Phase II trial of continuous low-dose temozolomide for patients with recurrent malignant glioma

Phase II trial of continuous low-dose temozolomide for patients with recurrent malignant gliomaAntonio Omuro,Timothy A.Chan,Lauren E.Abrey,Mustafa Khasraw,Anne S.Reiner,Thomas J.Kaley,Lisa M.DeAngelis,Andrew ssman,Craig P.Nolan,Igor T.Gavrilovic,Adilia Hormigo,Cynthia Salvant,Adriana Heguy,Andrew Kaufman,Jason T.Huse,Katherine S.Panageas,Andreas F.Hottinger,and Ingo MellinghoffDepartments of Neurology (A.O.,L.E.A.,M.K.,T.J.K.,L.M.D.,A.B.L.,C.P.N.,I.T.G.,A.H.,C.S.,A.F.H.,I.M.),Radiation Oncology (T.A.C.),Human Oncology and Pathogenesis Program (T.A.C.,A.H.,A.K.,I.M.),Epidemiology and Biostatistics (A.S.R.,K.S.P.),Pathology (J.T.H.),Memorial Sloan-Kettering Cancer Center,New York,New YorkPresent affiliation:Royal Melbourne and Geelong Hospitals,Australia (M.K.);University of Zurich,Switzerland (L.E.A.);Columbia University Medical Center,New York,New York (A.B.L.);University of Rochester Medical Center,Rochester,New York (A.H.);Centre Hospitalier Universitaire Vaudois and University of Lausanne,Switzerland (A.F.H.);Upstate Medical University,Syracuse,New York (S.C.)Background.In this phase II trial,we investigated the efficacy of a metronomic temozolomide schedule in the treatment of recurrent malignant gliomas (MGs).Methods.Eligible patients received daily temozolomide (50mg /m 2)continuously until progression.The primary endpoint was progression-free survival rate at 6months in the glioblastoma cohort (N ¼37).In an ex-ploratory analysis,10additional recurrent grade III MG patients were enrolled.Correlative studies included eval-uation of 76frequent mutations in glioblastoma (iPLEX assay,Sequenom)aiming at establishing the frequency of potentially “drugable”mutations in patients entering re-current MG clinical trials.Results.Among glioblastoma patients,median age was 56y;median Karnofsky Performance Score (KPS)was 80;62%of patients had been treated for ≥2recurrences,including 49%of patients having failed bevacizumab.Treatment was well tolerated;clinical benefit (complete response +partial response +stable disease)was seen in 10(36%)patients.Progression-free survival rate at 6months was 19%and median overall survival was 7months.Patients with previous bevacizumab exposure survived significantly less than bevacizumab-naive pa-tients (median overall survival: 4.3mo vs 13mo;hazard ratio ¼3.2;P ¼.001),but those patients had lower KPS (P ¼.04)and higher number of recurrences (P ,.0001).Mutations were found in 13of the 38MGs tested,including mutations of EGFR (N ¼10),IDH1(N ¼5),and ERBB2(N ¼1).Conclusions.In spite of a heavily pretreated population,including nearly half of patients having failed bevacizu-mab,the primary endpoint was met,suggesting that this regimen deserves further investigation.Results in bevaci-zumab-naive patients seemed particularly favorable,while results in bevacizumab-failing patients highlight the need to develop further treatment strategies for ad-vanced MG.Clinical identifier .NCT00498927(available at /ct2/show /NCT00498927)Keywords:bevacizumab,glioblastoma,malignant glioma,metronomic chemotherapy,temozolomide.Radiotherapy with concomitant temozolomide fol-lowed by adjuvant temozolomide is the standard of care for newly diagnosed glioblastoma,1butthe optimal regimen for recurrent disease has not been defined.Clinical trials in recurrent glioblastoma haveCorresponding Author:Antonio Omuro,MD,Memorial Sloan-Kettering Cancer Center,1275York Avenue,New York,NY 10065(omuroa@).Received August 13,2012;accepted October 17,2012.Neuro-Oncology 15(2):242–250,2013.doi:10.1093/neuonc /nos295NEURO-ONCOLOGYAdvance Access publication December 14,2012#The Author(s)2012.Published by Oxford University Press on behalf of the Society for Neuro-Oncology.All rights reserved.For permissions,please e-mail:journals.permissions@.at China Pharmaceutical University on December 5, 2016/Downloaded fromreported dismal outcomes,with objective response rates (ORRs)of5%–6%,6-month progression-free survival rates(PFS6)of9%–21%,and median overall survival (OS)of6–7months.2–6Treatment with numerous targeted and nontargeted agents has been attempted without success,with the exception of bevacizumab.7–9 This agent received accelerated FDA approval for recurrent glioblastoma,based on improved ORRs of 20%–26%seen in2phase II trials.8,9However,re-sponses tend to be short-lived(median:4months),and OS has been modest(median:8–10months).Metronomic temozolomide has emerged as a well-tolerated salvage approach in recurrent glioblastoma.10–15 Preclinical studies have suggested that metronomic temozolomide may result in increased activity through depletion of the O6-methylguanine-DNA methyltransfer-ase(MGMT)protein16and anti-angiogenic properties through targeting of endothelial cells.17,18A phase II study19using metronomic,daily temozolomide(50mg/ m2/d)for glioblastoma atfirst relapse investigated3pre-established cohorts:patients with progression before com-pletion of6adjuvant cycles(“early”group),patients with progression after6cycles while still on temozolomide(“ex-tended”group),and patients who discontinued temozolo-mide after completing6cycles without progression (“rechallenge”group).The extended group did not seem to benefit(PFS6:7%),but the early and rechallenge groups achieved PFS6of27%and36%,respectively. However,these results are difficult to interpret because there are no historical controls for either of these cohorts.Moreover,results in the early group could have represented inclusion of patients with pseudoprogression, while patients in the rechallenge group could have har-bored tumors with a different biology.It is also unclear how these results apply to current treatment trends in the United States,where temozolomide tends to be continued beyond6cycles and the use of bevacizumab is frequent.We report a phase II trial using a similar regimen of temozolomide50mg/m2daily but encompassing eligi-bility criteria similar to available historical controls and typical clinical trials in glioblastoma,4,20with no re-striction on timing of failure with respect to previous temozolomide use.We also performed an exploratory, comprehensive disease-specific mutational analysis to characterize the typical population of patients entering studies of recurrent malignant gliomas(MGs)from a molecular perspective.Patients and MethodsThis was a prospective,phase II trial conducted at Memorial Sloan-Kettering Cancer Center from July 2007to April2010.The main cohort comprised patients with progressive/recurrent glioblastoma;patients with recurrent World Health Organization grade III anaplas-tic astrocytoma,anaplastic oligoastrocytoma,or ana-plastic oligodendroglioma were enrolled in a separate, exploratory cohort.Inclusion criteria consisted of histologically con-firmed diagnosis of MG,previous exposure to temozolomide and radiotherapy,unequivocal tumor re-currence at enrollment,age≥18years,Karnofsky Performance Score(KPS)≥60,and adequate bonemarrow and organ function.Patients with other,concur-rent active malignancy or serious medical illness were excluded.There was no limit on number of previous re-currences or chemotherapy regimens.Treatment consisted of temozolomide given at a dose of50mg/m2daily,without interruption,until progression or development of unacceptable side effects.One cycle wasdefined as28days.Anti-emetic therapy and pneumocysto-sis prophylaxis were prescribed at the discretion of treating physicians.Treatment was held for toxicity grades≥3.Once resumed,the dose of temozolomide could bereduced by25%as clinically indicated,but if further reduc-tions were necessary,patients were removed from the study.Complete blood counts were repeated weekly,and compre-hensive chemistry panels were obtained monthly.MRIs andclinical exams were performed every2months.Responseswere evaluated using Macdonald criteria.21The studywas approved by the institutional review board;written in-formed consent was obtained from all patients.StatisticsThe primary endpoint was PFS6in glioblastoma pa-tients.A Simon2-stage design was used.In thefirststage,12patients were accrued.If at least one of thosepatients did not progress at the6-month mark,then an additional25patients(for a total of37)would beaccrued to the second stage;otherwise,the trial wouldbe terminated.At the end of the trial,if fewer than4pa-tients were progression free at6months,the studywould be declared negative.This design yields at leasta.90probability of a positive result if the true PFS6was at least20%and a.90probability of a negativeresult if the true PFS6was5%.Secondary endpoints were median PFS,OS,ORR,and toxicity as per the National Cancer Institute’sCommon Terminology Criteria of Adverse Eventsversion3.0.Survival was calculated from registrationdate(Kaplan–Meier methodology),and log-rank statis-tic was utilized for group comparisons.An additional10patients with grade III MG were enrolled in an explor-atory cohort,and a descriptive analysis was performed.MGMT Promoter Methylation Status and Evaluationof Relevant Glioblastoma MutationsAvailable formalin-fixed paraffin-embedded(FFPE)tissuesamples were evaluated for MGMT promoter methyla-tion status by real-time methylation-specific PCR as described previously,22performed by MDxHealthC.FFPE samples were also subjected to a comprehensive mu-tation screening using mass spectrometry genotyping/iPLEX assay(Sequenom)in genomic DNA,looking fora panel of76mutations described in glioblastoma (Supplementary material,Appendix S1).The iPLEXassay was performed as described by the manufacturer (Supplementary material,Appendix S2).Primers arelisted in Supplementary material,Appendix S3.Omuro et al.:Metronomic temozolomide for recurrent glioblastomaNEURO-ONCOLOGY†F E B R U A R Y2013243at China Pharmaceutical University on December 5, 2016/Downloaded fromResultsPatient CharacteristicsAccrual was completed as planned,with all37glioblas-toma patients enrolled,constituting the main study cohort.Ten additional grade III recurrent MG patients were accrued to the exploratory cohort.Patient charac-teristics are summarized in Table1.Among the glioblastoma patients,32(86%)had recurrence of a de novo glioblastoma and5(14%)had recurrent secondary glioblastoma(ie,initial diagnosis was of a grade2or3glioma,which recurred as a glio-blastoma histologically proven).Patients were heavily pretreated by the time of enrollment(Table1).All glioblastoma patients had re-ceived radiotherapy with concomitant temozolomide. Thirty(81%)patients had a history of tumor progres-sion diagnosed during adjuvant temozolomide,and7 (19%)had never progressed during temozolomide, having previously received a median of12cycles.The number of prior recurrences was1in14(38%)patients,2in11(30%),and3or more in12(32%).Other prior chemotherapies consisted of bevacizumab in18(49%) patients,other cytotoxic agents in9(24%),and experi-mental targeted therapies in8(21%).In all patients with bevacizumab exposure,bevacizumab had been given as salvage treatment,and all of those patients experienced progression while on bevacizumab.Glioblastoma Cohort—Response,PFS,and OS Patients who had measurable tumor and completed at least one cycle of treatment were considered evaluable for response(N¼28).A partial response(PR)was seen in3(11%)patients,stable disease(SD)in7 (25%),and progressive disease(PD)in18(64%);clini-cal benefit(CR+PR+SD)was seen in10(36%)pa-tients.Among the9patients considered nonevaluable for response,1had no measurable disease due to surgical resection and8did not complete thefirst cycle.All patients(N¼37)were included in the PFS and OS analysis,in an intent-to-treat fashion.A total of7pa-tients were progression free and alive at6months,andTable1.Patient characteristicsGlioblastoma(N537)Grade III MalignantGliomas(N510) GenderMen12(32%)6(60%) Women25(68%)4(40%)Median age(range)56(31–81)58(29–73) Median KPS(range)80(60–100)80(60–100) Histology at time of enrollmentDe novo glioblastoma32(86%)–Secondary glioblastoma5(14%)–Grade III anaplastic astrocytoma–4(40%) Grade III anaplastic oligodendroglioma–5(50%) Grade III anaplastic oligoastrocytoma–1(10%) Median time between current histologic diagnosisand enrollment(range)20mo(0.3–58)18mo(0.5–177) Number of previous recurrences114(38%)4(40%) 211(30%)3(30%)≥312(32%)3(30%) Prior salvage therapiesBevacizumab18(49%)0Other cytotoxic agents9(24%)4(40%) Experimental targeted therapies8(21%)3(30%) Progression while on previous adjuvant temozolomideProgression duringfirst6adjuvant cycles21(57%)NA Progression after.6adjuvant cycles9(24%)NA Completed adjuvant cycles without progression7(19%;median of12adjuvant cycles)NAMGMT promoter methylation statusMethylated3(8%)2(20%) Unmethylated20(54%)3(30%) Invalid MGMT results10(27%)4(40%) Tissue unavailable4(11%)1(10%) Abbreviation:NA,not applicable to grade3tumors(patients received variable chemotherapy regimens at diagnosis).Omuro et al.:Metronomic temozolomide for recurrent glioblastoma244NEURO-ONCOLOGY†F E B R U A R Y2013 at China Pharmaceutical University on December 5, 2016 /Downloaded fromtherefore the primary endpoint was met.PFS6was19% (N¼37;95%confidence interval[CI]:6–32),and median PFS was2months(95%CI:1–4)(Fig.1). Median OS was7months(95%CI:5–12),and1-year OS was35%(95%CI:20–51)(Fig.1).Median follow-up for survivors was19months.Given the previously reported poor prognosis observed after bevacizumab discontinuation/failure in retrospective studies,23–27we analyzed the impact of prior bevacizumab exposure on outcomes.Patients with bevacizumab failure survived significantly less(median OS:4.3months;6-months OS:28%)than bevacizumab-naive patients(median OS:13months;6-months OS: 84%);hazard ratio(HR)¼3.2;P¼.001(Fig.2).PFS6 in the bevacizumab-failure group was11.1%versus 26.3%in the bevacizumab-naive group(P¼.07) (Fig.3).Responses were seen in3/15(20%)evaluable bevacizumab-naive patients,and no responses were observed in the bevacizumab-failure group.Among the bevacizumab-naive group,bevacizumab was given to9 (47%)patients after progression on this trial.In an exploratory fashion,we sought to investigate whether differences in pretreatment characteristics could explain the differences in outcomes according to pre-vious bevacizumab exposure(Supplementary material, Appendix S4).In a post-hoc analysis,we evaluated the dis-tribution of potential prognostic factors in bevacizumab-naive versus bevacizumab-failure patients(categorical: Fisher’s exact test;continuous:Kruskal–Wallis test) and performed a multivariate analysis using a forward stepwise Cox proportional hazards regression model with entry and exit criteria of a¼0.20(Supplementary material,Appendix S4).Bevacizumab-failure patients had a higher number of previous progressions/ chemotherapy regimens(P,.0001)and lower KPS (P¼.04)than bevacizumab-naive patients.The median time from glioblastoma diagnosis was21months in bevacizumab-failure versus13months for bevacizumab-naive patients(P¼.18).Multivariate stepwise analysis suggested that the driving variable for decreased PFS was number of progressions/salvage chemotherapies (P¼.02),favored over other candidate variables,includ-ing bevacizumab exposure.Multivariate analysis of OS showed that number of progressions/salvage chemo-therapies(P¼.05)and KPS(P¼.05),but not prior bevacizumab exposure,were the driving variables for differences in survival.There were no differences in outcome according to previous history of progression on temozolomide(PFS: P¼.58;OS:P¼.18)or according to MGMT promoter methylation status(PFS:P¼.76;OS:P¼.14),although analysis is limited by the small number of patients in whom this could be determined accurately(Table1). Grade III Recurrent Malignant Glioma Exploratory CohortAmong the10patients with grade III MG,PFS6was 30%(95%CI:1.6–58.4);median PFS was3months. Median OS was18months;1-year OS was60%(95%Fig.1.OS and PFS(glioblastoma cohort,N¼37).PFS6:19%(95%CI:6–32);median PFS:2months;1-year OS:35%;median OS:7months.Fig.2.Glioblastoma cohort(N¼37):OS according to previoushistory of bevacizumab(BEV)treatment.Patients with previousBEV failure:median OS:4.3mo,6-mo OS:28%(95%CI:7–48);patients without BEV failure:median OS:13mo;6-monthOS:84%(95%CI:68–100);HR¼3.2;P¼.001.Fig.3.Glioblastoma cohort(N¼37):PFS according to previous bevacizumab(BEV)treatment.Patients with previous BEV failure:PFS6:11.1%(95%CI:0.0–25.6),median PFS:1.5mo;patientswithout bevacizumab failure:PFS6:26.3%(95%CI:6.5–46.1),median PFS:1.8mo;HR¼1.9;P¼.07.Omuro et al.:Metronomic temozolomide for recurrent glioblastomaNEURO-ONCOLOGY†F E B R U A R Y2013245at China Pharmaceutical University on December 5, 2016/Downloaded fromCI:30–90).One patient achieved PR,4achieved SD, and5achieved PD.ToxicityTreatment was well tolerated;no patient discontinued treatment as a result of toxicity,and there were no toxic deaths.Lymphopenia was the most frequent toxic-ity(grade3¼10patients,grade4¼1),but no lymphopenia-related infection was reported.The only other grade4toxicity was thrombocytopenia(n¼1). Grade3toxicities consisted of increased alanine amino-transferase(n¼2),fatigue(2),hyponatremia(2), hypokalemia(1),pulmonary embolism(1),neuropathy (1),and infection(1).Mutational AnalysisIn an exploratory analysis,we evaluated FFPE tissue from38patients(28glioblastomas and10grade III tumors)for the presence of76mutations previously de-scribed in gliomas using mass spectrometry genotyping/ iPLEX assay(Sequenom[Supplementary material, Appendix S1]).A total of9different mutations was found in13patients(Table2),including8(29%)glio-blastomas and5(50%)grade III MGs.Mutations were found in EGFR(N¼10),IDH1(N¼5),and ERBB2(N¼1).All IDH1mutations were found in grade III(N¼3)or secondary glioblastomas(N¼2) and none in recurrent primary glioblastomas.EGFR extracellular-domain mutations were found in both glio-blastomas(N¼7)and grade III gliomas(N¼3).None of the analyzed mutations involving phosphatidylinosi-tol3-kinase(PI3K),BRAF,or KRAS was found. Table2summarizes the mutations found,along with patient characteristics and treatment outcomes.DiscussionThis study was designed to test the efficacy of metro-nomic temozolomide in the typical population enrolled in clinical trials for recurrent glioblastoma,irrespective of prior treatments.The primary efficacy endpoint (based on pre–bevacizumab-era historical controls) was met,with19%PFS6,despite the inclusion of a heavily pretreated population,including nearly half of the patients with a history of bevacizumab failure.This regimen was therefore deemed active,warranting further investigation.Enrollment to this trial coincided with the rise of bev-acizumab for glioblastoma salvage therapy in the United States.Although this confounded the interpretation of our results in comparison with historical controls,it also provided the unique opportunity to evaluate the same cytotoxic regimen in patients who were bevacizu-mab naive or had failed bevacizumab.Results in bevacizumab-naive glioblastoma seemed particularly fa-vorable(PFS6:26%,median OS:13mo)and compara-ble to other active salvage treatments;activity was also observed in grade III tumors.Given the excellent toxicity profile and ease of drug administration,this regimencould potentially constitute a viable alternative to beva-cizumab and nitrosoureas as second-line treatment forglioblastoma and a suitable platform for combinationwith targeted agents.Conversely,bevacizumab-failurepatients achieved a much shorter PFS6of11%and median OS of4.3months,highlighting the needfor alternative therapies in this population.Several phase II trials testing intensified temozolo-mide schedules have been conducted(Table3),but theoptimal regimen remains to be defined.A direct compar-ison across trials is difficult,given the varying inclusioncriteria and study settings,but overall it appears thatsigns of activity were observed irrespective of theregimen used.A regimen of comparable efficacy usedtemozolomide150mg/m2given7/14days;toxicitieswere more frequent,likely reflecting the150%greatercumulative dose of this agent in comparison with ourregimen.13It must also be noted that an intensified adju-vant temozolomide schedule(75–100mg/m2×21d q 4wk)failed to improve survival in a recent phase IIItrial in newly diagnosed glioblastoma,28althoughthose results may not be applicable to recurrentdisease,given potential differences in chemosensitivityand patient selection.Dedicated randomized trials arethus needed to define the role and optimal metronomictemozolomide schedule in recurrent glioblastoma.Interestingly,results of trials combining daily temozolo-mide and bevacizumab29,30(Tables3and4)were disap-pointing and comparable or inferior to our single-agentresults.This raises the intriguing question of whetherbevacizumab could decrease the activity of temozolo-mide when given at low doses,perhaps by decreasingdrug penetration.31–34To our knowledge,this is thefirst prospective study re-porting outcomes in bevacizumab-failure patients inwhom bevacizumab was discontinued.Because of fearsof a rebound effect,bevacizumab is often continueddespite progression on the drug.23–27,35Three smallprospective studies in bevacizumab-failure patients usedcontinued bevacizumab with different cytotoxic agentsand found a median OS of3–6months(Table4).9,36,37The most favorable results were observed with combinedbevacizumab,carboplatin,and irinotecan(PFS6:16%;median OS:6mo),37but48%of patients in that studyhad failed bevacizumab as part offirst-line treatment,whereas the other studies included patients failingsalvage bevacizumab;moreover,that same drug combi-nation was not active in bevacizumab-naive patients.38Therefore,outcomes of continued bevacizumab regi-mens were poor and not superior to our results,suggest-ing that it is justifiable to discontinue bevacizumab afterbevacizumab failure39,40and that conducting clinicaltrials in that setting is feasible.Interestingly,our mul-tivariate analysis suggested that the poor outcomes inbevacizumab failures seemed to be at least partially ex-plained by poor KPS and greater number of progressions(Supplementary material,Appendix S4),although thepossibility that bevacizumab exposure may induce amore aggressive,invasive phenotype41,42cannot be en-tirely ruled out.Omuro et al.:Metronomic temozolomide for recurrent glioblastoma246NEURO-ONCOLOGY†F E B R U A R Y2013 at China Pharmaceutical University on December 5, 2016 /Downloaded fromFinally,we performed a Sequenom-based tissue anal-ysis in order to characterize the frequency of glioma-associated mutations43within patients who enroll in clinical trials for recurrent disease.A total of13(38%) patients displayed one or more mutations involving the EGFR,IDH1,and ERBB2genes,but overall theTable2.Mutations found in the patients enrolled in the trial with corresponding patient characteristics and clinical outcomes(N¼38)Pt Path MGMT Time from Diagnosis(mo)Prior BEV Response PFS(mo)OS(mo) EGFR_P596L1AA Unmethylated0.6No PD 1.637.332GBM Invalid30Yes NE0.370.373GBM Invalid22No SD7.417.24a AO Invalid 1.6No PD 1.838.95GBM Invalid55No PD 1.919.8 EGFR_C620Y6GBM Unmethylated17Yes SD 5.938.2 EGFR_G598V7GBM Unmethylated31Yes PD 1.5 2.4 EGFR_I91L8GBM Unmethylated17Yes NE 1.2 2.6 EGFR_T263P9AA Methylated26No SD8.0315.57 EGFR_V651M10a GBM(secondary)Methylated22No PR 5.4712.63 IDH1_R132L_H11AO Methylated163No SD 1.77 3.134a AO Invalid 1.6No PD 1.838.912AO Invalid25No SD 6.921.110a GBM(secondary)Methylated22No PR 5.4712.63 IDH1_R132C_G_S13GBM(secondary)Unmethylated10No NE0.4 1.7 ERBB2_L49H10a GBM(secondary)Methylated22No PR 5.4712.63Abbreviations:Pt,patient;Path,most recent pathology prior to enrollment;BEV,bevacizumab.a Patients with.1mutation found.Table3.Phase II trials of metronomic temozolomide schedules for glioblastomaReference N TMZ Daily Dose,Schedule ORR(%)PFS6(%)Median OS(mo) Khan112775mg/m2,42/70d14278Brandes123375mg/m2,21/28d93010Wick1364150mg/m2;7/14d15449Perry1050mg/m2,28/28d33Early progression on adjuvant TMZ327NA27Progression on extended TMZ07NA28Rechallenge1136NAKong153840–50m2,28/28d53213Abacioglu1416100mg/m2,21/28d7257Verhoeff301550mg/m2,28/28d+bevacizumab10mg/kg q21d NA74Desjardins293250mg/m2,28/28d+bevacizumab10mg/kg q14d28196This study3750mg/m2,28/28dAll patients11197Bevacizumab failures0114Bevacizumab naive202613 Abbreviations:TMZ,temozolomide;ORR,objective response rate(complete+partial responses);NA,not available.Omuro et al.:Metronomic temozolomide for recurrent glioblastomaNEURO-ONCOLOGY†F E B R U A R Y2013247 at China Pharmaceutical University on December 5, 2016 /Downloaded fromindividual frequency of tested mutations was low.The on-cogenic role of some of the identified extracellular-domain EGFR mutations has been validated in functional assays,44,45and testing EGFR inhibitors in those patients could be of interest.However,none of the33mutations involving the PI3K complex was found,suggesting that a very large number of recurrent patients would have to be screened in order to study a PI3K inhibitor in a trial re-stricted to PI3K mutated tumors.In addition,the wide variability in outcome observed in patients with IDH1 mutations was unexpected.These mutations have been reported to confer a better prognosis,46but the outcome of these patients in this trial was variable(OS:3–21 mo),likely influenced by pretreatment characteristics such as time from initial diagnosis and previous therapies (Table2).This suggests that even in trials for molecularly selected tumors,further patient selection and stratifica-tion based on pretreatment clinical characteristics may be necessary,which will add to the feasibility challenges that such trials will face.Of note,Sequenom-based meth-odology requires that point mutations be chosen and pre-specified;other panels have been proposed,47,48including mutations that are different from the ones we tested.The adoption of next-generation sequencing may overcome this limitation in the future.A few other limitations apply to this study.Our mul-tivariate analysis was performed post hoc and was based on a relatively small number of patients.We used Macdonald criteria for assessment of response,which allows for a comparison with historical controls;criteria of the Revised Assessment of Neuro-Oncology49have since been proposed and may define progression earlier.The median time between diagnosis and enroll-ment was unusually long(21mo),although the longest times were observed among bevacizumab-failure pa-tients,who fared poorly;therefore,selection bias does not seem to explain the activity observed.In summary,we provide further evidence that daily-dose temozolomide is a safe and active regimen in recur-rent glioblastoma.We also demonstrate the differences in outcomes between bevacizumab-naive and bevacizumab-failure patients in the prospective setting and provide historical controls that can be used for sample-size calculation and clinical trial interpretation in bevacizumab-failure patients.Ourfindings support the development of separate trials or stratification ac-cording to bevacizumab exposure but also highlight the prognostic importance of other variables,such as number of previous recurrences and KPS,for that purpose.Importantly,our results in bevacizumab fail-ures were comparable to trials of continuation of bevaci-zumab after progression,suggesting that one should not fear discontinuing bevacizumab for the purposes of pur-suing alternative treatments or clinical trials.Supplementary Material Supplementary material is available at Neuro-Oncology Journal online(http://neuro-oncology.oxfordjournals. org/).AcknowledgmentsPresented in part at the American Society of Clinical Oncology(ASCO)annual meeting2010and Society for Neuro-Oncology(SNO)annual meeting2010.Conflict of interest statement.L.E.A.is currently an employee of Hoffmann–La Roche.M.K.has received honoraria from Roche.A.O.has received compensation for consulting from Roche.A.B.L.has received com-pensation for consulting from Merck/Schering-Plough, BMS,Campus Bio,Cephalon,Eisai,Genentech,GSK, Imclone,Sigma Tau,and Abbott and reports research funding from Merck/Schering-Plough,Genentech, Keryx,Sigma Tau,Astra Zeneca,Medimmune,and Boehringer Ingelheim.FundingThis study was an investigator-initiated clinical trial funded by the Memorial Sloan-Kettering Cancer Center Department of Neurology Research and De-velopment Fund.Schering-Plough/Merck provided drug,partialfinancial support,and analysis of MGMT promoter methylation status.Table4.Outcomes of clinical trials conducted in recurrent glioblastoma patients with history of bevacizumab(BEV)failureReference Regimen N Patients who had Failed BEVas Part of First-LineTreatment PFS6(%)MedianOS(mo)ORRReardon36BEV+daily TMZ50mg/m2100030 BEV+VP-1650mg/m2(3wk on/1wkoff)1315%8 4.70 All pts2315%4 4.10 Reardon37BEV+carboplatin+CPT-112548%16 5.80 Kreisl9BEV+CPT-11190≤5a NA0 This study Daily TMZ50mg/m2,no BEV18011 4.30 Abbreviations:ORR,objective response rate(complete+partial responses);TMZ,temozolomide;VP-16,etoposide;NA,not available.a Only1patient received.3cycles.Omuro et al.:Metronomic temozolomide for recurrent glioblastoma248NEURO-ONCOLOGY†F E B R U A R Y2013 at China Pharmaceutical University on December 5, 2016 /Downloaded from。