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[CANCERRESEARCH56. 1737—1741,April 15,19961
Advances in Brief
Combination Gene Therapy for Oral Cancer in a Murine Model'
Bert W. O'Malley, Jr.,2 Keary A. Cope, Shu-Hsia Chen, Daqing Li, Mary R. Schwartz, and Savio L. C. Woo
Johns Hopkins University, Baltimore, MaryLand 21203 (B. W. 0., K. A. C.. D. LI. and Baylor College ofMedicine, Houston. Texas 77030 [S-H. C., M. R. S., S. L C. W.J
Abstract
Combinationtherapy involvingadenovirus-medlatedtransfer of thegenes for herpes thymidine kinase (tk) and murine interleukin 2 (mIL-2)was used to treat head and neck cancer In C3HIHeJ mice. Tumors weregenerated by transcutaneous Injection of S x@ murine squamouscarcinoma cells into the floor of the mouth of these syngeneic mice. After1 week, recombinant adenoviral vectorscontaining both therapeutic andcontrol genesin variouscombinationswere Injecteddirectly into theestablished tumors, and subsequently all mice were administered gancidovir twice daily (25 mg/kg) for 6 days.Animals receivingeither tk aloneor 1* + mIL-2 demonstratedsignificanttumor regressioncomparedtomIL-2 alone or control vector-treated mice (P < 0.008). MIce receivingboth tk + mIL-2, however, also demonstrated a significantly greaterregressionof tumors comparedto thosetreated with tk alone(P < 0.008),indicating a synergistic effect of the combination gene therapy. Thissynergismwas confirmed In survival studiesbecausetk + mIL-2-treatedmice showed increased survivals (P = 0.0002). ClinIcal and microscopicexam of regional surrounding tissues and distant orgaus showed no evidenceof cytotoxicity for representative animnic in each experimental group. Theseresults suggest that combination ik and mJL-2 gene therapy may provide a
powerful new modality for the treatment of head and neck cancer.
Introduction
Oral cavity andoral pharyngealtumorsare amongthe mostmorbidof human cancers and compromise the majority of the estimated42,800 new cases of head and neck cancer annually (1). Standard
therapy includes deforming radical surgical procedures coupled withradiotherapy and possibly chemotherapy. In addition to the severecosmetic deformity, surgical resection frequently results in significantfunctional deficits in speech, swallowing, and upper extremitystrength. Radiotherapy also brings substantial morbidity with mandible and laryngeal cartilage radionecrosis,soft tissue fibrosis, andmucosal atrophy, pain, and xerostomia (2). Chemotherapy regimensare not innocuous as well, and frequent complications from their use
include gastrointestinal, bone marrow, ototoxicity, and renal toxicity(2). Furthermore, the efficacy of adjuvant or induction chemotherapyin headandneckcancerremainsquestionable,with no clinical trial todate demonstrating improved survival (1). Despite years of investiga
tion of the treatment of head and neck cancer, outcomesremainconsistently poor with 2-year survivals rarely exceeding 30% inpatients with advanced stage III and IV disease(1, 3). Becauseof thedismal results of conventional therapy, a new thrust in treatmentstrategies has focused on the use of gene therapy. Two of the mostpromising strategies to date include the use of “suicidegenes―andcancer vaccines based on immunotherapies.
The herpessimplex virus tk@gene is a prototype“suicidegene―
Received2/6/96;accepted3/11/96.The costsof publicationof this article were defrayed in part by the paymentof page
charges.This article mustthereforebe herebymarkedadvertisementin accordancewith18U.S.C.Section1734solely to indicatethis fact.
I Supported in part by a grant from the Markey Foundation.
2 To whom requests for reprints should be addressed, at Department of Otolaryngology-HNS,JohnsHopkinsUniversity,P.0. Box 41402,Baltimore,MD 21203-6402.
3 The abbreviations used are: tk, thymidine kinase; GCV, ganciclovir, mIL-2, murine
interleukin2; ADV, adenovirus;RSV, Roussarcomavirus; ADV/RSV-tk, recombinant
becauseit encodesa viral enzyme that is foreign to mammalian cells,which will convert an inactive prodrug to a toxic product. Upon viraltransduction, the tk gene selectively kills dividing cells (cancer cells)by converting GCV into a phosphorylated compound that terminatesDNA synthesis(4). SinceGCV is an excellentsubstratefor viral ticsand a poor substrate for mammalian tics, concentrations can beachieved that are lethal to cells expressing tic but are nontoxic tonormal mammalian cells (5—7).An additional advantage of tk genetransfer is the presence of a “bystandereffect.―In both murine andhuman tumor models, surroundingnontransduceddividing cells arealso killed, presumably from the transfer of a toxic metabolite of GCVvia gap junctions or endocytosis of apoptotic vesicles from virallytransduced dying tumor cells (8, 9).
Recently,the evolution of cancervaccineresearchhas led to thetransplantation of genetically modified tumor cells that generate cytokines locally and elicit an antitumor immune response. The mostcommon strategies developed in animal models involve the transduction or transfectionof establishedtumor cell lines with variouscytokine genes in vitro, followed by the implantation of these modifiedcells into immune competent animals (10, 11). Several studies haveshownthat the transductionof murine tumor cells with the genesformIL-2 (10, 12) have resulted in the rejection of these tumor cells afterimplantation into syngeneic hosts. In addition to the local tumoricidalinflammatory effect, injection of cytokine-secreting tumor cells gencrates systemic immunological responses capable of protecting animals from subsequentchallenge with the same tumor at a distant site(11).
For the suicidegene therapy and cytokine vaccine strategies,theuseof a recombinantadenoviralvectorsystemhasmany advantages.Unlike the low efficiency of previously described retroviral systems(13, 14), adenoviral vectors have very high titers and efficiency fordirect delivery into cancer cells in vivo (15). As a safety factor for thesuicide gene therapy strategy, the rapidly dividing tumor cells will beselectively targeted for adenoviral transduction over the surroundingnormal tissues. For cytokine therapies, the use of recombinant adenoviral vectors should prove valuable in eliminating the need for dailylocal cytokine suspension injections while still providing the important paracrine cytokine function. This will also greatly reduce themoderate to severe toxicities including fever, chills, headaches,andcapillary leak syndrome associatedwith systemic mIL-2 delivery (16).With respectto cancervaccinestrategies,theadenoviralvectorsystemwould eliminate the time-consuming and costly procedures of harvesting tumor specimens, isolating cancer cells, culturing cells, treating with radiationand genetransferin vitro, and then re-introducingthe engineered tumor cells back into the patients as a vaccine (11).
Adenoviral vectors containing the tic and mIL-2 genes can also beincorporated into a single injection procedure into primary tumors toassessthe potential benefit of both a direct cytotoxic and immuneenhancing gene therapy strategy. For thesereasons,we have chosen toevaluate single gene versus combination adenoviral gene transfer
ADV vectorcontainingthe tk genedrivenby theRS―/long terminalrepeat;ADV/RSV@-gal,samevectorasabovebut containingthe genefor @-gatsctosidase;SCC, squamous
cell carcinoma;MOl, ratio of viral plaque-formingunits to tumor cell.
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COMBINATIONGENE THERAPY FOR ORAL CANCER
In Vitro Experiments. The 5CC line, 5CC VII, was used in all experiments. Originally, SCC VII arose spontaneously in C3H/HCJ mice and has
subsequentlybeenpropagatedin vivo (19). The cells wereculturedin T-75tissue culture flasks (Corning) containing 30 ml of RPMI 1640 (Sigma Chemical Co.), 12% bovine calf serum, 1% penicillin-streptomycin, and 1% L
glutamine. Cells were maintained in 10% CO2 incubators.
In Vivo Treatment and Survival Experiments. All animal experimentswere performed on C3HIHeJ mice (Jackson Laboratories) using sterile technique under a laminar flow hood. Mice 6—10weeks old were anesthetized
using the inhalational agent Metophane, and a 0.1-nil suspension of 5 X l0@SCC VII cells in HBSS were injected directly into the floor of the mouth. Theanimals were then maintained in standard housing conditions.
Six days after cell implantation,mice were anesthetizedwith 0.5 ml ofavertin at a concentration of 20 mg/mI with the depth of anesthesia determined
by toe pinch. A skin incision was made in the lower neck, and surgicaldissection revealed the established floor of mouth tumors. Tumors were
measured in three dimensions with calipers. Using a lOO-pJ syringe (Hamilton,Reno, NV) and 26-gauge needle, 1.0 X LO@total plaque-forming units of either
ADV/RSV-tk, ADVfRSV-@3-ga1control, ADV/RSV-tk + ADVIRSV-mJL-2(2.0 X 10@),or ADVIRSV-mIL-2 (2.0 X 108) + ADV/RSV-@3-gal in 60 ,.dsolution were injected directly into the tumors. Neck incisions were closedwith 4—0silk suture (Ethicon). Eighteen h after adenoviral injection, the micewere administered GCV i.p. at a regimen of 25 mg/kg twice daily. Mice weresacrificed on the seventh day after adenoviral delivery, the neck incision wasreopened, and the tumor sites were examined. Residual tumor masses weremeasured, and specimens including normal surrounding tissues were sent forroutine histology. The pathologists were blinded to the treatment regimensuntil after interpretation of the results. The significance of outcomes was
determinedby Mann-Whitneyanalysisand was further confirmedby Dunnets's test of multiple comparisons.
A survival experimentwas performedunder the sameconditions, andanimalswereevaluateddaily. Tumor size was assessedevery 3 daysusingcalipermeasurements.The significanceof survivaloutcomeswasdeterminedusing identical statistical analysis as in the preceding experiments.
Histology. At the time of necropsy, harvested tumor, surrounding tissues,and distant organs were placed in 10% buffered formalin for fixation. Thespecimens were then embedded in paraffin, sectioned, and stained with H&E.
Results
Efficacy of Adenoviral Transduction in Vitro. ADV/RSV-tk wasdelivered to SCC VII cells in vitro to assessthe efficacy of cancer cellregression after subsequent administration of GCV (Fig. 1A) After
ADV/RSV-tk transduced cells (%)
Fig. 1. A, ADV/RSV-tk transduction of SCC VII squamous carcinoma cells in vitrofollowed by treatmentof eitherGCV at 10 @sg/ml(U) or PBScontrol (U). Cell survivalwasassessed3 daysaftertransduction,andGCV administrationresultedin 93—98%tumor $P'O.OScell killing with MOls rangingfrom 25 to 100.B, bystandereffectdepictedaftervarious _________proportionsof ADvIRSV-tk-transducedcells (at a MOl of 100)weremixed with nontransduced 5CC VII cells. Four days after subsequentadministration of GCV (@) or PBS(U). parental tumor cell toxicity reached 90—100%at low ratios of transduced cells.
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tk+IL 2 tic B-gal+IL 2 B-gal
Treatment Group
Fig.2. Box andwhiskerplot of residualtumorsizesasmeasuredby concurnngclinicalandhistopathologicalinterpretationin animalsaftervariousgenetherapytreatments.Thenumbersof animalsin eachgroupwere 11(tk + mlL-2), 11(tk), 7 ((3-gal+ mIL-2), and7 (gS-gal). The range of data points is depicted by the vertical lines, the boxes representthe middle 50% of data points, and the bisecting lines show the median values. Statisticalsignificancewasassessedby Mann-Whitneyanalysisandconfirmedby Dunnett'stestofmultiple comparisons.
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using tk and mouse mIL-2 vectors in a recently developed immuno
competent murine model for head and neck SCC. This study investigates the efficacy and synergism of this combination gene therapystrategy on primary tumor regression and animal survival.
Materials and Methods
Construction of Recombinant Adenoviral Vectors. Construction of a
replication-defective adenoviral vector containing the tk gene under transcnptional controls of the RSV long terminal repeat (ADVIRSV-:k) has been
reported previously (17, 18). A replication-defective adenoviral vector contaming the mIL-2 cDNA under the transcriptional control of the RSV longterminal repeat promoter (ADVIRSV-mIL-2) was similarly constructed and
plaque purified. The viral titer (plaque-forming units/mI) was determined byplaque assay.
1738
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COMBINATIONGENE THERAPY FOR ORAL CANCER
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Fig. 3. Photomicrographsof tumors from various treatment groups.A, completelyviable tumor in control animalafterADV/RSV-@-galtransductionwith OCV,demonstratingpoorly differentiated SCC (H&E, X400). B, ADV/RSVtk + mIL-2 and GCV-treated tumor showing extensive zonalnecrosis(rightto left) with inflammatoryinfiltrate (X200). C,anotherADV/RSv-tk+ mlL-2andGCV-treatedtumordepicting interminglednecrosis,swollendying cells with pyknotic nuclei, someviable tumor cells, and inflammatoryinfiltrate (X400).
1739
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COMBINATION GENE THERAPY FOR ORAL CANCER
transduction with ADVIRSV-tk and GCV treatment, less than 10%viable cancercells remainedat a MOl of 25 and 98% cell deathwasachieved at a MO! of 100. ADV/RSV transduction followed by PBSas a control, however, showed no cell death. The presence of abystandereffect was exemplified by mixing ADVIRSV-tk transducedSCC VU cells with nontransducedtumor cells in variousconcentrations. Only 3—33%ADV/RSV-tk transduced 5CC VII cells wererequired to confer GCV toxicity to 90—100%of the parental tumorcells in vitro (Fig. 1B). Efficacy of the replication-defective ADV/RSV-mlL-2 vector at an MO! of 20 had been established previously invitro on B 16 cells using a T-cell proliferation assay on conditionedmedia (data not shown).
Regression of Established SCCs after Combination Gene Therapy in Vivo.After implantationof 5 X l0@tumorcells into 36animals, the mice showed rapid tumor growth in the floor of themouthover 5 to 7 days.Therewere no signsof cachexiaduring thisperiod, and all animals appearedclinically healthy at the time ofadenovirus injection. Four experimental groups were designed asfollows: group 1, ADVIRSV-@3-ga1control virus injection ((3-gal);group 2, ADV/RSV-mlL-2 + ADV/RSV-f3-gal ((3-gal + mIL-2);group 3, ADV/RSV-tk alone (tk); and group 4, ADVIRSV-tk + ADV/RSV-mIL-2 (tk + mIL-2). After direct intratumoral viral delivery, allanimals were treated with GCV at 25 mg/kg twice daily for 6 days andthenwere sacrificedon the seventhday after adenoviraldelivery. Anyresidual tumors were measured and harvested for histopathologicalevaluation. All animals showed no signs of cachexia or change ineating habits during the treatment period, and all were clinicallyhealthy at the time of necropsy.
Pretreatment tumor volumes ranged from 70—160mm3 with anoverall averagesizeof 122.5mm3.There were no significant variations in pretreatment tumor sizes between each tumor group, asdeterminedby Mann-Whitney analysis (data not shown). Animalstreated with either tk or combination tk + mJL-2 demonstrated significant tumor regression as compared to the large tumors found inmIL-2 alone or (3-gal control injection mice (P < 0.008; Fig. 2). Threeof the combinationtic+ mJL-2-treatedanimalshad nearcompletetumor eradication with residual necrotic masses less than 2 mm3;however, none of the remaining groupsdemonstratedsuchdramaticeffects. Overall, the combinationof tk + mIL-2 showedthe greatesteffect on tumor regression and was significantly better than tk alone(P < 0.008). Although the tumor sizes in the (3-gal + mIL-2 animalsshowed less tumor progressionthan the (3-gal alone controls, theywere not significantly different based on 95% confidence limits.
Histopathological analysis revealed poorly differentiated 5CC in
residual tumors and controls. There was substantial tumor necrosis,including regionsof zonal necrosisabuttingareasof mixed necroticand viable cells noted especially in the tk + mIL-2 and tk alonetreatment groups (Fig. 3). The residual tumors had low levels ofmacrophage infiltrates and variable levels of lymphocyte infiltrationnoted more commonly in the mice receiving mIL-2 gene transfer.Distant organs including lung, liver, bowel, and kidneys were exammed from threeanimalsin eachexperimentalgroup,and no evidenceof necrosisor cytotoxicity or viral inclusionbodieswere seen(datanot shown).
Survival Experiment, To evaluate the long-term effects of combination gene therapy, a survival experiment was performed on 38animalswith established5CC VII tumors using identical treatmentregimensas in the precedingexperiment.The effects of adenovirustreatment were assessedby visual inspection, palpation, and calipermeasurements of tumors three times a week (Fig. 4.). Survival wascalculated in days after original adenovirus injection, and end pointwas assessedby either animal demiseor when tumorsincreasedto 2cm in any dimension (cachexia is present in all animals with tumors
I@ Ip@2
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1740
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Treatment Group
Fig.4. Box andwhiskerplot for animalsurvivalaftervariousgenetherapytreatmentsThere were I I mice in the tk + mIL-2 group, and the remaininggroupscontained9animalseach.The synergismof combinationtherapywas depictedby the significantsurvival ofcombination tk + mIL-2-treated animals over the tk-alone group(P = 0.0002).Both tk + ntIL-2 and tk alone demonstrated significant survival over the remaining groups.Statistical determinations were performed using Mann-Whitney analysis.
of this size), asrequiredby the Institution'sAnimal Care Committee.Mice receiving ik + mJL-2 combination therapy demonstrated significant survival over both the tk alone and remaining groups(P = 0.0002). The tk alone-treated animals showed significant survival over the (3-gal + mIL-2 and control (3-gal-alone group. Animalsreceiving (3-gal + mIL-2 showed no significant survival over thecontrol group.
Discussion
These experiments demonstrate three important aspectsand effectsof adenovirus-mediatedgenetransferon 5CC regressionin a murineanimal model:
(a) Initial in vitro studies established that tk gene delivery andGcv administrationprovedirectlycytotoxicto SCCsandinitiatea bystander effect, whereby surrounding nontransducedcells aredestroyed.Only 3—33%of transducedtumor cells were required tokill 90—100%of the total tumor cells in culture. The presence ofthis effect is a major strength of this suicide gene therapy strategyand an essential therapeutic component when considering actualclinical application. Known limited diffusion of adenovirus afterdirect in vivo injection (17, 18) could greatly reduce therapeuticbenefit in larger human tumors. Incorporation of a bystandereffect, however, may circumvent unforeseen limitations of viraltiters, delivery volumes, or actual accessto complete tumor injection in the human patient.
(b)ThedeliveryoftktoestablishedtumorsinvivofollowedbyGCV administration results in significant tumor regression and improved survivalsover animals treated with adenoviruscarrying themurine mIL-2 gene alone as well as control animals. This findingshows the strength of tic over a cytokine approach when consideringsingle gene therapy for cancer treatment. Upon considering the lack ofefficacy of mlL-2 alone despite a notable increasedinflammatoryinfiltrate in mJL-2-treated animals, issues of injection volume or totalviral particle delivery cannot be raised becauseeach of thesevariableswereconstantamongthetreatmentgroups.Furthermore,the viral titerfor mIL-2 delivered to the murine tumors was at the high end of safedosing basedon previous toxicity studiesfor this vector (data notshown). In this tumor model, the use of mlL-2 alone as a adenoviralstrategy paralleling classic cancer vaccine approaches is not an acceptablechoiceof therapeuticregimens.
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COMBINATION GENE ThERAPY FOR ORAL CANCER
5. Moolten, F. L. Tumor chemosensitivity conferred by inserted herpes thymidine kinasegenes:paradigmfor a prospectivecancercontrol strategy.Cancer.Res.,46: 5276—5281,1986.
6. Moolten,F. L., andWells,J.M. Curabilityoftumorsbearingherpesthymidinekinasegenestransferredby retroviral vectors.J. Natl. Cancer Inst., 82: 297—300,1990.
7. St. Clair, M. H., Lambe,C. U., andFurman,P. A. Inhibition by ganciclovirof cellgrowth and DNA synthesisof cells biochemically transformed with herpesvirusgenetic information. Antimicrob. AgentsChemother.,31: 844—889, 1987.
8. Freeman,S.M., Abboud,C.N., Whartenby,K. A., Koeplin,D. S.,Moolten,F.L., andAbraham, G. N. The “bystandereffect―:tumor regression when a fraction of thetumor massis geneticallymodified.CancerRes.,53: 5274—5283,1993.
9. Wan, L. B., Parysek,L. M., Warnick, R., and Stambrook,P. J. In vitro evidencethatmetabolic cooperation is responsible for the bystander effect observed with HS'v' tkretroviral gene therapy.Hum. Gene Ther., 4: 725—731,1993.
10. Fearon, E. R., Pardoll, D. M., Itaya, R., Golumbek, P., Levitsky, H., and Simons,J.Interleukin-2productionby tumorcells bypassesT helperfunction in thegenerationof an antitumorresponse.Cell, 60: 397—403,1990.
11. Dranoff, G., Jaffee,E., Lazenby, A., Golumbek, P., Levitsky, H., Brose,K., Jackson,V., Hirofumi, H., Pardoll, D. M., and Mulligan, R. C. Vaccination with irradiatedtumorcellsengineeredto secretemurinegranulocyte-macrophagecolony-stimulatingfactor stimulatespotent,specific,and long lastinganti-tumorimmunity. Proc.Natl.Acad. Sci. USA, 90: 3539—3543,1993.
12. Gansbacher,B., Zier, K., Daniels, B., Cronin, K., Bannerji, R., and Gilboa, E.lnterleukin-2 genetransfer into tumor cells abrogatestumorigenicity and inducesprotective immunity. J. Exp. Med., 172: 1217—1224,1990.
13. Culver, K. W., Ram,Z., Wallbridge,S., Ishii, H., Oldfield, E. H., andBlaese,R. M.In vivo gene transfer with retroviral vector-producer cells for treatment of expenmental brain tumors. Science (Washington DC), 256: 1550—1552,1992.
14. Oldfield, E. H., Ram, Z., Culver, K. W., Blaese, R. M., DeVroom, H. L., andAnderson, W. F. Gene therapy for the treatment of brain tumors using intra-tumoraltransductionwith thethymidinekinasegeneandintravenousganciclovir.Hum.GeneTher.,4: 39—69,1993.
15. Stratford-Pemcaudet,L. D., Levrero,M., Chasse,J. F., Pemcaudet,M., andBriand.P. Hum.GeneTher.,1: 241—256,1990.
16. West, W. H., Taver, K. W., Yannelli, J. R., Marshall, G. D., Orr, D. W., Thurman,G.B.. and Oldham,R. K. Constant-infusionrecombinantinterleukin-2in adoptiveimmunotherapyof advancedcancer.N. EngI.J. Med.,316: 898—905,1987.
17. Chen,S-H., Shine,D., Goodman,J. C., Grossman,R. G., andWoo, S. L. C. Genetherapyfor braintumors:regressionof experimentalgliomasby adenovirus-mediatedgenetransfer.Proc.Nail. Acad.Sci. USA, 91: 3054—3057,1994.
18. O'Malley. B. W., Jr., Chen,S-H., Schwartz,M. R., andWoo, S. L. C. Adenovirusmediatedgenetherapyfor humanheadand neck squamouscell cancerin a nudemousemodel. Cancer Res., 55: 1080—1085,1995.
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20. Chen,S-H.,Li. Chen,X. H., Wang,Y., Kosai,K-I., Finegold,M. J., Rich,S.S.,andWoo, S. L. C. Combinationgenetherapyfor liver metastasisof colon carcinomainvivo. Proc. Natl. Acad. Sci. USA, 92: 2577—2581,1995.
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(c) The most important aspect of this work is the demonstration ofa synergisticeffect after combinationtk and mJL-2 gene transferonprimary oral cancer. Although a recent report by Chen et al. (20)described a similar short-term effect of combined tk and mJL-2 therapy on regressionof metastaticcolon carcinoma,this previousstudydid not demonstrate a survival advantage (20). In the oral cancermodel, the combining of tk and mIL-2 followed by GCV administration was more effective than tk alone, mJL-2 alone, and (3-gal viruscontrols for both short-term and survival studies. This work introducesa merger between a strong direct in vivo gene therapy strategy with astandard ex vivo cancer vaccine (immune-enhancing) modality for thetreatment of primary head and neck cancer. The results suggest thatthe presence of cell debris or local tumor cytotoxic effects (from tkand GCV) provides a medium from which the local expressionofmIL-2 can activate a significant antitumor effect. A more detailedinvestigation of the immune response generated by the combinationtherapyis in progress,and additionalstudiesmay allow full interpretation of the observed synergism. This work establishes efficacy ontumor regression and survival and provides a foundation for advancing combination gene therapy for the treatment of head and neckcancer.
Acknowledgments
We thank Lois Brandon for her expert technical assistance with tissue
cultureexperimentsandAnnenaWalkerof theDermatopathologyLaboratoryat Baylor College of Medicine for preparation of histological slides.
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3. Ervin, T. J., Clark, J. R., Weichselbaum,R. R., Gallon, B. G., Miller, D., Fabian,R. L., Posner,M. R., Norris,C. M., Tuttle,S. A., Schoenfeld,D. A., Price,K. N., andFrei, E., Ill. An analysisof inductionandadjuvantchemotherapyin the multidisciplinary treatmentof squamous-cellcarcinomaof the headand neck. J. Clin. Oncol.,5: 10—20,1987.
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1996;56:1737-1741. Cancer Res Bert W. O'Malley, Jr., Keary A. Cope, Shu-Hsia Chen, et al. Combination Gene Therapy for Oral Cancer in a Murine Model
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