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Award Menber: DAMDI7-92-1-0029, TIME: HMolecslar Epideniclogy of Prostate Cences PRINCIPAL INVESTIGATOR: Brace J. Trock, Ph.D vere Hopking University Baltimore, Marylara 21205 CORIRACTENS ORGANTZA! REPORT 1 a ry 234 TOE OF FEDGRT: Annual PREPARES FOR: J.S. Atty Medicel Zeseaxch and Materiel Command Fort Detrick, Merylend 21702-5012 DISTRIBUTION STATIVENT: Approved for Public Relecser Distribucion oxtinited The views, opinions ard/or findings contained in this report are thoce of the author ‘a! and should net be conetrued ap an official Department of tre amy position, palicy ar decision unless so designazed by other documentation Farin Approved REPORT DOCUMENTATION PAGE OMA No, 070188 RAR gaa racuauigae eye aaiae jose een ie = ane danuaey 2004 Saul |dvan 2002 = 31 Dac 2002) TE a TE SAE Tas dolecder Foldniclagy of Prostate cance Syeany-o9-1-0020 Sewer Reve, 2.3 HERE ORGAO 7 FERS ORAL ON NARA] ATO TEREST Fos Ropkinsr University Beltivaro, tarvlexd 22205 fay Medics] Research aad xateriel Somnand Detrick, tazyland 21702-5012 1 20061028 137 | soproved for Prslic Release; Jistrizution UnLim‘ced 7 ABSTRACT Darina 700 Wr] us objective of this case-control study 4¢ to determine whether oxidative stress, specifically lipid peroxidation, 1s « rick factor for prostate vuncct. Furthermore, the stusy will iavestiyaue wlth oxidyeive exrees mediates En association betwesn high dietary far intake snd prostate cancor risk. specitically, tue study will caspare malonsialdehyae (hon) in cerum, ap a marker of Lipid perewidation, nd dcoxyguaioyine maloadialdensde (te WOA! in ceripneral lywphecyLew and promare timor ag a Marler of acidative CNR domege- These sesociations wil. be adjusted by dietaxy weagures uf Tot and antiocidagt intake, ae well ae cecum moasurce of specific tatty acids ond antioxidante. tmminee veda cycling oF andiuyens as recently been suown to be a source cf reaccive daygen species, serus androgen, Jevele will also be sorrofated ith WDA, GG-HDA, and risk, Ef an aeeucintion between Lipid perexidacicn and prostate cancer ick is demorstrated, it cowla lead t9 new utiategies for prevention jetperially chemoprevention) and markers that could @sfine men Bt bigh riek. Uhia report cavers activities since the yeast we JZokna Hopkins Sohoo) of Medicine in 2001 owed from Geargennwn University to TE SORRET tite Peae-consrol proszate Lipid peroxidet!on, maloadinldety Wsclassitied | __ondatted ee Unolaveitied tel ~ Vrock, Bruce J AMD! 7-00-1-002 FOREWOR (Opinions, interpretations, conclusions and recommentatons are those ofthe author and] are not nesessarily endarsed by the (18. Army. ‘Where copyrighted! material is quoted, permiston has been cbtained to use such materi __.., Where material from documents designated for timited distribution i quote, permission as ‘Sblained to use the materia, Gitafions of eoramersial organizations and tratle names in this repent do mot cousctute ww ‘iliai Depaurent of Army endorsement or ppreval ofthe producls or services ofthese organizations. NAL In conducting rescateh using animale, the investigaton(s) auhered tothe “Guide forthe Cave 5nd Use of Laboratory Animals,” prepared by the Commiltee on Cara and Use of LaboraeryAmmals ofthe Institute of Laborstory Resources, National Research Council (NEM Publication No. 56-23, Revised 1985), For the protection oF human sobjecis, she inveclgator{s) adhered lo poticies of applicable Federal Law 45 CER 46. NA. iu conducting research utilizing recombinant DNA technology, ch investigaton( ‘caren guidelines promulgated by the National Institutes of Health, ‘NAL Ie conducting research utilivAng recoubinant DNA, the investisaor(s) adhered to he NIA Guidelines Zur Research Involving Recombinant DNA Molevales, [NA_ Inthe conduct of research involving harutis onganistas, che investigater(e) adhered tothe CDC-NIET Guide for Biosefety in Mucrobioloyieal and Bivmnedi¢ul Tabxrataies, a PI- Signe Buse LO bute bo ¥f08 “Trock, Bruce J AMD! 7-00-1-0020 . Report Documentation Page ~ |. Table of Contents TABLE OF CONTENTS Front Cover — Foreword Antreduction —- Body Comelusion = == References Appendix Listing List oF abbreviate und acronyne Meeting abstracts during repring periud —- Publicalin using reporting penal Manuscripts ie'preparation ‘Persemnel receiving pay ftom this negotialed effort ‘trock, Bruce I. DAMDI7-00-1-0020 INTRODUCTION ‘The objoctive of this ense-comtol study is to determine whether exidative damage is arisk factor for prostate cancet, and whether this mechanism mcdiates the association between dietary ft and rosie cancer risk. Specifically, cases and controls wilt be compared! with respect to rnalondialdehyde (MDA) in serum as a measure of oxidative sess, and see yguamosine malondialdehyde (4C-MD.) in peripheral lymphocytes aud prostate tumor samples 2s a measure ‘of onidative DNA daraage. In addition to Ihese measures, dietary intake of fats and specific fatty acids, and of antioxidants willbe conskdcrod as patel effect coli Gers or confounding facto, sas will serum antioxidant eves “This teport covers activities since une project uansfetrod from Georgetown University lo Johns ‘Hopkins School af Medicine ip 2001. There was a long period of insctvity while the studly jprotoral anil consent linia wore boing negotiated betwecn the Department of Defense, the ‘Principal hrvestgator, and Jehns Hopkins Schoot of Modicine. ‘The issues were successfully resolved and the study was re-activated in Jon 2003. Since that time, progress has been rapid, ‘We have enrolled 223 prostate eanecr carcs and 165 contrls fiom Tohns Hoplons Hospital and Joluns Hopkins Bayview Medical Center. Added to tbe patients reruited ftom Georgetown Taiversiey, the Washinglom, DC Veterans Adrainistration Medical Center, and Washington TTospital there are a total of 320 eases and 250 controls, of whom 13% are African Ataxtica. Seram, plasma and fymphocytes art available font 89% of these pation and eplidemiotogy’diecary questionnaire data are available for 85% ‘The population enrolled in this case-control study formed the besis for & projest in the NCI funded Johns Hopkins Prostate Specialized Program of Rescarch Excellence (SPORR) svar {SPORE Grant #2 P50 CA38236-10, “Project &: DNA polymorphisms in genes affecting levels of onidative sires in prostate cells: population siudies of association with prostate cancer risk") ‘lt stely will uli nthe existing casc-control dalasel and analy a series of single imeleotide polymonphisia (SNPS) associatod with generation oF or regponse to vxidative stress, tu determine whether any are risk actors for prostate cancer, or modify the risk associa with cosidative sess, ‘An uditional small pit project has been initiated with Dr. Prakash Rao af the Laiversty of Sou Alabatna, This study will moesure leptin in the seram of a subset of prostate cancer cases and controls from the parent study, Leptin is a potentlal risk factor for prostate caicer that may also modify the associnlion of diet with isk, ‘These samples are currently being analyzed. arin the mainder ofthe Fung period ws wil continu to call pacts, and bon he anulyies ol malonlaldehyde (MA), doonsguanosine malondialdehyde (AGMDAY, serum Nounmes and snionidans, and dictry dra, We expect to complete ese analyses in ely 2005 and eb marserps for publication. Troek, Bruce J, DAMD!7-00-1-0020 BODY Study Progress “This section wilh doseribe the fsltowing: {a} original sty objectives {0) he change in institotions duc lo the Principal Investigators move ta another aniversity {¢) delays in e-activarng the study at the now institution related primarily to approval ofthe consent form (al) progres since the sty was re-activated (6) additional ongoing reseurch thal was spun-aff at the original grant (0 plans for concluding the stady, Study Objectives, ‘Task J. ‘To complete enrollment of prostate cancer cascs scheduled to undergo prostatectomy, and benign urologic surgery controls from urology clinics at Johns Hopkins University School of Medlizine (HUN, Georgetown Tniversity (GU), the Veteran's Administration Medical Center (VA) in Washisgloa, DC, and the Washinglon Hospital Center (WIIC), This facludes callection of serum, plasma, tymphocytes, epideraiolagie and dietary intake data, and (from eases) paratfin- embedded tomas tissue. Task 2. To mesure te flowing biornarke + Serum ralondisklchyele + Matondiatdclyele decrryguanosine adducts in pariphera! blood! Iyanphocytes + Matondiatdchyde deoxyguanosine adducts in prostate tumor tissue = Complete serura tay acid profile = Serum anGixilunts ineluling alpha-tocopherol (vitamin E) and carotenoids = Serum androgens and relsied horraoncs ox metnboliles, including testosterone, dihydrotestosterone, 3a andzostancdial glucuronidc, ond SHBG Task 3. Ta conduct avase-cureat study withthe shove datato compare the levels of ipid peroxidation Biomarkers in curcs with controls, (9 determine the foflorsing = Whether tpi poronidtion lovels modify te waociaton of iotary fat with prostate cancer sk ‘Whether lipid peroxidation biomaukers in serum (MDA) or lymphocytes (€UMDA ducts) provide a good estimate ofthe extent of oxidative DNA damage i postu tumors (QGMDA adduct in tumor) = Whether ip perowidation levels are function of ary fat and androgens, ‘Teook, Bruce J DAMDI7-#0-1-0020 Change in Tastitutions. Tn April 2001 the Principal investigate, Dr. Bruce Track, moved from Georgetown University (GU) to Johns Hopking University (THU). ‘The plan at thot time was to ‘continue to encod patients ftom the origins) enrollment sles at GU, the Veteran's Administration ‘Musical Center (VA) in Washington, DC, and Washingtun Hospital Center (WHC), and ta also bogin enrolling patients fm TUT [Delays in reactivating the smdy. All research nofivity ant ucceptance of new IRB applicalions at TAU were suspended in Tuly 2001 due to the death of » subject inn roscarch stuely completely torclaed to Dr. Troek's cesearch, This suspension was lifted in Oeloher 2001, and SHO urranged for an oulsice institutional review board, Western Institutional Reviow Board (WIRE) foassist in reviewing new applications. Around the same time. in Scptember 2001, Dr. ‘Track ‘was notified by De. Angela Howaal (USAMRAA) that the original informed consent form for this study bad never boon approved, nr that the study bad not ben authorized to enroll patients, Dr, Howard's letter requested that all stuly activities immediately be suspended. Dr. Track ‘conaplied and suspended shidy activities. The problesn had been that De. Trock hud newer acocived a loter from USAMRAA, dtl Seplember 1999 (12 the dime of the oeigial grant vingd while at Georgetown) requesting modification fo his proposed consent form before the study could to authorized ( hogin. At that tio, a study nuarbee was subsequently assigned aru the grant finds wore released ta Georgetown University (GU), 50 Dr. Track assumed thatthe study had been approved te begin, and he begim carollicg patierts while at GU. Following Dr, Howard's September 2001 letter, a long sercs of communications took place, during which Dr, ‘Trock provided evidence that no research subjects hae! been harmed in any way, and the entire gant proposal, study protocol, and consent form wore ro-roviewed, ‘This proccss involved ‘communication with Dr, Howard und ler successors, wclvding Dr. Adrienne King, Sacelia Heller, Christine Helm, and Shirley Roach, of AMDEX Corperation and the USAMRAA Office of Regulatory Compliance and Quality. Uhisnately, everything bad been approved except for a clase the consent form concerning payment for medical care needed in the event Uhat study subject was injured while participating in the sludy. Fventully chs was resolved to the sallsaction af USAMRAA, Johns Hopkins University, and Dr. Track. Tho final eonscat foren and study protocel were approved by WIRB (WIR Protocol Number 20011642). The study was finally authorized by USAMRAA to beein in Jamvary2003. This entire Human Subjects Revlew process was astigaed TISRRB Log Number A-09282, and all communlcatfons during that pragess are referenced to that eumnber. ‘Frogress since re-activation of the sluly. ARermaving to JHU, the original intent wus fo confine to enzall atthe thre original sites (GU, VA, and WITC), as well as beginning enrollnent at JHU. However, because the pationt velume for general nralogy an prostate cancer at JHU far exceeds that of GU: and WHIC combined, and demographics are similar at afl sites, it was decile thal enrollment at GLE and WAG could be stopped without adversely affecting study 7 Trock, Bruce 3. DAMDI7-00-1-0020 acerual goals or atudy composition. This step was taken becanse there was only one Research ‘Nurse funded by the grant, and it was not feusble for her to actively recrait at 4 sles that wen ‘over 40 miles apart (e.g, Washington LC to Rallimore}. Tarollenent af tbe VA will continuc, since 7506 of postal emscer patients there are Aftican American, Tihs will provide the ‘opportunity 1 examine edie differences in risk associated with oxidative stress. ‘The Brady Urelogzea! Institute at JHU is one of the largest clinical erology programs in the ‘world, Anmually, spprexiovately 1,000 men undergo radical prostatectomy for prostate cancer, and mors than 2000 men ure seen for other urological conditions. We initially began recruitment ‘of cases only, to establish ihe infrastructure and relations withthe attending uralagiss to insure ‘Hat enrollment would not interfere with clinical practice. The reerutmenl process requires the [Recearch Nutoe ta review clinic appoinanen schedules end surgery schedules np to one month in ‘advunce, idently potentially eligible patients based an age, scx, and indication for clinie visit ar ‘agnosis, and when possible review the patio maedical record for eligibility eiteia using the ITU Elecuonic Patient Record. Following implemeptation of HIPAA, access to this type of jisformation without pri paticut assent was no longer permissible so we apptied for ant received a ITPA A Putial Waiver of Aha ization to pemit eiviclans to provide this inforroation tus. The justification for the Waiver is that idextlying and contacting eligible study subjects weld not he Feaible via other means. We have had ne complaints from patents that their information sass provided without their prior assent, It is important to note that the Waiver of Authorization is aot a waiver ofthe requirement for consent. Tt ocly provides ur the ‘means to contact patients, inform tem about the snidy, aud then see whether they are interested in Tearniag taore about the study and nltimately, going through the informed consent proces, ‘The Research Nurse sends each patient a packet with a cover letter explaining the study, a short series of questions to sereen far eligibility, the consent form, and Ge epidemilogy sl ict questionnaires. When the interval hetween huing notified about a polenitally ligibie patent and he patients clinis iste (oe short to mail the packel, the Research nurse calls the subject, and if they express willingness to consider the stay, she urrunyes ln meet thn prior to their appointment fo explain the stady’ wnd obtain informed consent from those willing to patiipate Blood samples are obtained at the elinical phlebotomy station for paticuts who are already scheatuled for a bload draw roquitod for their elinle vist, or by the Research Nuive for palcals ‘mot scheduled fir ather blo work Ouee this process was going smoothly we began to focus on control recruitment as well. We soon realized that despite the very large patient volumes soon by the Brady Unslogical Yntitute a large fraction of patients being seen for reasens other than prostate cancer Ge, potential controls) were being soon for olher urologic euneers(e-. bladder, kidney}, or had prior history of otbor ‘eancera (including non-vrologic cancers), and wore (his, ineligible, Relatively fewer patieuts ia the target ege-rangs forthe study were being seen for yeneral urology problems (e.g. kidney stones, nonmalignant urinary tact obstruction, nephrepathies, voiding dysfunction) because such paients are offen treated by community wologists rather than going toa large referral cere. ‘Trock, Brace J DAMDI7-00-1-0020 ‘The requiroment thal eanifals have documented PSA <2.5 and a noumal DRE also reducod the ‘mumber of eligible patients, Bocaue we tealizad that a moch yreater effort was required to sere urology elinie schedules for potentially sligible paticts, und the grant id not provide funding to perabit us to hive an additional nerse for this task, we temporarily suspended ‘exrollment af euses to focus efforts on control rcerwjlment. We also began to actively recruit controls from the Jobs Tlopking Bayview Medical Center, « hospital several miles away fio the main Johus Hopkins Hospital. This hospital provides more routine erfogy care han the urology cline a the main hospitel ands a good source uf contols. ‘We curently enrall v0 different types of controls, Patients coming fr soutine wsblogy eae unrelated to malignancy und with co history of canoer form the fist group (*urue contrls") However, despite the fur thal these patents will have had axevent (within 2 years) PSA «2.5 gin! anda normal DRE, some of theve patients will harbor undetceted prostate cancer (Thompson 200), For thie reason we also include among the controls men who have ad a recent negative prostate biopsy, and are nol considered to be at “high risk” of being a false nogitive fie. PSACR ng/ml, no evideneo of prastats intraepithelial neoptasia (PIN) oF atypia on ‘igpsy. and normal digital rectal scar. These men have about a 20% chance of having prostate ‘cancer missod by the biopsy (False negative). We follow thesc men for one year fo idenify any ‘ah have a subsequent biopsy that ilentfies eancer; snet men would be switehod from the control popalation ta the ease population ‘The following tahle summuacizes out recruitment of eases and comtnals to date, shoving resus from the different clinical sites: Tse (o=120) Toate fe=B5) nny negative: 39, crue control 126_| 97 | Wiopsy neg 279 | biopsy negatives 105s emerak 122] ___295 "_" [biopry negativer 95, Truc conteot 54 as a 3 5 i 7 | ‘Trock, Bruce J. DAMDI7-00-1-0020 17% of subjoce ure AGiean Ametican, with a somewhat higher proportion among cases (20%) thm controls (13%), Note thal the nuenbersinchaded inthe breakdown of ethnicities do not add ‘up © the otal mumer of subjects — wo do not have ethuic data in the database for some individuals for whom we have not yet reccived their questionnaire. Senn, plasma and Iymphooytes are available from 8% of crralled patients and epideuniotogy‘diecary questionnaire daa are available for 83%, We are continuing In cemtactpalients who Pave not yet ture thee ‘questionnaires to obtaln additional data, In addition lo the vases soy in the able seals have ‘questiniaire date wid blood sinples floun 15 patients with premalignant lesions, ie. subjects ‘with PIN, HGPIN, and alypia. These may fest because if oxidative damage truly is 8 risk fhctor for prosate cancer, these may exhibil levels intermediate between those of cancer ceases und cout, ‘Additional reneorch spun off the parent ezant. ‘Two ongoing projects have been based on the ‘existing case-control stuly and are Tesurthed below. 1, Johns Hopkins Prostate Cancer SPORE (grant #2.PS0 CA54236-10): “Project 5: DNA polyzmovphisms in genes atfecting levels of oxidative sires i pructate cells: population studics ‘of association with prostate cancer risk.” ‘This study was funded hy the National Cancer Institote in Sepleraber 2003 as ane of five reseatch prajeets in the Tone Hopkine Prostate Cancer SPORE iraat. The stady will use the current eaze-contol population. Fifty genes known to be sesaclated with production of reactive oxygen species (ROS), detoxification of ROS, or repair of DNA damage due to ROS wil be evaluated. For each of the cmnlidate genes targeted for unalyia, we will genotype anprosimately B to 12 single aveleotiec polymorphisms (SNP) per ‘get in he cases und controls frorn the pareal study. These will be used to idewtify prostate “euncer rsk-rnliTying genes hy performing assnciation analyses of genotype ftequencies in cases amt controls. We wall corral this data wilh Ure data on dietary intake, biomarkers of oxidative stross, oxidative damage, and antioxidents from the parent study to determine which genes modify tho association of those exposures with risk, Finally, the associations identified in the ‘case-control population will be validatod by oxaranting two independent sluly populations: & cohort study in Washington County, MD (Kathy Helzlsower, PI), and an Aftican Amicriean casc- ‘contte] smdy popalation collosted at Howard Lniversity (Rick Kittles, PD). 2. Serum leptin and prostate cancer risk. This pilot study isa collaboration with De. Prakash Rap at the University of South Alabama Qhesity is a potential sisk factor for prostate cancer, and is closoly corrlated with dictary fatty acid intake, a potential source of oxtduive cress, CCireulating leptin levels are conelated with obesity and androgen activity, and the prostate ‘contains receptors or leptin. Stadies én vitro have shown that leptin stinulates prostate cancer cell proliferation (Somasunidar 2004), hut epidemiologic studies have been few and inconsistent (Statin 2001; Stalin 2003), We deciced ta examine leptin in a pilot prostate cancer case- control study to determine whether (a) teptin was associated with oxidative stress, apd (b) 10