (n ¼ 10), or three treatment groups (n ¼ 10 for each group). Micebearing human neuroblastoma [SK-N-BE(2) and CHLA-20] xenografts were treated for 2 weeks with evofosfamide (50 mg/kg; every day � 5 days/week, i.p.)or topotecan (1 mg/kg; every day � 5 days/week, orally) as single agents or in combination. The shaded area indicates the period of drug treatment. Tumorgrowth curveswere plottedwith the tumor volumes at different time points. One-wayANOVAwas used to compare tumor volumesbetween experimental groups atdifferent time points during the experimental period (� , P < 0.05; �� , P < 0.01). Data are expressed as mean � SEM. After 1 cycle of therapy, the combinationtreatment with evofosfamide and topotecan led to complete tumor regression in both SK-N-BE(2) and CHLA-20 xenograft models. In the CHLA-20 model, tumorrecurrence was observed in the combined treatment group. On day 55, we rechallenged the tumor bearing animals with the original treatment dosage andschedule on day 55 (identified by shaded area between day 55 and 69).All the relapsed tumors remained responsive to combined treatment. B, recurrence-freeinterval curveswere estimated in CHLA-20 xenograft model by the Kaplan–Meier method and compared between topotecan and combination of evofosfamide andtopotecan treatment using the log-rank (Mantel–Cox) analysis. The tumor-free interval was defined from the end of treatment to the first appearance ofa palpable mammary tumor at least 100mm3 in size. � , P < 0.05. C, mouse body weight wasmeasured in CHLA-20 tumor-bearingmice in different treatment groupsand control animals. Percentage of body weight loss was calculated and plotted to monitor the potential drug toxicity. D, Kaplan–Meier survival curves ofthe SK-N-BE(2) metastatic tumor model. NOD/SCID mice (n ¼ 8/group) were injected with 1 � 106 SK-N-BE(2) cells intravenously. Fourteen days after tumor cellinoculation, mice were treated for 2 weeks with control vehicle, evofosfamide, topotecan, or the combination of evofosfamide and topotecanwith the same dose asindicated above. Statistical differences between the various groups were compared pair-wise using log-rank (Mantel–Cox) analysis. � , P < 0.05.

Zhang et al.





Fourteen days after tumor cell inoculation, we initiated drugtreatment with evofosfamide and topotecan as single agents orcombined treatment. As shown in Fig. 2D, evofosfamide ortopotecan treatment showed a substantial increase in animalsurvival compared with the control mice with a median survivalof 22.5 days for the control group, 25 days for the evofosfamidegroup, and 36 days for the topotecan group. However, treatmentwith a combination of evofosfamide and topotecan had thegreatest impact on animal survival (P < 0.01) with a mediansurvival of 46 days.

Topotecan and evofosfamide delays tumor growth inrhabdomyosarcoma models

To test the antitumor activity of evofosfamide and topotecan inrhabdomyosarcoma, we chose an alveolar rhabdomyosarcomacell line (RH4) and an embryonal rhabdomyosarcoma cell line(RD) to generate xenograft models. Tumor bearing NOD/SCIDmice were randomized to therapy with evofosfamide, topotecan,or combined therapy. In both RH4 and RD xenograft models,both evofosfamide and topotecan monotherapy significantlydelayed tumor growth with 2 weeks of treatment (Fig. 3). Com-pared with neuroblastoma models, single-agent evofosfamidewas more effective in rhabdomyosarcoma models with partialtumor regression in all treated xenografts. The combination oftopotecan and evofosfamide were superior to the respective drugsgiven alone at different time points from day 26 to day 30 (P <0.01; Fig. 3). There was no significant drug-related toxicity in anyof the treatment arms.

Colocalization of cell apoptosis and tumor hypoxia withevofosfamide and topotecan treatment

As combined treatment with evofosfamide and topotecansignificantly improved antitumor activity both in vitro and in vivo,we have particular interest to examine its potential mechanism ofaction in vivo. To this end, tumor cell apoptosis was assessed bycleaved caspase-3 immunoreactivity analysis in our xenograftmodels. On day 5 of drug treatment, RH4 xenografts from

different regimens were harvested for immunohistochemicalanalysis (Fig. 4A). Hypoxic regions were identified as the area ofpimonidazole-positive staining. Apoptotic cells were detected byanti-cleaved caspase-3 antibody. Apoptosis was quantified as thepercentage of positive cleaved caspase-3 staining area using Ima-geJ software (Fig. 4B). As shown in Fig. 4A and B, evofosfamideinduced tumor cell apoptosis in hypoxic regions, with a remark-ably higher percentage of cleaved caspase-3–positive cells inhypoxic regions compared with normoxic regions (Student t test,P < 0.01). On the contrary, topotecan inducedmore extensive cellapoptosis in normoxic regions than hypoxic regions (P < 0.05).With the combination treatment, significant tumor cell apoptosiswas observed throughout the tumor, in both normoxic andhypoxic areas.

Decreased cell proliferation with evofosfamide and topotecancombined treatment in vivo

We further evaluated tumor cell proliferation in differenttreatment groups by immunostaining of the proliferationmarker,Ki67. As shown in Fig. 4C and D, the Ki67þ proliferating tumorcell population wasmainly located in normoxic regions. After the4-day in vivo treatment, single-agent topotecan showed a limitedeffect on cell proliferation as measured by Ki67 positivity. Sur-prisingly, decreased cell proliferation was observed in evofosfa-mide-treated tumors in normoxic regions with significantly lowerpercentage of Ki67 positive cells than control tumors (P < 0.05),suggesting a hypoxia-independent bystander effect. Combinedevofosfamide and topotecan treatment led to a further decrease inthe Ki67þ-proliferating tumor cell population compared withsingle-agent evofosfamide group (combined group vs. single-agent topotecan, P < 0.01; combined group vs. single-agentevofosfamide, P < 0.05).

DiscussionTopotecan has demonstrated efficacy in preclinical and clinical

studies in neuroblastoma and rhabdomyosarcoma. However,total eradication of highly tumorigenic populations of tumor

Figure 3.Effects of evofosfamide and topotecan on the growth of rhabdomyosarcoma xenografts. A total of 1� 106 cellswere implanted subcutaneously into SCID/SCIDmice.Once tumors were palpable (about 0.25 cm3), mice were randomized into vehicle control group (n ¼ 10) or three treatment arms (n ¼ 10 for each group).RH4 andRD xenograftswere treatedwith evofosfamide (50mg/kg; every day� 5 days/week, i.p.) or topotecan (1mg/kg; every day� 5 days/week, orally) as singleagents or in combination. The shaded area indicates the period of drug treatment. Tumor growth was monitored as described above. One-way ANOVA wasused to compare tumor volumes between experimental groups at different timepoints during the experimental period (� ,P<0.05; ��,P<0.01). Data are expressed asmean � SEM.






cells is required to achieve relapse-free survival in patients withcancer. Incomplete distribution of chemotherapeutics to poorlyperfused areas of primary tumors and metastatic sites is a majorhindrance in complete eradication of these tumor cells. Thesesparsely vascularized areas are characterized by hypoxia whichconfers drug and radiation resistance, and a proangiogenic andinvasive phenotype to tumor cells. Despite the advent of novelchemotherapies, complete eradication of tumor cells cannot beaccomplished until hypoxic tumor cells are targeted. Therefore,targeting hypoxic zone of solid tumor may reverse drug resis-tance and make cytotoxic agent more effective. In this study, weare proposing new strategies to improve the efficacy of chemo-therapy by incorporating a novel agent targeting tumor hypoxicregions.

HAPs, which deliver the cytotoxic payload in hypoxic zones,have been developed to solve this issue. PR-104, an earlier HAP,was found to have a steep dose–response relationship in apanel of pediatric tumor xenograft models, which raises safetyconcerns in pediatric patients (42). Evofosfamide, however, hasdemonstrated survival benefit in various adult cancers withoutcausing prohibitively additive toxicity with conventional che-motherapy in clinical trials (16). Evofosfamide shares thesimilar active metabolites as ifofosfamide, but it is preferen-tially activated in hypoxic tissues and does not have the sametoxic metabolites as ifosfamide. It is anticipated that evofosfa-mide should have less hematologic, renal, and CNS toxicitythan ifosfamide. There is no direct clinical studies comparingevofosfamide with ifosfamide, but evofosfamide shows

Figure 4.Localization of tumor cell apoptosis,proliferation, and tumor hypoxia afterevofosfamide and topotecantreatment. RH4 xenografts fromdifferent regimens were harvested 4days after drug treatment forimmunohistochemical analysis.Hypoxic regions were identified as thearea of pimonidazole-positive staining(circled area). A, apoptotic cells weredetected by anti-cleaved caspase-3antibody. B, Ten high-power fields pertreatment arm were quantified for cellapoptosis in both tumor hypoxic andnormoxic regions expressed bypercentage of positive cleavedcaspase-3 staining area using ImageJsoftware. Statistical analysis was doneby t test (� , P < 0.05; �� , P < 0.01).Error bars, SD. (Continued on thefollowing page.)

Zhang et al.





superior efficacy and less toxicity than ifosfamide in preclinicallung carcinoma models (43). In our xenograft models, weobserved tumor growth inhibition with single-agent evofosfa-mide, but no significant antitumor effect was achieved in theCHLA-20 model with ifosfamide (50 mg/kg; every day � 5days/week, i.p.; Supplementary Fig. S1). In clinical trials(NCT02047500), evofosfamide is administered at a dose rang-ing from 170 to 340 mg/m2. This could be converted to 32–64mg/kg in the tumor bearing mice, calculated by the pharma-cokinetic data from the clinical studies and preclinical mousestudies (44). In this study, we tested evofosfamide at the doseof 50 mg/kg, as single agent and in combination with topotecanin two of the most common pediatric extracranial solid tumors,neuroblastoma and rhabdomyosarcoma. To our knowledge,this is the first study evaluating the effectiveness of evofosfa-mide in pediatric tumors.

Our observation that single-agent topotecan ismore effective indelaying tumor growth in neuroblastoma than in rhabdomyo-sarcoma is in agreement with our previous finding (23). Fromourobservations in vitro, evofosfamide showed more activity in neu-roblastoma cell lines than in rhabdomyosarcoma cells. However,in vivo, rhabdomyosarcoma xenografts were more sensitive toevofosfamide than neuroblastoma xenografts. In this study, wecannot simply translate in vitro observation to in vivo activity. Invitro, we simulated tumor hypoxia by incubating tumor cellsinside the hypoxia chamber. Therefore, 100% of cell populationwas under hypoxia and exposed to activated evofosfamide. In vivo,the percentage of hypoxic tumor cells varies among differenttumor types (45, 46). In vivo tumor microenvironment is alsomore complex in which HAP activation would be affected bymany other factors, including the duration and the intensity ofoxidative stress, tumor angiogenesis, drug penetration in different

Figure 4.(Continued. ) C, immunohistochemicalstaining of Ki67 was performed toidentify proliferating cells in differentgroups of xenografts. D, cellproliferation in normoxic regions wasquantified by percentage of positiveKi67 staining area using ImageJsoftware. Statistical analysis was doneby t test (� , P < 0.05; �� , P < 0.01). Dataare means � SEM.






solid tumor types, and acquired drug resistance under prolongedhypoxic conditions. Although it was beyond the scope of thisstudy to examine the tumor microenvironment among differenttumor types, it remains an important area for our futureinvestigation.

From our study, evofosfamide showed enhanced cytotoxicityunder overnight and 3-day (Supplementary Fig. S2) prolongedhypoxia compared with short-term (2-hour) hypoxia exposure.Whenwe test the effect of evofosfamide and its combination withtopotecan on five neuroblastoma and three rhabdomyosarcomacell lines under normoxia, short-term hypoxia and prolongedhypoxia, it is also under overnight hypoxia (1% O2) that addingtopotecan significantly lowered IC50 of evofosfamide in mosttumor cell line, except SK-N-BE(2). However, in the SK-N-BE(2)xenograft model, combined therapy achieved significant antitu-mor effects in vivo. Combined treatment induced complete tumorregression in all treated animals, which was superior to evofosfa-mide or topotecan single-agent treatment. This is likely becauseSK-N-BE(2) xenografts have more severe and more chronic hyp-oxic environment than in vitro cell culture (1% O2, overnight),which could more effectively activate evofosfamide to exert itscytotoxic activity in vivo.

In all the neuroblastoma and rhabdomyosarcoma xenograftmodels tested, combined treatment showed superior antitumoreffects compared with single-agent treatments, and inducedtumor regression after a 2-week treatment period. The advantageof the combination regimen was also approved in the metastaticneuroblastoma model. Our experimental metastatic model hasbeen developed to simulate residual disease in neuroblastoma.Residual disease refers to disseminated tumor cells which surviveafter therapy (47). More than 50% of children with high-riskneuroblastoma develop recurrence due to the presence of min-imal residual disease. In our metastatic model derived from adisseminated MYCN-amplified cell line, mice treated with com-bined topotecan and evofosfamide had extended survival com-pared with single-agent topotecan treatment. This result is signif-icant in promoting new therapies for neuroblastoma minimalresidual disease or refractory neuroblastoma by combining HAPswith existing chemotherapeutic drugs. We believe that incorpo-ratingHAPs, such as evofosfamide, could potentially eradicate thetumor cells hiding in hypoxic zones which are largely responsiblefor eventual relapse and metastasis. Eventually, this new regimenmight prolong progression-free survival in high-risk neuroblas-toma patients.

In this study, we were able to assess the correlation betweentumor hypoxia and tumor cell apoptosis with immunostaining ofhypoxic marker pimonidazole and apoptoic marker cleaved cas-pase-3. As expected, in evofosfamide-treated tumors, apoptoticcells were mostly confined to hypoxic zones while those intopotecan-treated tumors were located in normoxic areas. Homo-geneous population of tumor cells undergoing apoptosis wasobserved in bothnormoxic andhypoxic regions,which supportedthe mechanism of synergistic efficacy of the combination therapyin vivo. In a previous study, the accessibility of topotecan, doxo-rubicin, and mitoxantrone was found to decrease with increasingdistance from functional blood vessels in breast cancer xenografts(48). Clearly, poor drug penetration into solid tumors limits drugefficacy. In our study, we achieved tumor cytotoxicity in bothnormoxic and hypoxic regions by combining evofosfamide withtopotecan, which explains the superior efficacy of our combina-tion regimen in all tumor models.

An ideal HAP requires selective activation in hypoxic regions aswell as good penetration of multiple cell layers to exert a localcytotoxic bystander effect. Using a multicellular tumor spheroidand multicellular layer (MCL) coculture model systems, Mengand colleagues (49) showed that evofosfamide has penetrabilityand bystander effect. In our study, with evofosfamide single-agenttreatment, we observed decreased cell proliferation even at nor-moxic regions. This phenomenon confirms that once activated inhypoxic tissues, evofosfamide releases Br-IPM which can diffuseinto surrounding oxygenated regions of the tumor and kill cells inthose regions via a "bystander effect."

In summary, topotecan and evofosfamide demonstrated anti-tumor activities in our preclinical models of various subtypes ofneuroblastoma and rhabdomyosarcoma. Compared with singleagents, combination treatment induces more tumor regression,delays tumor relapse, and enhances animal survival in our pre-clinical tumor models. In this study, we explored the mechanismof action with combined evofosfamide/topotecan therapy. Weobserved increased reactive oxygen species (ROS) under hypoxiccondition (1% O2, overnight; Supplementary Fig. S3). We alsofound that adding topotecan enhanced the oxidative stress inrhabdomyosarcoma cells under hypoxic condition (Supplemen-tary Fig. S4). In vivo, evofosfamide showed significant cytotoxicityagainst hypoxic tumor cells, while topotecan plays complemen-tary roles by targeting tumor cells residing at normoxic regions.The ability of this combination to target cells in both normoxicand hypoxic tumor zones, as demonstrated by immunofluores-cence, provides a proof-of-principle for its superior efficacy. Thesepreclinical data support the development of a clinical trial in high-risk neuroblastoma and rhabdomyosarcoma.

Disclosure of Potential Conflicts of InterestS. Baruchel reports receiving commercial research grants fromMerck TH302.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Zhang, S. BaruchelDevelopment of methodology: L. Zhang, P.S. Thorner, S. BaruchelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Zhang, P. Marrano, P.S. Thorner, S. BaruchelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Zhang, P. Marrano, B. Wu, S. Kumar, P.S. Thorner,S. BaruchelWriting, review, and/or revision of the manuscript: L. Zhang, P. Marrano,S. Kumar, P.S. Thorner, S. BaruchelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Zhang, P. Marrano, B. Wu, S. BaruchelStudy supervision: L. Zhang, S. Baruchel

The costs of publication of this articlewere defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Grant SupportThis work was supported by Threshold Pharmaceuticals, Merck-Serono,

James Birrell Neuroblastoma Research Fund and CJ Memorial Fund/Sick KidsFoundation.

The costs of publication of this article were defrayed in part by the paymentofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 31, 2015; revised December 4, 2015; accepted December 17,2015; published OnlineFirst December 30, 2015.

Zhang et al.





References1. Howman-Giles R, Shaw PJ, Uren RF, Chung DK. Neuroblastoma and other

neuroendocrine tumors. Semin Nucl Med 2007;37:286–302.2. Ora I, Eggert A. Progress in treatment and risk stratification of neuroblas-

toma: impact on future clinical and basic research. Semin Cancer Biol2011;21:217–28.

3. Huh WW, Skapek SX. Childhood rhabdomyosarcoma: new insight onbiology and treatment. Curr Oncol Rep 2010;12:402–10.

4. Vaupel P. The role of hypoxia-induced factors in tumor progression.Oncologist 2004;9(Suppl 5):10–7.

5. Durand RE. The influence of microenvironmental factors during cancertherapy. In Vivo 1994;8:691–702.

6. Brown JM. Tumor hypoxia, drug resistance, and metastases. J Natl CancerInst 1990;82:338–9.

7. Das B, Tsuchida R, Malkin D, Koren G, Baruchel S, Yeger H. Hypoxiaenhances tumor stemness by increasing the invasive and tumorigenic sidepopulation fraction. Stem Cells 2008;26:1818–30.

8. Das B, Yeger H, Tsuchida R, Torkin R, GeeMF, Thorner PS, et al. A hypoxia-driven vascular endothelial growth factor/Flt1 autocrine loop interactswithhypoxia-inducible factor-1alpha through mitogen-activated proteinkinase/extracellular signal-regulated kinase 1/2 pathway in neuroblasto-ma. Cancer Res 2005;65:7267–75.

9. Jogi A, Ora I, Nilsson H, Lindeheim A, Makino Y, Poellinger L, et al.Hypoxia alters gene expression in human neuroblastoma cells toward animmature and neural crest-like phenotype. Proc Natl Acad Sci U S A2002;99:7021–6.

10. Kilic M, Kasperczyk H, Fulda S, Debatin KM. Role of hypoxia induciblefactor-1 alpha in modulation of apoptosis resistance. Oncogene2007;26:2027–38.

11. Kilic-Eren M, Boylu T, Tabor V. Targeting PI3K/Akt represses Hypoxiainducible factor-1alpha activation and sensitizes Rhabdomyosarcoma andEwing's sarcoma cells for apoptosis. Cancer Cell Int 2013;13:36.

12. Teicher BA. Hypoxia and drug resistance. Cancer Metastasis Rev 1994;13:139–68.

13. Duan JX, Jiao H, Kaizerman J, Stanton T, Evans JW, Lan L, et al. Potent andhighly selective hypoxia-activated achiral phosphoramidate mustards asanticancer drugs. J Med Chem 2008;51:2412–20.

14. Sun JD, Liu Q, Wang J, Ahluwalia D, Ferraro D, Wang Y, et al. Selectivetumor hypoxia targeting by hypoxia-activated prodrug TH-302 inhibitstumor growth in preclinical models of cancer. Clin Cancer Res 2011;18:758–70.

15. LiuQ, Sun JD,Wang J, Ahluwalia D, Baker AF, Cranmer LD, et al. TH-302, ahypoxia-activated prodrug with broad in vivo preclinical combinationtherapy efficacy: optimization of dosing regimens and schedules. CancerChemother Pharmacol 2012;69:1487–98.

16. Chawla SP, Cranmer LD, Van Tine BA, Reed DR, Okuno SH, ButrynskiJE, et al. Phase II study of the safety and antitumor activity of thehypoxia-activated prodrug TH-302 in combination with doxorubicin inpatients with advanced soft tissue sarcoma. Am J Clin Oncol 2014;32:3299–306.

17. Nitschke R, Parkhurst J, Sullivan J, Harris MB, Bernstein M, Pratt C.Topotecan in pediatric patients with recurrent and progressive solidtumors: a Pediatric Oncology Group phase II study. Am J Pediatr HematolOncol 1998;20:315–8.

18. LondonWB, Frantz CN, Campbell LA, Seeger RC, Brumback BA, Cohn SL,et al. Phase II randomized comparison of topotecan plus cyclophospha-mide versus topotecan alone in children with recurrent or refractoryneuroblastoma: a Children's Oncology Group study. Am J Clin Oncol2010;28:3808–15.

19. Farhat R, Raad R, Khoury NJ, Feghaly J, Eid T, Muwakkit S, et al. Cyclo-phosphamide and topotecan as first-line salvage therapy in patients withrelapsedEwing sarcoma at a single institution. Am JPediatrHematolOncol2013;35:356–60.

20. WalterhouseDO, Lyden ER, Breitfeld PP,Qualman SJ,WharamMD,MeyerWH. Efficacy of topotecan and cyclophosphamide given in a phase IIwindow trial in children with newly diagnosed metastatic rhabdomyosar-coma: a Children's Oncology Group study. Am J Clin Oncol 2004;22:1398–403.

21. Ashraf K, Shaikh F, Gibson P, Baruchel S, Irwin MS. Treatment withtopotecan plus cyclophosphamide in children with first relapse of neuro-blastoma. Pediatr Blood Cancer 2013;60:1636–41.

22. Soffer SZ, Kim E, Moore JT, Huang J, Yokoi A, Manley C, et al. Novel use ofan established agent: Topotecan is anti-angiogenic in experimental Wilmstumor. J Pediatr Surg 2001;36:1781–4.

23. Kumar S, Mokhtari RB, Sheikh R, Wu B, Zhang L, Xu P, et al. Metronomicoral topotecan with pazopanib is an active antiangiogenic regimen inmouse models of aggressive pediatric solid tumor. Clin Cancer Res2011;17:5656–67.

24. PanP, Li Y, YuH, SunH,HouT.Molecular principle of topotecan resistanceby topoisomerase I mutations through molecular modeling approaches. JChem Inf Model 2013;53:997–1006.

25. Hendricks CB, Rowinsky EK, Grochow LB, Donehower RC, Kaufmann SH.Effect of P-glycoprotein expression on the accumulation and cytotoxicity oftopotecan (SK&F 104864), a new camptothecin analogue. Cancer Res1992;52:2268–78.

26. Leggas M, Adachi M, Scheffer GL, Sun D, Wielinga P, Du G, et al. Mrp4confers resistance to topotecan and protects the brain from chemotherapy.Mol Cell Biol 2004;24:7612–21.

27. Hashimoto K, Man S, Xu P, Cruz-Munoz W, Tang T, Kumar R, et al. Potentpreclinical impact of metronomic low-dose oral topotecan combined withthe antiangiogenic drugpazopanib for the treatment of ovarian cancer.MolCancer Ther 2010;9:996–1006.

28. Merritt WM, Nick AM, Carroll AR, Lu C, Matsuo K, Dumble M, et al.Bridging the gap between cytotoxic and biologic therapy withmetronomictopotecan and pazopanib in ovarian cancer. Mol Cancer Ther 2010;9:985–95.

29. Minturn JE, Janss AJ, Fisher PG, Allen JC, Patti R, Phillips PC, et al. A phase IIstudy ofmetronomic oral topotecan for recurrent childhood brain tumors.Pediatr Blood Cancer 2011;56:39–44.

30. Romiti A, Cox MC, Sarcina I, Di Rocco R, D'Antonio C, Barucca V, et al.Metronomic chemotherapy for cancer treatment: a decade of clinicalstudies. Cancer Chemother Pharmacol 2013;72:13–33.

31. Rodriguez-Perales S,Martinez-Ramirez A, de Andres SA, Valle L, UriosteM,Benitez J, et al. Molecular cytogenetic characterization of rhabdomyosar-coma cell lines. Cancer Genet Cytogenet 2004;148:35–43.

32. Seeger RC, Siegel SE, Sidell N. Neuroblastoma: clinical perspectives,monoclonal antibodies, and retinoic acid. Ann Intern Med 1982;97:873–84.

33. Barnes EN, Biedler JL, Spengler BA, Lyser KM. The fine structure ofcontinuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC. In Vitro 1981;17:619–31.

34. Ciccarone V, Spengler BA, Meyers MB, Biedler JL, Ross RA. Phenotypicdiversification in humanneuroblastoma cells: expression of distinct neuralcrest lineages. Cancer Res 1989;49:219–25.

35. Keshelava N, Seeger RC, Groshen S, Reynolds CP. Drug resistance patternsof human neuroblastoma cell lines derived from patients at differentphases of therapy. Cancer Res 1998;58:5396–405.

36. Masters JR, Thomson JA, Daly-Burns B, Reid YA, Dirks WG, Packer P, et al.Short tandem repeat profiling provides an international reference standardfor human cell lines. Proc Natl Acad Sci U S A 2001;98:8012–7.

37. Zhang L, Smith KM, Chong AL, Stempak D, Yeger H, Marrano P, et al. Invivo antitumor and antimetastatic activity of sunitinib in preclinicalneuroblastoma mouse model. Neoplasia 2009;11:426–35.

38. Zhang L, Marrano P, Kumar S, Leadley M, Elias E, Thorner P, et al. Nab-paclitaxel is an active drug in preclinical model of pediatric solid tumors.Clin Cancer Res 2013;19:5972–83.

39. Prichard MN, Shipman CJr. A three-dimensional model to analyze drug-drug interactions. Antiviral Res 1990;14:181–205.

40. Herben VM, Rosing H, ten Bokkel Huinink WW, van Zomeren DM,Batchelor D, Doyle E, et al. Oral topotecan: bioavailablity and effect offood co-administration. Br J Cancer 1999;80:1380–6.

41. Ghobrial IM, Laubach JP, Raje N, Armand P, Schlossman RL, Boswell EN,et al. Phase 1 study Of TH-302, an investigational hypoxia-targeted drug,and dexamethasone In Patients With Relapsed/Refractory Multiple Mye-loma. ASH 2013 AnnualMeeting; December 7-10, 2013; NewOrleans, LA.

42. Houghton PJ, Lock R, CarolH,Morton CL, PhelpsD, Gorlick R, et al. Initialtesting of the hypoxia-activated prodrug PR-104 by the pediatric preclinicaltesting program. Pediatr Blood Cancer 2011;57:443–53.

43. Ahluwalia D SJ, Liu Q, Ferraro DJ, Wang Y, Lewis JG, et al. TH-302, a novelhypoxia-activated prodrug, shows superior efficacy and less toxicity thanifosfamide (IFOS) in metastatic and ectopic human lung carcinoma






models [abstract]. In: Proceedings of the AACR Annual Meeting; 2009Apr 18–22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr4517.

44. JungD, Lin L, JiaoH,Cai X,Duan JX,MatteucciM. Pharmacokinetics of TH-302: a hypoxically activated prodrug of bromo-isophosphoramide mus-tard in mice, rats, dogs and monkeys. Cancer Chemother Pharmacol2012;69:643–54.

45. Reinhold HS, DeBree C. Tumour cure rate and cell survival of a trans-plantable rat rhabdomyosarcoma following x-irradiation. Eur J Cancer1968;4:367–74.

46. Kallman RF. The phenomenon of reoxygenation and its implications forfractionated radiotherapy. Radiology 1972;105:135–42.

47. Reynolds CP.Detection and treatment ofminimal residual disease in high-risk neuroblastoma. Pediatr Transplant 2004;8(Suppl 5):56–66.

48. Patel KJ, Tredan O, Tannock IF. Distribution of the anticancer drugsdoxorubicin, mitoxantrone and topotecan in tumors and normal tissues.Cancer Chemother Pharmacol 2013;72:127–38.

49. Meng F, Evans JW, Bhupathi D, BanicaM, Lan L, Lorente G, et al. Molecularand cellular pharmacology of the hypoxia-activated prodrug TH-302. MolCancer Ther 2012;11:740–51.


Zhang et al.




2016;22:2697-2708. Published OnlineFirst December 30, 2015.Clin Cancer Res Libo Zhang, Paula Marrano, Bing Wu, et al. Rhabdomyosarcoma Preclinical ModelsHypoxia-Activated Prodrug, Evofosfamide, in Neuroblastoma and Combined Antitumor Therapy with Metronomic Topotecan and

Updated version

10.1158/1078-0432.CCR-15-1853doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2016/06/02/1078-0432.CCR-15-1853.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/22/11/2697.full#ref-list-1

This article cites 47 articles, 13 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/22/11/2697.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/22/11/2697To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-1853

http://clincancerres.aacrjournals.org/content/suppl/2016/06/02/1078-0432.CCR-15-1853.DC1

http://clincancerres.aacrjournals.org/content/22/11/2697.full#ref-list-1

http://clincancerres.aacrjournals.org/content/22/11/2697.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/22/11/2697


combined antitumor therapy with metronomic...

Documents