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A natural cytokine mixture (IRX-2) and interference with immune suppression induce immune mobilization and regression of head and neck cancer

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A natural cytokine mixture (IRX-2) and interference with immune suppression induce immune mobilization and regression of head and neck cancer
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  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 Britain9081Ð9450:87 ,08[99¦9[99 PII] S9081Ð9450"86#99948Ð2 A NATURAL CYTOKINE MIXTURE "IRX!1# AND INTERFERENCE WITHIMMUNE SUPPRESSION INDUCE IMMUNE MOBILIZATION ANDREGRESSION 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:M 1 #\ 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 "0131 2 77 mm 2 # 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\ if abnormal\ 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 etal  [\ 0885#[ Tumorin_ltrating lymphocytes "TILs# have been dem!onstrated in most H+N SCC "Wolf  etal  [\ 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#[Inaddition\anumberoftumor!associatedanti!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 of immune rejection of tumor with long!term palliationin 1 patients with recurrent disease and prolongeddisease!freesurvivalina thirdpatientwhowastreatedprior 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 methodsPatients [ 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 peripheralbloodlymphocytesstimulatedbyphytohemagglutinin"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 " ³ 9[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\interferongamma"IFN! g #\colony!stimulating factors "CSF#\ for granulocytes "G#\ andmacrophages "M# and tumor necrosis factor! a "TNF a #\ 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:M 1 # 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 Tabs TM [ 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\LosAngeles\CA#[Thesecountsweredonebefore 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 " 2 standard 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 of this 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 of SCC[ Of those who received biopsies at the NCI "04patients#\ the specimen showed on average 70 2 2)solid tumor and 09 2 2) stroma with 8 2 1) 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[Followingtheimmunotherapy\07ofthe19patientsshowed 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 " 2 S[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 tumorreduction\ tumor fragmentation and increased lymphoid in_ltration are ³ 9[90[ increase in _ltration of lymphocytes and other cells"8Ð31)# "Fig[ 0# ð P   values  ³ 9[90Ł[ The increasedin_ltration in the responders was mainly accountedfor by lymphocytes "78 2 1)# involving het!erochromatic lymphoblasts "49 2 6)# and plasmacells "28 2 6)#[ The remaining cells "00)# were mac!rophages and granulocytes without notable largegranular lymphocytes and with occasional giant cells"4 patients#[ Immunohistochemistry showed meanaveragesof40 2 7)T!cells\19 2 1)B!cells\and18)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 Althoughthesepatientshavenotbeenstudiedfurther\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 mm 2 "mean0131 2 77#[ These patients had lower levels of bothCD3 and CD7 lymphocytes "285 2 4 and 179 2 53\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 of the 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 of increased 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 "Cf Hadden\ 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 etal  [\ 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 of a 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 of immune 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# akeratin pearl previously tumor!embedded\ now surrounded by giant cells[
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