Int[ J[ Immunopharmac[\ Vol[ 08\ No[ 00:01\ pp[ 508Ð516\ 0886\ Pergamon Þ 0887 International Society for ImmunopharmacologyPublished by Elsevier Science Ltd[ Printed in Great Britain

9081Ð9450:87 ,08[99¦9[99

PII] S9081Ð9450"86#99948Ð2

A NATURAL CYTOKINE MIXTURE "IRX!1# AND INTERFERENCE WITHIMMUNE SUPPRESSION INDUCE IMMUNE MOBILIZATION AND

REGRESSION OF HEAD AND NECK CANCER

EMMA VERASTEGUI\�& JOSE LUIS BARRERA\$& JUAN ZINSER\�& ROXANA DEL RIO\&ABELARDO MENESES\%& JAIME DE LA GARZA& and JOHN W[ HADDEN'

� Department of Medicine\ $ Department of Surgery\ Head and Neck Division\% Department of Pathology\ & Instituto Nacional de Cancerologia\ Mexico\ D[F[ ^

' Division of Immunopharmacology\ Department of Internal Medicine\ University of South FloridaCollege of Medicine\ Tampa\ Florida\ U[S[A[

"Received 7 July 0886 and in _nal form 20 July 0886#

Abstract*Prior studies indicate that combination immunotherapy of squamous cell cancer "SCC# of head andneck "H+N# with cytokines is feasible "Hadden et al[\ 0883#[ To induce immune regression of H+N SCC 19stage IIÐIV patients received 2 weeks prior to surgery low dose cyclophosphamide "299 mg:M1#\ then 09 dailyperilymphatic injections of a natural cytokine mixture "IRX!1# "049 units of IL!1 equivalence# and daily oralindomethacin and zinc[

Tumor responses\ T!lymphocyte and subset counts\ and toxicity were monitored[ Six patients had majorclinical responses "both complete ðCRŁ and partial ðPRŁ# without major toxicity[ Five of 19 patients werelymphocytopenic "0131277 mm2# prior to treatment and the immunotherapy induced marked signi_cantincreases in total lymphocyte counts\ CD2¦ T!cells\ and both CD3¦ and CD7¦ T!cells as well as apopulation of CD2¦\ CD3−\ and CD7− lymphocytes[

The post treatment specimen of 07:19 patients showed histologically tumor fragmentation\ overall reductionand di}use in_ltration with lymphocytes and plasma cells[ Histologic tumor reductions in these patientsaveraged 33) and the lymphoid in_ltration increased 3[6 fold from 8Ð31)[ The immune in_ltration of thetumor re~ects varying degrees of both T! and B!cells and indicates immunization to the tumor[ The immu!nization achieved may improve clinical control of H+N SCC by improving the possibility that surgicalresection of advanced loco!regional disease will leave no viable tumor[ Þ 0887 International Society forImmunopharmacology[

Keywords] head and neck cancer\ immunotherapy\ natural cytokine mixture\ interleukins\ immune regression[

Squamous cell carcinoma of the head and neck "H+NSCC# arises in a background of cellular immunede_ciency with decreased T!cell number and function\macrophage:monocyte dysfunction\ and anergy"Katz\ 0882 ^ Hadden\ 0884a#[ Humoral immunity\ ifabnormal\ involves elevated IgA levels "Katz\ 0872#[These indicators of altered immunity are prog!nostically signi_cant indicating that immunity isimportant in the outcome of H+N SCC to con!ventional therapy "Hadden\ 0884a ^ Richtsmeier\0886#[ A variety of immunosuppressive factorsderived from the tumor and from the immune systemitself have been implicated in the cellular immunede_ciency "Hadden\ 0884 ^ Young et al[\ 0885#[ Tumorin_ltrating lymphocytes "TILs# have been dem!onstrated in most H+N SCC "Wolf et al[\ 0875# ^ TILsare known to re~ect paralyzed T!cells with capability\when restored with interleukin 1 "IL!1#\ to react with

508

speci_c cytotoxicity to autologous tumor "Heo et al[\0876#[ In addition\ a number of tumor!associated anti!gens have been linked to H+N SCC "Cf Hadden\0884a ^ Richtsmeier\ 0886#[

Initial experimental immunotherapy e}orts haveyielded some positive results "Cf Hadden\ 0884a# ^however\ no particular immunotherapy has gainedgeneral clinical acceptance[ In 0877\ Cortesina et al["0877# observed partial "PR# and complete "CR#responses in 5 out of 09 H+N SCC patients givenperilymphatic injections of a low dose of a naturalinterleukin 1 mixture "199 units:day×09#[

Attempts to con_rm these observations with higherdoses or with recombinant IL!1 "rIL!1# in varyingdoses yielded less convincing and even poor results"Cortesina et al[\ 0880 ^ Mattijssen et al[\ 0880 ^ Corte!sina et al[\ 0883 ^ Vlock et al[\ 0883#[ Hadden et al["0883# employed a natural IL!1 mixture "NCM# with

E[ VERASTEGUI et al[519

low dose cyclophosphamide\ indomethacin\ and zincin 3 H+N SCC patients and observed evidence ofimmune rejection of tumor with long!term palliationin 1 patients with recurrent disease and prolongeddisease!free survival in a third patient who was treatedprior to surgery[

In an e}ort to extend these _ndings\ an open studyof this treatment strategy was performed in 19 H+NSCC patients who were treated with the 2!week cycleof combination immunotherapy prior to surgery atthe National Institute of Cancerology "NCI# in Mex!ico City "Barrera et al[\ 0885\ 0886#[ This reportemphasizes the immunologic _ndings in these patientsand provides evidence for immune restoration as acentral feature of this therapy[

EXPERIMENTAL PROCEDURES

Patients\ materials and methods

Patients[ Twenty patients with H+N SCC of theskin\ sinus\ oral cavity\ oro!pharynx\ hypopharynx\or larynx consented to participate in this study whichwas approved by the Research Committee and Insti!tutional Review Board of the Mexican NCI and bythe Mexican Federal Drug Regulatory Authority"Mexican FDA#[ All patients met the following cri!teria ] biopsy proven SCC of the stated areas with orwithout locoregional extension but without distantmetastasis ^ no prior therapy ^ no other signi_cant dis!eases ^ and a positive skin test to intracutaneousadministration of the NCM[ With two exceptions\ thepatients were considered to be surgical candidates andunderwent surgery and radiotherapy following theimmunotherapy[

Materials and methods[[ The natural cytokine mix!ture IRX!1 was prepared from human peripheralblood lymphocytes stimulated by phytohemagglutinin"PHA# under serum!free medium "X!Vivo!09\BioWhittaker\ Walkersville\ MD# as described"Hadden et al[\ 0884#[ Two batches prepared from 5blood donors each and screened by the Blood Bankfor\ among other\ hepatitis B and C\ human T!cellleukemia virus I and II and AIDS "HIV# were made\_ltered "9[1 m#\ and bottled[ The _nal preparation wasendotoxin!free "³9[4 EU:ml# by limulus Lysate assay"BioWhittaker#\ sterile in 1 culture media\ and HIV!negative by quantitative polymerase chain reaction"PCR# "SmithKline Beecham\ Tampa#[ Each batchcontained approximately 049 units of IL!1:ml byELISA "R+D Systems\ Minneapolis\ MN# and 539units of IL!1 activity by bioassay "Gillis et al[\ 0867#re~ecting activity of other cytokines in the mixtureincluding pico to nanomolar quantities of Il!0\ IL!5\IL!7\ IL!09\ IL!01\ interferon gamma "IFN!g#\ colony!

stimulating factors "CSF#\ for granulocytes "G#\ andmacrophages "M# and tumor necrosis factor!a"TNFa#\ but free of Il!2\ IL!3\ IL!4\ and Il!6[ The vials"09 ml# were stored frozen "−69>C# until use[ Eachpatient who quali_ed and signed the consent formsreceived an IRX!1 skin test which\ if positive at 13 h\allowed entry to the protocol[ On the protocol\ eachpatient received an initial low dose of cyclo!phosphamide "299 mg:M1# by intravenous infusion\09 daily 0 ml injections administered at the insertionof the sternocleidomastoid muscle as described"Cortesina et al[\ 0877 ^ Hadden et al[\ 0883#\ anddaily oral indomethacin "14 mg thrice daily# and zincgluconate 54 mg daily as Stress TabsTM[ The patientswere followed carefully for signs of toxicity andchange in tumor by physical examination[ All patientshad a complete WBC count\ HIV serology\ and theroutine pre!surgical laboratory and clinical work!up[A battery of skin tests consisted of the intracutaneousadministration of IRX!1\ puri_ed protein derivative"PPD ^ Lederle\ Pearl River\ NY#\ and the MerieuxMultitest skin test device "Merieux\ Lyon\ France#[Total T!cell counts and subsets from peripheral bloodmononuclear cells were done by indirect immu!no~uorescence using monoclonal antibodies directedagainst CD2\ CD3 and CD7\ "Dako\ Carpinteria\CA#[ The samples were read in a FacSort "BectonDickinson\ Los Angeles\ CA#[ These counts were donebefore and after the completion of immunotherapy"Day 9 and Day 02#[

Routine surgical pathology was performed on allpre!treatment and post!treatment specimen biopsies[To quantitate the tumor:stroma:lymphocyte relation!ships\ a typical biopsy section containing tumor wasselected under low power and the quantity of tumorwas expressed as a percent of the total[ The remainingstroma was evaluated for the percent of the area hav!ing lymphocytes[ The surgical specimen was similarlyevaluated and percent areas ascribed to the respectivechanges[ The tumors were further evaluated withrespect to the percent which was solid like the biopsyand the percent fragmented with interspersed\ in_l!trating lymphocytes and in~ammatory cells[ The per!cents of stroma and the area heavily in_ltrated byleukocytes were estimated and the in_ltrating lym!phocytes\ plasma cells\ granulocytes\ and mac!rophages were estimated as a percent of the totalin_ltrating cells[ Immunohistology for T!cells "CD34#and B!cells "CD19# was performed on para_n!_xedsurgical specimens by using the horse radish per!oxidase method "Dako\ Carpinteria\ CA#[ Thesevalues were charted on a data sheet and the valuesaveraged to express the data in a semiquantitativemanner "2standard error of the mean#[

Immune Regression of H+N Cancer 510

RESULTS

Most patients were treated on an outpatient basis[Nine patients had SCC of the larynx or associatedtissues ^ eleven had other H+N locations includingone skin of the cheek[ One patient "tongue# had acomplete response "CR#[ Five had partial responses"PR\ ×49) tumor reduction#[ Two patients "sinusSCC# progressed clinically[ In general\ the treatmentprotocol was well!tolerated[ The clinical features ofthis trial will be described in detail elsewhere "manu!script in preparation#[

Patholo`y

All patients had pretreatment biopsies at the NCIor the referring hospital to con_rm the diagnosis ofSCC[ Of those who received biopsies at the NCI "04patients#\ the specimen showed on average 7022)solid tumor and 0922) stroma with 821) lym!phocytes in the stroma[ These tumor!associated lym!phocytes "TALs# were\ in the majority\ small\euchromatic\ nonactivated\ and scattered in thestroma at low density[

Following the immunotherapy\ 07 of the 19 patientsshowed histological changes in the areas of tumorinvolvement indicating a mean tumor reduction from70) solid to 06) solid and 17) fragmented and an

Fig[ 0[ Mean areas "2S[E[M[# of solid tumor\ fragmented tumor\ stroma and lymphoid in_ltration from histological slidesof 04 patients with H+N SCC prior to treatment and 07 responders post!treatment were evaluated[ The P values for tumor

reduction\ tumor fragmentation and increased lymphoid in_ltration are ³9[90[

increase in _ltration of lymphocytes and other cells"8Ð31)# "Fig[ 0# ðP values ³9[90Ł[ The increasedin_ltration in the responders was mainly accountedfor by lymphocytes "7821)# involving het!erochromatic lymphoblasts "4926)# and plasmacells "2826)#[ The remaining cells "00)# were mac!rophages and granulocytes without notable largegranular lymphocytes and with occasional giant cells"4 patients#[ Immunohistochemistry showed meanaverages of 4027) T!cells\ 1921) B!cells\ and 18)plasma cells[ Edema and necrosis were not prominentfeatures and these are included in stroma in Fig[ 0[Notable is the fact that the changes were generallydi}use throughout the area rather than being restric!ted to the periphery of the tumor[ Figure 1 shows arepresentative pretreatment biopsy "Fig[ 1a# and posttreatment histology from _ve representativeresponders "Fig[ 1BÐ1F#[ The pathology _ndings willbe published in detail elsewhere "Arch[ Pathol[ + Lab[Med[\ in press#[

Immunolo`ic studies

Prior to admission to these studies\ all patients weregiven an intracutaneous skin test with the IRX!1[Approximately one in four patients presenting withloco!regional H+N SCC showed no skin test reactionto the IRX!1 and had to be excluded from the study[

E[ VERASTEGUI et al[511

Although these patients have not been studied further\several had normal lymphocyte counts re~ecting alack of T!lymphocytopenia[ Anergy to intradermalcytokine challenge in the absence of T!lym!phocytopenia suggests a marked e}erent limb defectof cellular immune response involving the monocyteand deserves further analysis[ Of the 19 patientsadmitted to the protocol\ the average skin testresponse was 9[4Ð0[9 cm[ This response is\ on average\considerably less than those observed in melanomaand breast cancer patients "mean response 0[4Ð1[9cm#[ Responses to the Merieux multitest were absentin all patients re~ecting a high prevalence of anergy[Only 0 of 19 patients was positive to PPD in a popu!lation which is generally ×89) positive[

Lymphocyte counts\ total T!cell counts "CD2#\ T!cell subsets "CD3 + CD7# were normal in 04 of thepatients on entry to the study[ In these patients nosigni_cant impact of the immunotherapy on thesecounts was observed "data not shown#[ Five patientshad total lymphocyte counts below 0499 mm2 "mean0131277#[ These patients had lower levels of bothCD3 and CD7 lymphocytes "28524 and 179253\respectively ^ CD3 ] CD7 ratio of 0[3# with no cor!relation with clinical response[ Treatment with theimmunotherapy induced signi_cant increases in totallymphocyte counts\ total T!cell counts\ CD3 and CD7"Figs 2\ 3 ^ P ³ 9[90 by paired student t!test#[ Inter!estingly\ there was a signi_cant increase in a CD2¦but low or absent CD3 ] CD7 population "Fig[ 3#\perhaps re~ecting new T!cells from thymus[ In onepatient\ later studies of these changes showed furtherincreases in CD3¦ and CD7¦ T!cells and a loss ofthe CD2¦\ CD3− and CD7− population "Fig[4A\B# suggesting these increases may re~ect durableimmune reconstitution[

The massive in_ltration of mostly T!lymphocytesinto the tumors of these patients in conjunction withevidence of correction of T!lymphocytopenia in 4 pat!ients indicate a marked mobilization of T!lym!phocytes and perhaps increased thymopoiesis[

DISCUSSION

This study con_rms that this protocol using per!ilymphatic\ low dose natural cytokine mixture\ lowdose cyclophosphamide\ indomethacin\ and zincinduces marked alterations of H+N SCC consistentwith immune rejection[ Signi_cant tumor reductionwas noted clinically in 5 patients "0 CR ^ 4 PR ] 29)response rate#[ Overall\ 07 of 19 patients respondedhistologically "89)# to the treatment with increasedlymphoid in_ltration and tumor fragmentation withan average reduction of tumor of 33) "P ³ 9[90#[

The rationale for this form of immunotherapy isdiverse[ For a number of years we have exploredmeans to promote thymopoiesis immunopharma!cologically in murine models of immunode_ciencyincluding neonates and hydrocortisone!induced thy!mic involution in aged mice "Hadden\ et al[\ 0878\0882\ 0884#[ In all of those studies we found evidencethat NCM but not recombinant IL!1\ rIL!0\ or thecombination promoted T!cell development and func!tion[ We observed that low doses of NCM "49 units:mouse# induced signi_cant increases in peripheral T!cells of both subsets\ presumably the result ofincreased thymopoiesis "Hadden et al[\ 0881\ 0884#[

We also showed that zinc replenishment in agedstressed mice increased T!lymphocyte function butnot number "Saha et al[\ 0884#[ Zinc is known tobe critical for cellular immune function through theaction of the thymic hormone zinc!thymulin "CfHadden\ 0884b# and zinc replenishment restores nor!mal thymic morphology and cellularity in aged mice"Mocchegiani et al[\ 0884#[

Patients with H+N SCC present with signi_cantcellular immune defects often in association with poornutrition and zinc de_ciency "Hadden\ 0884a ^ Richts!meier\ 0886#[ The use of NCM in conjunction withzinc was speci_cally designed to address this immu!node_ciency[ In our previous study with 3 patientswith H+N SCC "Hadden et al[\ 0883#\ 2 of which werelymphocytopenic\ we observed marked increases inCD2\ CD3\ and CD7 T!lymphocyte counts[ In thepresent study\ we observed the same in 4 lym!phocytopenic patients[ Thus 7:7 patients having T!lymphocytopenia showed marked correction of T!lymphocyte levels toward normal[ The presence ofa signi_cant increase in a CD2¦\ CD3−\ CD7−population suggests immature T!cells in the circu!lation\ perhaps akin to recent thymus emigreesobserved in rat "Mojcik et al[\ 0880#[ E}orts toincrease T!lymphocyte counts in humans with humanimmunode_ciency virus "HIV# infection have beendisappointing "Hadden\ 0885# and signi_cantincreases have only been observed\ to the degree seenin our studies\ when patients were given large toxicdoses of Il!1 "millions of units# by infusion over longperiods "Kovacs et al[\ 0884#[ Correction of T!lym!phocytopenia observed in our studies is not thoughtto occur in the adult cancer patient "Mackell et al[\0884# and we have studies in progress to furtherelucidate this process and to study its functional sig!ni_cance on the expression of cellular immunity inthese patients[

The cellular immune defects of patients with H+NSCC are also associated with marked evidence ofimmune suppression by a variety of serum factors\

Immune Regression of H+N Cancer 512

)

)CMYK Page 512

(a) (b)

(c) (d)

(e) (f)

Fig[ 1[ Photomicrographs of a representative pre!treatment biopsy of H+N SCC "A# and post!treatment surgical histologies"BÐF# of _ve representative responders showing tumor reduction\ fragmentation and lymphoid in_ltration[ Note in "D# a

keratin pearl previously tumor!embedded\ now surrounded by giant cells[

E[ VERASTEGUI et al[513

Fig[ 2[ Total lymphocyte and CD2¦ T!cell counts of _ve lymphocytopenic patients pre! and post!treatment[

tumor derived factors\ immune components includingacute phase reactants\ antigen!antibody complexes\ P!04!like peptides\ transforming growth factor!b "TGF!b#\ IL!09\ and prostaglandin "PGE1# "Hadden\ 0884a ^Young et al[\ 0885#[ Also\ suppressor cells have beendetected in the circulation of patients with H+N SCC"Berlinger\ 0873#[ The use of low dose cyclo!

Fig[ 3[ CD3¦ CD7¦\ and CD3−\ CD7− subset popu!lations of CD2 positive T!cells pre! and post!treatment are

presented[

phosphamide and indomethacin as methods of con!trasuppression to obviate these immunosuppressivein~uences has an extensive rationale "Cf Hadden\0884a# and provided the basis for their use in theprevious and present studies "Hadden et al[\ 0883 ^Barrera et al[\ 0885\ 0886#[ Their use presumablyaccounted for the ability of a restored and mobilizedimmune system to attack more e}ectively the cancersof these patients[

The clinical use of regionally administered naturalinterleukin 1 was pioneered by Cortesina et al[ "0877#[The foundation for the regional use comes from avariety of e}orts to employ interleukins e}ectively[Regional administration of an IRX!1 equivalent hasresulted in enhanced immunization "Kelly et al[\ 0861#and co!presentation of cytokines with tumor cellswithout transfection provides an e}ective and simplemeans of inducing tumor immunity in mice "Playfairet al[\ 0889 ^ Forni et al[\ 0883#[ Local administrationof interferons or IL!1 in H+N SCC historically metwith some success "Hadden\ 0884a#[ The use of naturalIL!1 "a mixture# by Cortesina yielded partial or com!plete responses in 5 of 19 patients with H+N SCC"Cortesina et al[\ 0877 ^ Cortesina et al[\ 0880#[ Theseresponses were not evaluated pathologically[ Theresponses lasted from 1Ð5 months ^ however\ repeatedtreatment following recurrence was not e}ective[ Fol!low!up studies with recombinant IL!1 treatmentshowed 4 CR and PR responses in 68 patients "Mat!tijssen et al[\ 0880 ^ Cortesina et al[\ 0883 ^ Vlock et al[\

Immune Regression of H+N Cancer 514

(a)

(b)

Fig[ 4[ a\ b[ Total lymphocyte\ CD2¦\ CD3¦\ CD7¦\ and CD3−\ CD7− T!lymphocyte levels of a single patient analyzedpre!treatment "Day 9#\ post!treatment "Day 02#\ and 06 days later "Day 29# are presented[

0883#[ Histological evaluation in such patients showedevidence in some patients of tumor necrosis and in_l!tration with CD2¦ lymphocytes or natural killer cellswith or without granulocytes "Whiteside et al[\ 0882 ^Valente et al[\ 0889#[ In general\ these results wereinterpreted to re~ect non!speci_c stimulation leadingto transient clinical responses without immunologic

memory "Cortesina et al[\ 0880 ^ Cortesina et al[\0883#[

The modi_cation of the Cortesina et al[ "0877# pro!tocol by us was designed to capitalize on the principlesof immunorestoration "IRX!1¦zinc#\ contra!suppression "low dose cyclophosphamide and indo!methacin#\ as well as adjuvancy "regional IRX!1 and

E[ VERASTEGUI et al[515

tumor antigen# discussed above[ The evidence for atumor!speci_c response to account for the tumorreduction and fragmentation in these H+N SCC pat!ients are several[ The responses were elicited not byinjection into the tumor but at a distance[ The pres!ence of immune reactions {{upstream|| from the lymphnodes draining the tumor implies hematogenousspread of the reaction i[e[ the in_ltrating lymphocytes[The response\ once initiated by the perilymphaticinterleukins\ continued in the period after the IRX!1injection and prior to surgery\ approximately 00 dayslater[ This re~ects a self!perpetuating\ autonomousreaction as seen with immunity[ Such reactions arenot seen with rIL!12LAK therapy which involvesnonspeci_c mechanisms[ The IRX!1 employed con!tained a variety of interleukins and cytokines andnotably contained those which promote TH0

responses "IL!0\ IL!1\ IFN!g\ and IL!01# and lackedthose promoting TH1 responses "Il!2\ IL!3\ + IL!4#and would likely promote a cellular immune response[That an immunization has taken place is also re~ectedin the regional lymph nodes[ Where analyzed thesenodes showed reactive hyperplasia as would beexpected from an immunization[ It has recently beencon_rmed that lymph nodes are the site of e}ectiveimmunization rather than the tumor itself\ under thesecircumstances "Maass et al[\ 0884#[

From an histological standpoint\ the fragmentationand reduction of tumor in the absence of overallenlargement re~ect a replacement of tumor by animmune reaction which is capable of killing tumorcells[ Clearly\ both cellular and humoral componentsof the immune response are involved since both T!lymphocytes and B!lymphocytes participate[ No sig!

REFERENCES

Barrera\ J[\ Verastegui\ E[\ Zinser\ J[\ Meneses\ A[\ de La Garza\ J[ and Hadden\ J[ W[ "0885# Natural cytokine mixture andcontra!immune suppression induce immune regression of head and neck cancer "H+N#[ 3th Int|l Congress Head +Neck Cancer\ Toronto\ Canada\ July 14Ð20\ "abstract è205\ p[ 626#[

Barrera\ J[\ Verastegui\ E[\ Meneses\ A[\ Zinser\ J[\ de La Garza\ J[ and Hadden\ J[ W[ "0886# Neoadjuvant treatment withnatural cytokine mixture of squamous cell carcinoma of head and neck[ 6th Int|l Congress on AntiCancer Treatment\Paris\ France\ Feb 2Ð5\ "abstract è006#[

Berlinger\ N[ T[ "0873# De_cient immunity in head and neck cancer due to excessive monocyte production of prostaglandins[Laryn`oscope 83\ 0396Ð0300[

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Coto\ J[ A[\ Hadden\ E[ M[\ Sauro\ M[ D[ et al[ "0881# Interleukin 0 regulates secretion of zinc!thymulin by human thymicepithelial cells and its action of T!lymphocyte proliferation and nuclear kinase C[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 78\6641Ð6645[

Cortesina\ G[\ DeStefani\ A[\ Galeazzi\ E[\ Cavallo\ G[ P[\ Jemma\ C[\ Giovarelli\ M[\ Vai\ S[ and Forni\ G[ "0880# Interleukin!1 injected around tumor!draining lymph nodes in head and neck cancer[ Head + Neck 02\ 014Ð020[

Cortesina\ G[\ DeStefani\ A[\ Giovarelli\ M[\ Barioglio\ M[ G[\ Cavallo\ G[ P[\ Jemma\ C[ and Forni\ G[ "0877# Treatmentof recurrent squamous cell carcinoma of the head and neck with low doses of interleukin!1 injected perilymphatically[Cancer 51\ 1371Ð1374[

Cortesina\ G[\ DeStefani\ A[\ Galeazzi\ E[\ Cavallo\ G[ P[\ Badellino\ F[\ Margarino\ G[\ Jemma\ C[ and Forni\ G[ "0883#Temporary regression of recurrent squamous cell carcinoma of the head and neck is achieved with a low but not witha high dose of recombinant interleukin 1 injected perilymphatically[ Br[ J[ Cancer 58\ 461Ð465[

ni_cant numbers of large granular lymphocytes wereobserved by routine staining in the histologicalsections\ indicating this is not a phenomenon involv!ing natural killer cells and lymphokine activated killer"LAK# cells[ Thus\ from an immunological stand!point\ we interpret these data to re~ect an anti!tumorimmune process[ The current study con_rms that ahigh frequency "89)# of patients have a signi_cantdegree of histological response to a 2 week treatmentwith this protocol and that major clinical responsesalso occur[ The signi_cance of the histologicalresponses in relation to the clinical responses remainto be determined and cellular subpopulations andcytotoxic responses need to be studied[

The speci_city of this immune reaction clearly liesin the presence of antigens on H+N SCC capable ofeliciting immune rejection[ The evidence for antigenson these cancer cells capable of eliciting both humoraland cellular immunity is extensive "Hadden\ 0884a ^Richstmeier\ 0886#[ Candidate antigens include mucincore and carbohydrate antigens "like sialyl Tn Lewisantigen#\ MAGE antigens "particularly 0\ 1 and 2#\Epstein Barr virus "EBV#\ p42\ perhaps papillomavirus antigens and various uncharacterized squamouscell antigens[ We have no information which of thesemay be involved in the responses observed in the pre!sent studies but intend to pursue them with cyto!toxicity studies with T!cell clones derived from thesepatients "Cf Boon et al[\ 0884#[ Such antigens willprovide the basis for immune detection\ immunemonitoring\ and immunotherapy in the future[

Acknowled`ement*This work was supported by a grantfrom ImmunoRx\ Inc[\ Sarasota\ Florida[

Immune Regression of H+N Cancer 516

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