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2009;15:7719-7725. Published OnlineFirst December 8, 2009.Clin Cancer Res

John Nemunaitis, Gary Clayman, Sanjiv S. Agarwala, et al.

Squamous Cell Carcinoma of the Head and NeckGene Therapy Efficacy in Recurrentp53Biomarkers Predict

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Cancer Therapy: Clinical

Biomarkers Predict p53Gene Therapy Efficacy in Recurrent

Squamous Cell Carcinoma of the Head and Neck

John Nemunaitis,1 Gary Clayman,2 Sanjiv S. Agarwala,4 William Hrushesky,5 James R. Wells,5

Charles Moore,6 John Hamm,7 George Yoo,8 Jose Baselga,9 Barbara A. Murphy,10

Kerstin A. Menander,3 Laura L. Licato,3 Sunil Chada,3 Robert D. Gibbons,11 Magali Olivier,12

Pierre Hainaut,12 Jack A. Roth,2 Robert E. Sobol,3 and W. Jarrard Goodwin13

Abstract Purpose: Most recurrent squamous cell carcinomas of the head and neck have a dys-functional p53 tumor suppressor pathway contributing to treatment resistance. We hy-

pothesized that tumor p53 biomarkers may predict the efficacy of normal p53 delivered

by gene therapy in these patients.

Experimental Design:Tumor p53 biomarkers were evaluated in 116 patients, including

29 treated with methotrexate in a phase III randomized controlled trial. Profiles favor-

able for p53 gene therapy efficacy were hypothesized to have either normal p53 gene

sequences or low-level p53 protein expression, whereas unfavorable p53 inhibitor pro-

files were predicted to have high-level expression of mutated p53 that can inhibit nor-mal p53 protein function.

Results: A statistically significant increase in tumor responses was observed for pa-

tients with favorable p53 efficacy profiles compared with those with unfavorable p53

inhibitor profiles [phase I/II trials: favorable (34 of 46, 74%) versus unfavorable (1 of

5, 20%), P= 0.0290; phase III trial: favorable (17 of 24, 71%) versus unfavorable (2 of

11, 18%), P = 0.0088]. In the phase III trial, there was statistically significant increased

time to progression (TTP) and survival following p53 gene therapy in patients with fa-

vorable p53 profiles compared with unfavorable p53 inhibitor profiles (median TTP,

2.7 months versus 1.4 months, P = 0.0121; median survival, 7.2 months versus

2.7 months,P< 0.0001). In contrast, the biomarker profiles predictive of p53 gene ther-

apy efficacy did not predict methotrexate response, TTP, or survival outcomes.

Conclusions:These results indicate that tumor p53 biomarker profiles may predict p53

gene therapy efficacy in recurrent squamous cell carcinoma of the head and neck. (ClinCancer Res 2009;15(24):771925)

Recurrent, late-stage squamous cell carcinoma of the head andneck (SCCHN) is typically incurable. The vast majority of pa-tients do not respond to standard therapies, which may resultin toxicity (13). In addition, conventional treatments providemedian survivals of only 4 to 6 months and there is a clear needfor novel therapies (13).

Thep53 tumor suppressor pathway is dysfunctional in virtu-ally all cases of recurrent SCCHN (47). Hence, p53 gene ther-apy to restore p53 function is a logical targeted treatment forthis disease (8, 9). Adenoviral p53 gene therapy (Advexin) usesa replication-defective adenovirus to deliver and express normalp53 following local intratumoral administration (8, 9).

Authors' Affiliations: 1

Mary Crowley Cancer Research Centers, Dallas,Texas; 2The University of Texas M.D. Anderson Cancer Center; 3Introgen

Therapeutics, Houston, Texas; 4St. Luke's Cancer Center, Bethlehem,

Pennsylvania; 5WJB Dorn Veterans Affairs Medical Center, Columbia,

South Carolina; 6Emory University School of Medicine, Atlanta, Georgia;7University of Louisville, Louisville, Kentucky; 8Wayne State University,

Detroit, Michigan; 9Hospital General Universitario Vall d' Hebron,

Barcelona, Spain; 10Vanderbilt-Ingram Cancer Center, Nashville,

Tennessee; 11University of Illinois, Chicago, Illinois; 12IARC, Lyon, France;

and 13University of Miami Sylvester Cancer Center, Miami, Florida

Received 4/23/09; revised 9/3/09; accepted 9/4/09; published OnlineFirst

12/8/09.

Grant support: Introgen Therapeutics, Inc.

The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertise-

mentin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

R.E. Sobol and W.J. Goodwin contributed equally to this work.

Other members of the ADVEXIN p53 Gene Therapy Head and Neck Cancer

Study Group (Investigators): D. Adkins, Washington University School of

Medicine, St. Louis, MO; R. Axelrod, Thomas Jefferson University,

Philadelphia, PA; M. Biakhov, N.A. Semashko Central Clinical Hospital of

Russian Ministry of Railways, Moscow, Russia; J. Bier, Virchow-Klinikum

Medical Fakultat der Humboldt-Universitat Mund-, Kiefer und Gesichtschir-

urgie, Berlin, Germany; R. Breau, University of Kansas, Little Rock, AR; B.

Brockstein, Evanston Hospital Kellog Cancer Center, Evanston,IL; B. Burkey,

Vanderbilt University Medical Center, Nashville, TN; M. Constenla, Com-

plexo Hospital de Pontevedra, Ponteverda, Spain; W. Flood, Milton S.

Hershey Medical Center, Hershey, PA; E. Diaz-Rubio, Hospital Universitario

San Carlos, Madrid, Spain; A. Dietz, HNO-Universitatsklinik and Poliklinik

Leiter, Heidelberg, Germany; D. Esser, Helios Klinikum, Berlin, Germany; J.

7719 Clin Cancer Res 2009;15(24) December 15, 2009www.aacrjournals.org


http://clincancerres.aacrjournals.org/http://clincancerres.aacrjournals.org/http://clincancerres.aacrjournals.org/http://clincancerres.aacrjournals.org/

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Knowledge of the mechanisms tumors use to inhibit p53(813) supported the development of p53 biomarker profilesto predict the efficacy of p53 gene therapy. Before analysis, wehypothesized that tumor p53 profiles favorable for gene ther-apy efficacy would have wild-type p53 gene configurations

where additional normal p53 may overcome p53 inhibitioncaused by upregulation of the p53 inhibitors HDM2 andHDM4. We further hypothesized that tumor p53 profiles un-

favorable for p53 gene therapy may be characterized by high-level expression of mutated p53 proteins that are associatedwith dominant-negative p53-inhibitory effects, which mayinactivate the normal p53 delivered by gene therapy.

The National Cancer Institute and the Food and Drug Ad-ministration proposed the Oncology Biomarker QualificationInitiative to accelerate cancer drug development and to guidepatient care by identifying biomarkers that define patient popu-lations most likely to benefit from a specific therapy (14, 15).Biomarkers predictive of therapeutic efficacy are particularly im-portant for patients with short life expectancies, as the limited

time for empirical selection may result in the failure to admin-ister a beneficial therapy.

These considerations lead to the incorporation of predictivep53 biomarkers in clinical trials to evaluate the benefit ofp53gene therapy in patients with recurrent SCCHN. The results ofthese trials indicate the utility of tumor p53 biomarkers to iden-tify patients most likely to benefit fromp53 gene therapy.

Materials and Methods

Study design. Tumor p53 biomarker profiles were evaluated in

116 patients enrolled in adenoviral p53 gene therapy clinical trials.

The training tumor p53 biomarker data set involved 51 patients eval-

uated for tumor response who were treated in trials INT-002 (phase

I/II) and T201 (phase II) as described (9). The validation set of 65

patients was evaluated in a multicenter, multinational, open-label,

prospectively randomized, phase III study of adenoviral p53 genetherapy (Advexin) and methotrexate (control agent) in recurrent

SCCHN patients who had previously been exposed to platinum or

taxane therapy in the recurrent disease setting. Eligible patients in

the phase III trial had histologically confirmed SCCHN with cytolog-ically confirmed recurrence after first-line therapy administered with

curative intent (at least 50 Gy radiotherapy and/or surgery with orwithout initial chemotherapy). All patients were required to have

had at least one prior platinum or taxane chemotherapy regimen.

All human investigations were done after approval by a local Human

Investigations Committee and in accordance with assurances ap-proved by required regulatory agencies. In the phase III trial, patients

were randomly assigned to receive either adenoviral p53 gene thera-

py administered intratumorally on days 1 and 3 of each week at a

daily dose of 2 1012 viral particles or methotrexate administered

i.v. once weekly at a starting dose of 40 mg/m2. Each treatment cyclewas 3 wk (21 d). The primary efficacy end point was survival and

the secondary efficacy end point was tumor growth control response

based on comparisons of pretreatment and posttreatment computer-

ized tomography or magnetic resonance imaging scans of treated le-

sions. Response was defined as either stable disease or tumor sizereduction by Southwest Oncology Group criteria (16) that were as-

sessed following two cycles of treatment. In the phase III biomarker

study, one patient in each of the p53 gene therapy and methotrexate

treatment groups was not evaluable for treatment response. For

agents such as adenoviral p53 that are known to induce cell cycle

arrest and senescence, tumor response definitions based on tumorgrowth control are more appropriate for defining tumor responses

predictive of increased survival than response definitions based solely

on reduction in tumor size (17, 18).Tumor p53 biomarker evaluations. The p53 biomarker profiles pre-

dictive ofp53 gene therapy effects were based on mechanisms of tumor

Germa, Institut Catala d'Oncologia, Barcelona, Spain; V. Guillem, InstitutoValenciano de Oncologia, Valencia, Spain; I. Gulidov, Medical Radiological

ResearchCenter RAMS,Obninsk KalugaRegion, Russia;T. Guthrie, University

of Florida-Jacksonville, Jacksonville, FL; J. Medina, University of Oklahoma

Health SciencesCenter, Oklahoma City, OK; B. Murphy, Vanderbilt University

MedicalCenter, Nashville, TN;M. Kane, University of ColoradoCancer Center,

Aurora,CO; A. Kawecki,Kinika Nowotworow Glowyi Szyi, Warsaw,Poland; G.

Krempl, University of Oklahoma, Oklahoma City, OK; A. Law, Geisinger

Medical Center, Danville, PA; M. Levine, Cancer Center at GBMC, Baltimore,

MD;M. Litwiniuk,Katedra i KlinikaOnkologiiAm, Poznan, Poland;A. Makhson,

Moscow City Oncology Hospital, Moscow, Russia; K. Mardaleishvili, National

Cancer Center ofGeorgia, Tbilisi,Georgia;E. Matyakin, N.N.BlohkinOncology

Research Center RAMS, Moscow, Russia; C. Nathan, Louisiana State

University Medical Center, Shreveport, LA; C. Peschel, Klinikum Rechts der

Isar der TU Munchen III, Munchen, Germany; P. Poliakov, M.F. Vladimirsky

Moscow Regional Clinical Research Institute (MONIK), Moscow, Russia; V.

Protsyk, Ukrainian Research Institute of Oncology, Kiev, Ukraine; F. Rosen,University of Illinois at Chicago, Chicago, IL;S. Schantz,New YorkEye and Ear

Infirmary, New York, NY; J. Schipper, Klinikum der Albert-Ludwigs-Universi-

tat, Freiburg, Germany; D. Schuller, Ohio State University Medical Center,

Columbus, OH; J. Spiro, Universityof Connecticut Health Center, Farmington,

CT; D. Trask, University of Iowa Hospital and Clinics, Iowa City, IA; J. Trigo,

Hospital Valle de Hebron, Barcelona, Spain; D. Villaret, University of Florida,

Gainesville, FL; J. Weber, USC Norris Cancer Center, Los Angeles, CA; R.

Weisman, University of California, San Diego Cancer Center, La Jolla, CA; S.

Williamson, University of Kansas Cancer Center, Kansas City, KS; R. Zitsch,

University of Missouri Health Sciences Center, Columbia, MO.

Requests for reprints: John Nemunaitis, Mary Crowley Cancer Research

Centers, 1700 Pacific Avenue, Suite 1100, Dallas, TX 75201. Phone: 214-

658-1964; Fax: 214-658-1992; E-mail: [email protected].

F2009 American Association for Cancer Research.

doi:10.1158/1078-0432.CCR-09-1044

Translational Relevance

This study ofp53gene therapy in recurrent squa-

mous cell carcinoma of the head and neck (SCCHN)

describes novel biomarkers predictive ofp53gene

therapy efficacy that may guide individualized pa-

tient treatment. The tumorp53biomarkers predictive

of efficacy were based on known molecular mechan-isms of tumor p53 inactivation. The p53 biomarker

analyses of survival and tumor response end points

suggest that p53 gene therapy and methotrexate

have therapeutic efficacy in different and comple-

mentary groups of recurrent SCCHN patients refrac-

tory to platinum or taxanes. Hence, these p53

biomarker profiles permit the personalized selection

of potentially efficacious p53gene therapy or meth-

otrexate treatment for recurrent SCCHN patients.

Further studies will be required to determine if

the tumor p53 biomarker profiles predictive of p53

gene therapy efficacy in recurrent SCCHN will also

be useful to guide p53 gene therapy of other tu-

mors that are known to have similar p53molecular

abnormalities.

7720Clin Cancer Res 2009;15(24) December 15, 2009 www.aacrjournals.org




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p53 inactivation (4, 913). p53 biomarker profiling involved determi-

nation of mutation byp53 gene sequencing and p53 protein expression

by immunohistochemistry (9). The tumor p53 profiles favorable and

unfavorable forp53 gene therapy efficacy are summarized in Table 1.

Tumor p53 profiles were determined in a blinded manner withoutknowledge of the clinical results. Mutational p53 gene sequence analy-

sis was done by BRT Laboratories, Inc. Genomic DNA preparation was

carried out using the QIAamp kit (Qiagen, Inc.) for frozen tumor tissue

samples or by standard phenol/chloroform extraction for paraffin-em-

bedded tissues. Mutational screening was carried out by amplificationof all samples using Multiplex primers (Affymetrix, Inc.). The resultant

PCR products were analyzed using the Affymetrixp53 GeneChip, which

encompasses all coding exons, 2 to 11, as well as immediate flanking

regions. Confirmation of the results was carried out by sequencing of

the particular locations indicated by gene chip screening analysis or all

exons in the cases where no mutation was detected using the gene chip.Immunohistochemical staining was done at QualTek Molecular

Laboratories following Good Laboratory Practices. The DO-7 mono-

clonal antibody (Lab Vision Corp.) was used for p53 detection. This

antibody binds at the NH2 terminus ofp53 and thus can be used todetect both wild-type and mutant protein. The characteristics of the

monoclonal antibodies specific for HDM2 and HDM4 have been de-

scribed previously (4). Criteria for positive p53 overexpression were

nuclear staining in 20% of tumor cells, whereas the criteria for

HDM2 and HDM4 positivity were >10% as described (4). Samplesfrom patients in trial INT-002 were analyzed as reported (9). The cri-teria for immunohistochemistry positivity were consistent with previ-

ously published studies (4, 9) and the prospectively defined values

developed for the phase III registration clinical trial. In the majority

of positive cases, >50% of the tumor cells expressed the biomarker.Comparison of the study patients' p53 tumor profile characteristics

was made with the IARC TP53 databas e for European and North

American patients with SCCHN (19).Statistical analysis. All statistical analyses were prospectively speci-

fied. Time to progression (TTP) and survival curves were generated us-

ing the Kaplan-Meier method (19). Comparisons between treatment

arms were made with the log-rank test (20). Cox regression analysis

was used to evaluate the interactions of treatment and tumor p53 bio-

marker profiles in terms of survival (21). Fisher's exact test was used tocompare overall tumor response rates, and logistic regression analysis

was used to evaluate the interactions of treatment and p53 biomarker

profiles in terms of tumor response (21). A Cox proportional hazards

regression model was used to assess the prognostic ability of treatment

while adjusting for the effect of important covariates (21, 22).

Results

Patient demographic and clinical characteristics. The demo-graphic and clinical characteristics of the 116 patients evaluatedfor tumor p53 profiles in comparison with the intent to treatpopulations are listed in Supplementary Tables S1 to S4. There

were no statistically significant differences in the demographicand clinical characteristics between patients with and withouttumor p53 profile data (Supplementary Tables S1-S4). Thephase IIII trial was initiated on April 24, 2001 and closed toenrollment on April 30, 2008.

Table 2. Tumor responses and p53 biomarkers predictive ofp53 gene therapy efficacy

Study/treatment

population

Tumor p53 profile for p53

gene therapy efficacy

% Tumor growth

control*

P

Phase I/II trials

p53 gene therapy Favorable 74% (34/46) 0.0290

Unfavorable 20% (1/5)

Phase III trial

p53 gene therapy Favorable 71% (17/24) 0.0088


Methotrexate Favorable 50% (11/22) 0.1965


*All patients with reductions in tumor size had favorable p53 profiles. Of the phase III trial patients who received p53 gene therapy, one had a

complete response, five patients had >50% tumor reduction, eight patients had tumor reductions ranging between >10% and 50% tumor reduction, two patients had tumor reductions

ranging between >10% and

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p53 tumor biomarker profile frequencies and expression of the

p53inhibitors HDM2 and HDM4. Tumor samples were evalu-ated forp53 gene sequence and for expression of p53 and thep53-inhibitory proteins HDM2 and HDM4 (also known as mdm2 andmdm4).The vast majority of cases (>90%)had evidence for inhib-ited p53 either by the presence of a mutated p53genotype or byoverexpression of the p53 inhibitors HDM2 or HDM4. Elevatedlevels of HDM2 or HDM4 were observed in >90% of patients witheither normal or mutatedp53 gene sequences. The majority ofcases (72%) had tumor p53 profiles favorable forp53gene ther-apy efficacy, which were characterized by either wild-type p53genotypes or low-level expression of mutated p53 by immunohis-

tochemical staining. The remaining patients had tumor p53inhibitor profiles unfavorable forp53gene therapy efficacy char-acterized by high-level expression of mutated p53.p53 tumor biomarker profiles and tumor response. Tumor

p53 biomarker analysis in the training data set involved pa-tients receiving p53 gene therapy in phase I/II clinical trialsand revealed a statistically significant increase in tumor re-sponses for patients with favorable p53 biomarker profilescompared with unfavorable profiles (Table 2). Tumor re-sponses were observed in 74% (34 of 46) of p53 gene thera-pytreated patients with favorable tumor p53 profilescompared with only 20% (1 of 5) of patients with unfavorableprofiles (P= 0.0290, Fisher's exact test).

The tumor response results forp53 gene therapytreated pa-tients were validated in the phase III trial. Tumor growth con-trol response following p5 3 therapy was retained as anindependent prognostic factor for survival in Cox proportionalhazards multivariate analysis of known prognostic variables,

with a significantly decreased risk of death for patients with tu-mor growth control responses compared with nonresponders[hazard ratio (HR), 0.17; 95% confidence interval (95% CI),0.08-0.36; P< 0.0001]. There was a statistically significant in-crease in tumor responses for patients with favorable p53 effi-cacy profiles compared with those with unfavorable profiles[favorable (17 of 24, 71%) versus unfavorable (2 of 11,

18%);P= 0.0088; Table 2]. In contrast, the p53 profiles predic-tive ofp53 gene therapy efficacy did not predict methotrexateresponse outcomes. Opposite to p53 gene therapy, methotrex-ate responders had a lower percentage of p53 profiles favorablefor p53 gene therapy efficacy and a higher proportion of re-sponders with unfavorable p53 profiles, although the difference

was not statistically significant [favorable (11 of 22, 50%) ver-sus unfavorable (5 of 6, 83%);P= 0.1965; ref. 2]. The treatmentby p53 biomarker profile interaction was statistically significantby logistic regression analysis (P= 0.0069) and indicates thatp53gene therapy and methotrexate tumor responses were asso-ciated with different and complimentary groups of recurrentSCCHN patients (Table 2).

Fig. 1. A, tumor p53 biomarker efficacy profilespredict TTP following p53gene therapy in recurrentSCCHN. B, p53 biomarker profiles favorable andunfavorable for p53gene therapy efficacy donot predict TTP following methotrexate treatment inrecurrent SCCHN.





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p53 tumor biomarker profiles, TTP, and survival. Consistentwith the tumor response results presented above, the favorablep53 biomarker profiles associated with increased tumor re-sponses were also significantly related to increased TTP andsurvival following p53 gene therapy. There was a statisticallysignificant increased TTP following p53 gene therapy for pa-tients with p53 favorable profiles compared with those withunfavorable p53 inhibitor profiles (median TTP, 2.7 months

versus 1.4 months; P= 0.0121; Fig. 1A). There was a statisti-cally significant increased survival following p53 gene therapyfor patients with p53 favorable profiles compared with those

with unfavorable profiles (median survival, 7.2 months versus2.7 months; P< 0.0001, log-rank test; Fig. 2A). The p53 pro-files predictive of p53 gene therapy treatment effects were re-tained as an independent prognostic factor for survival in Coxproportional hazards multivariate analysis of known prognos-tic variables, with a significantly decreased risk of death forpatients with favorable Advexin efficacy profiles (HR, 0.15;95% CI, 0.06-0.39; P= 0.0001) compared with patients withunfavorable profiles.

In contrast and consistent with the tumor response results,

the p53 profiles predictive of p53 gene therapy efficacy didnot predict methotrexate TTP or survival outcomes. There wasno statistical difference between the median TTP or median sur-

vival of methotrexate-treated patients with favorable versus un-

favorable tumor p53 profiles for p53 gene therapy efficacy(median TTP, 2.3 months versus 2.5 months, P = 0.3819,Fig. 1B; median survival, 4.3 months versus 5.9 months,P= 0.5701, Fig. 2B). Comparison of the intent to treat popula-tion without consideration of tumor p53 biomarker profile sta-tus revealed no statistically significant difference in the survivalbetween thep53gene therapy and methotrexate-treated patients(median survival, 4.4 versus 6.1 months; P= 0.236).

However, as shown in Table 3, there was a statistically signif-icant difference in survival outcomes by Cox regression analysisfor patients treated with p53gene therapy versus methotrexatebased on p53 biomarker profiles. There was a statistically signif-icant treatment effect by tumor p53 profile interaction in termsof overall survival (P= 0.0215). Specifically for overall survival,the HR for the interaction was 0.249 (95% CI, 0.076-0.815), in-dicating that for patients with a favorable tumor p53 profile,there was significantly increased survival for treatment withp53 gene therapy. The HR for the effect of treatment (2.941;95% CI, 1.057-8.183) revealed that for patients with an unfa-

vorable tumor p53 profile, there was increased survival formethotrexate (P= 0.0388). These findings were consistent with

the p53 biomarker tumor response results and indicate thatp53gene therapy and methotrexate survival benefits were associatedwith different and complimentary groups of recurrent SCCHNpatients (Table 3).

Fig. 2. A, tumor p53 biomarker efficacy profilespredict p53 gene therapy survival benefit inrecurrent SCCHN. B, p53 biomarker profilesfavorable and unfavorable for p53 gene therapyefficacy do not predict methotrexate outcomein recurrent SCCHN.


Efficacy Biomarkers for p53 Gene Therapy in SCCHN



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Discussion

Knowledge of the genetic defects responsible for cancer pa-thology provides the opportunity to develop targeted moleculartreatments with specific therapeutic advantages. In addition topermitting the design of rational therapy addressing a funda-mental molecular pathway, targeted treatments also allow de-

velopment of companion predictive diagnostic biomarkers toguide the most appropriate application of these therapies.

These principles were exemplified in the present study describ-

ing the development ofp53 gene therapy for the treatment ofpatients with recurrent SCCHN characterized by a high frequen-cy of inhibited p53 function.

The results presented in this report indicate that tumor p53biomarker profiles can identify recurrent, refractory SCCHN pa-tients most likely to benefit from targeted p53 gene therapy

with statistically significant increased tumor responses, TTP,and survival. These p53 profiles were based on knowledge ofthe mechanisms tumors use to block p53 activity. In tumors

with normal p53 gene sequences, p53 may be inactivated byupregulation of the p53 inhibitors HDM2 and/or HDM4 asshown in our study and reported by other investigators (4,10). The combination of normal p53 delivered by gene therapyand endogenous wild-type p53 produced by these tumorsseems sufficient to overcome this form of p53 inhibition, andtumors with wild-type p53 gene profiles were found to be fa-

vorable forp53 gene therapy efficacy.Another major mechanism of p53 inactivation is through

gene mutations that result in the loss of p53 function. A similarfrequency of favorable p53 profiles (64.4%) was observed inthe IARC TP53 database for SCCHN cancers from Europe andNorthern America with both p53 immunohistochemistry andgene sequence information (n= 141). Consistent with the IARC

TP53 database (19), the vast majority ofp53 mutations in ourstudy (88%) were found in the DNA binding domain (exons 5-8), which may be associated with dominant-negative activitythat can inhibit normal p53 (1013). The majority of missense

mutations observed in our study population are defective fortransactivation (21 of 25, 84%) according to classification avail-able in the IARC TP53 database, and the majority of base sub-stitutions were G>T or from A:T bases (18 of 25, 72%), which isa pattern of mutation typical of tobacco and alcohol exposure(19). Several studies have shown a correlation between the con-centration of mutantp53 molecules and decreased p53 tran-scriptional activity of normal p5 3 (10, 12, 13). Theseobservations predict that tumors with low and high levels, re-spectively, of mutated p53 protein expression would have fa-

vo ra bl e an d un fa vo ra bl e pr of il es fo r p5 3 gene therapyefficacy. In our study, tumor p53 profiles with high-level expres-sion of mutated p53 were unfavorable forp53 gene therapy ef-

ficacy and patients with these unfavorable tumor p53 inhibitorprofiles had statistically significant decreased tumor responses,

TTP, and survival compared with patients with favorable tumorp53 profiles The ability of these molecular biomarkers to directtargeted p53 gene therapy was supported by consistent resultsamong several types of efficacy parameters and statistical anal-

yses reproduced in two sets of data from independently doneclinical trials. Our study patients had at least one lesion amena-ble to intratumoral injection and seemed to share similar p53characteristics representative of the recurrent SCCHN popula-

tion based on comparison with the IARC TP53 database for Eu-ropean and North American patients with SCCHN (19).The tumor p53 profiles predictive ofp53 gene therapy effects

did not similarly predict methotrexate efficacy outcomes, andthere was no statistical difference between tumor responses,

TTP, or survival of methotrexate-treated patients with p53 tu-mor profiles favorable or unfavorable forp53 gene therapy ef-ficacy. Hence, the p53 tumor profiles used in the study are notgeneral prognostic markers. This result is not surprising for themethotrexate-treated patients, as these predictive biomarkers

were developed based on known mechanisms of expectedp53 gene therapy efficacy.

Hence, our findings showed the ability of molecular bio-markers to direct efficacy of the p53 therapy to which they aretargeted. However, our results also illustrate the unexpectedutility of targeted biomarkers to guide the clinically beneficialapplication of other treatments. Patients with p53 profiles un-favorable for gene therapy efficacy had a statistically significantincrease in tumor growth control and survival when treated

with methotrexate. It was uncertain from previous clinical stud-ies whether methotrexate efficacy was affected by p53 function-al status (23). Our findings are consistent with in vitro studiesshowing therapeutic activity of methotrexate in tumors withp53 mutations (24). The clinical results of our study extendthese observations and indicate that p53 gene therapy andmethotrexate are efficacious in different and complementarygroups of recurrent head and neck cancer patients that can

be identified by tumor p53 biomarker profiles.The results of our study encourage the identification of pre-

dictive molecular markers to guide cancer patient management(14, 15). The survival outcomes comparing p53 gene therapyand methotrexate in the overall intent to treat populations

were not statistically significant. An important result of ourstudy is that traditional trial designs without predictive bio-markers for target-specific therapeutics may fail to identifyclinically beneficial therapies when the efficacy of the com-pared treatments occurs in different patient populations. Ourfindings also show that molecular biomarkers of targeted ther-apies may also prove useful in guiding the application of oth-er treatments, which have efficacy in patient populations

Table 3. Interaction of treatment and tumor p53 profiles in terms of survival by Cox regression analysis

Phase III trial

treatment

Tumor p53 profile for p53

gene therapy efficacy

Median survival

(mo)

HR (95% CI) P

p53 gene therapy Favorable 7.2 0.249 (0.076-0.815) Overall survival

P= 0.0215Methotrexate Favorable 4.3

p53 gene therapy Unfavorable 2.7 2.941 (1.057-8.183)

Methotrexate Unfavorable 5.9





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where the targeted treatment is not efficacious. As illustratedby our findings, pairing of treatments for comparison in clin-ical trials with different mechanisms of action combined withthe performance of predictive biomarkers may result in theability to direct potentially efficacious individualized treat-ment for both therapies. These results show useful principlesand the benefits of incorporating molecular biomarkers in thedesign of cancer clinical trials.

In conclusion, the results of this study have implications forthe treatment of patients with recurrent SCCHN. The data sug-gest thatp53 gene therapy is efficacious for tumors with favor-able p53 biomarker profiles and that p53 gene therapy mayrepresent a new treatment option for patients whose tumorsare refractory to standard therapies. In addition, the p53 bio-marker analyses of survival and tumor response indicate thatp53 gene therapy and methotrexate have therapeutic efficacyin different and complementary groups of recurrent SCCHN pa-tients refractory to platinum or taxanes. Hence, these p53 bio-marker profiles permit the personalized selection of potentiallyefficacious p53 gene therapy or methotrexate treatment for re-

current SCCHN patients. Further studies will be required to de-termine if the tumor p53 biomarker profiles predictive ofp53gene therapy efficacy in recurrent SCCHN will also be useful toguidep53 gene therapy of other tumors that are known to havesimilar p53 molecular abnormalities.

Disclosure of Potential Conflicts of Interest

J. Nemunaitis, G. Clayman, W. Hrushesky, J. Wells, C. Moore, J. Hamm,G. Yoo, J. Baselga, J.A. Roth, B.A. Murphy, and W.J. Goodwin have re-

ceived research funding from Introgen Therapeutics; K.A. Menander, L.L.

Licato, S. Chada, and R.E. Sobol hold employment or leadership positions

with, as well as stock in, Introgen Therapeutics; R.D. Gibbons, J.A. Roth,

and R.E Sobol are consultants for Introgen Therapeutics.

Acknowledgments

We thank Drs. Arlene Forastiere, Carol Prives, David Sidransky, Bert

Vogelstein, and Everett Vokes for helpful suggestions; Susan Mill, Tresha

Goldsmith, and Martha French for their assistance in preparing the manu-

script; and Sara Koehler, Wesley Gage, Dr. Hector Battifora, and Dr. Frank

Lynch for assistance with the immunohistochemistry studies.

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Efficacy Biomarkers for p53 Gene Therapy in SCCHN

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