DTIC ADA425768: Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer From Benign Breast Lesions

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Awerd Nuebes: DAMDL7-00-1-020a

TITEE: Populacion Based Resesamen: of MHC Class T Antigens Down
Regulazion a2 Markers of Increased Risk for Develontent
and 2zogzession of Breas: Cancer from Benign Breast

Lesions

PRINCIPAL INVESTIGATOR: Marie J. Worsham, Ph.D.

CONTRACTING ORGANIZATION: Henry Ford Health systom
Detroit, Michigan 48202-2689

REPORT DATR: January 2004

TYPR OF REPORT: fAmeuel

PREPARED FOR: 1.8. Amy Vedical Research and Muteriel Conmaud
Fort Detrick, Maryland 21702-5022

DISTRIBUTION STATEMENT: Approved for Fublic Release;
Distribution Unlind ted

che views, opinions and/or findinge convained in thia report are
those of the authsr(s) and should act he construed ag a2 official
Departmont of the Army position, policy or decision unless so
designated by other documentation.

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PopulaLior toised aseeaenent of mi clase 1 antigens Dem — | Salmi7-Do-1-0263
Regulation ab Markevs af Zacruseud Rick for Dowsiepanne an
Progression of Breast Cancer From. Berign Breast Lesions
5 ATAORT
Marie J, Worshar, Ph.2 ;
7 PERFOR OREANER TOW TAREE) AND ADDRESSES ‘SRR GRBATEATION 1
Renry Tors Healt syater ‘eponr auaaaee
Detroit, xzenigen 48202-2669
Site, mvorehatehihs ory
V.8, Amy Medical Research and Materiel Comare
Fort vetrick, Maryland 21702-sp12
1 BUPERERTARY HOTES
Graginal nowsains color plates: ALL ONC reproductions wi: be in black aud white
Fb, IS TBUTION J AVARABENY SATEEN Eb, TROT CODE

approved for Public Release; Disweitutsom Unlimited

TS AS THCT aa 20 WRT
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tet wovtslity raze. The nocd far aftoctive Ehorapies Le yrvwal: Te overall sim of this necoarch
Bropsecl iz to racognize early nackere of disease nad Hutie interaction with other cpiacatcl
Fisk dsctor: that con orcva ae lek dadicwsore for sabeeqeent develspeene of Brees? cancer fem
peetancerous Joslonay ond ae yrogusetic markers fox paoqrescic= fiom prinesy to agtontarte asco
‘The aojor histeceapatiniliey cemiex (iic) clas: i molecles gen fount oh the cele aehbrane of ail
eels Ip the toy wad are involved in tngeceallices commalearions ard an eomolex dscerectione With
he dane syatien. Canoes evils wich reanosd or aereant 1H scleculee Dave Dew stim to twede
sites cf the tray. anmut holt + ail Breast cancers have coraiete lees cf vodaced covel of Bae claca
Tholeccies and tate finding asa beat aevosined with anceonced tumor Srvanivenrat acd ape
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Clinical stgalticance of abacnasl Mi clare I aplecutes ip procanactoss cad ingeatee ovorgt lsatona
fe movkera of Limuslogicel svente thar ecild eféact suryival, releceion, and extasowen ot
Brccanceroue celle, and tasiz subsoguent progression bo cease cael Ucter Wie lonaee Could slau
Sort more anuewcstve "mare end thee contrinite t0 aslectien Of appcusiate teestiansa is iediviiced

Wajor hiatorenpat:bilicy complex (MC) edann I eateries, aisens ef
Erogression navkers, yurvivel, beast cancer TE TREE TORE
Yi, SECURITY CTARSIBCARON | 76 FEGURTTY CLAESIGATION | 18 SECURTY CLASSRGNTON —| 2. TARTAN OF ABSTRACT
OF aeront ‘arnas pace oraasreacr
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EW ESTEE Sinead Fapy a ae FT
Table of Contents.

Table of Contents,

Introduction. 4
Body.

Key Research Accomplishments 5
Reportable Outcomes. 6
Conclustons.

Rofereness....

Appendices.
INTRODUCTION
thas boon known for some tune that malignant cransformation of cals is frequently assoviated
‘with ebeormalities in tae expression of MC class tautigens (1). ‘These abnormalities uppest to play 4
role inthe clinical course ofthe disease (1) neta have a negative efect on the outcome of I sell-based
imamotherapy for maliguane disensss €, 3), lv breast lesions examined for expression a MIC chs
approxicnntly aalf (51%) of carcinomas had 99 almorma Ty los content af LAA, -B, ad
dlolerminnnts 4), Dawn regutaticm ol M.A fase [catigens in breast carcinomas may be more Eequent
thar previously repacted suggesting that alteracions af HLA elass | call represen: an :mportacr step
anseiated with tumor :avasion prcviding tumor cells with che ability to escape recognition by T-
Iymphnoyies (8), The evercl alm of our research i t better define tho rale of MHC class I antiga: Yoss
and ils ieraction with ater histe-pattologic and epidowciological factors thy: can seve a risk inlcators
in the progression af primary breast cancer to metastatic di

BODY: Statement of Work
TASK I:

Tn womens with printry und metastalc lesions af the breast tn detormine whether ILA Class L
antigen lis: ed owsn regulation ix greater i hese with lage stage and merastrie disease than in women
‘with early gage disease (monihs 1-48): to determire whether among women with concurrent

scenanpfamtie Fesions aad breast tutiocs HLA, Class | anrigon loss or dow. cepnlation is more freqcent in

‘he Lame chan ia ce pro-neoplastie lesion (1-S4): association with bistonarkolog'c characteristics of the

lesions, ineludiag esttogen and progesterone receptor status (months I-54); and discase surviva. (1-8)

‘3; Begin consiraction afthe breast canoor eohor 300 eases)"

Pathologss De Raju and the JI will begin scroonng breast can:
ceases for detineation into Stage. aed forthe presence of
‘eunuire;Tesions ef henign proliferative ans! cancer Tesions, tage her
swith nome] breast issue, We will dean appnopriate farms to tocond
Aletopatholgical ané clinical daca based on our current KIM project
forens amel instru (Insirumerts selon in origin] pra}

bi: Retrieval of T & F slides for ses

Review al sites

4: selection ef tumor blocks and sectioning af tissce for
immunohistachem stey assays

© epi HLA class f imucunonssays. as stides become available

#— Cortinue consiruetion nf ie breast cancer eohont, dle concurrent

Tesi cob ae
Reviaw osen (PRE dl

istopallloyie data aatheriig. See Patholog.”
:numents Scotion) for hisopattologic,

parameters

Bcomirug HLA chess Tizamumessiys as adil cases are enteral
into the eghort

hs Anmual reparis willbe wailten

i Tniticl manusercs on che PISD cohort will hewriton
Tinatized Pathology Review Foum (PRE) has been convoited icto the Ieleform version
far alectencie data entey. Electronic deta oncy has been accoraplished for 203 sases, We
‘expect his cumber to tnereaso substantially doing 2004

‘Ths Far over 139 pathology reports have been shieinel._ A (inelicad data base ar 1128
‘roast cancer ytape Rewitry pulients haa been cotr pleted totaling 6338 cases

3, TAT slides hve heen retrieve! fv the pathlagy archives for ea additonal 540 cases,
ora total oF 1250 eases:

4, 30fballsinnal cases have heen reviewee by the pathologist on PRE forms fora tal of
£950 case

5. Selectioc of tamer blocks cempleted for an additional 200 eases ringing the toil eves
7 thus tar

6 Sectioning ¢f ussus for immunchisrachorsisory assays: completed for 700 cases

7. HLAclass 1 immesonssays: conmpleted for 319 wases

8 ‘The finalized HLA Imuscuohistachemistry Form has boun cunvertal into the Teeter,
version far slect"onie cata entry Appenalix #1)

9, Mechva abslrction lm: ecnnpletll 1 483 cases

TASK 2

na analysis ané repoxt witing (months 56-69)

2. Final snalysis of epidemiologics. tisk factor det, hislopo:hiloy cal and einical data and
HLA expression rests wil he performed.

eA Final report and adione) manuscripts on the brcast cancer cohort
‘willbe prepared

Brosress: PENDING

REY RESEARCH ACCOMPLISHVENTS,

> The Tenry Tard Teall: Systea Tuswir Registty data, whic: isthe sommes af our sendy
alu staeting than 198] through 2100 was velidared aed verted for vital patient
information agsinst tho SEER daca (2X03 progress tera). We hove so fur acqu'ved a lotal
palientdatuhase oF 4,900 yale nd verified breiol cancer vases, The verification sad
validation of wur consructel hreast cancer coo, camap:eted as part of our 2003 progress
goal has heen Foclawed up wich a complete database coustrction of all pathology repo
‘umnbers (sucgical pach mumbsrs) nambering 3139, Thos a crite tisk ws the FIFTS Tursor
Aegiszy docs ot indicate dhe pathology report uuimbers, The Tatter is iraperative far ay
tissue amalysie which requ-res‘nforenalom of che path murnber und the speci tsave block.
A raxjornccecaplishiner! has heen Fe careful reieceva’, and daca ancy of pathology reports
Taran updated database of 6338 patients,

4 Mimuscript Publication 'Subrnissions
See Reportable Qurcsines eli
Completed a total 92314 eases Zor HLA. assessment
REPORTABLE OLITCOMES
1, MANUSCRIPTSRFPORTS

A; DOWNREGULATION OF IILA-A AND BW6, BUT NOT BW,
AL.LOSPRCITICITIES IN LEUKEMIC CELLS. AN ESCAPE MECHANISM
FROM CTL AND NK ATTACK?

Geangigh (e200 Amen Sooty of Henuoloy, ocd Fit Eton Paps
prepulse waline Decewiher 4, 2408; DOL 10,1 82bload-2003-07-2:
fAppendis 42)

Bi HLA ANTIGEN EXPRESSION INBREAST CANCER: A MULTICENTRIC STUDY
UTILIZING FORMALIN-FIXED PARAFTINIZED TISSUES. Mt |. Worstont,R.
Namevnt, U. Rijul SR. Walaa, T. Cabverz °F. Garrido SE. A. Repaaky",
Hiyloer"M, FeensteeMVeedaasdan’, MSchipper’, Milam’, SFemonc!, Cancer
Cenetcs Revearch, Depatntent of Pathology. Henry Kate Heal Systoms, Detrovt, MI,
448202. LSA? Untfoumed Serves Univ. ofthe Health S'gnces, Methesla, MD 20814
LSA, Heap, Laiv., Virgen de is Nitwes, Canad, Spin, Raswall Park Cwcer [atat,
Bufale. NY L4263,° Univ, Moup, Chrecht, The Net erland
Sumi io: Braast Corea Reseach (Appendix #3]

(| SUBMITTED REPORT TO THE “HLA AND CANCER” COMPONENT OF
LB" INTERNATIONAL BISTOCOMPATTBILITY WORKSIOP, 8.2402
LOSS OF RFTFROZYGOSITY ATTITTA TCI API AND 18921 (Appendix #4)

CONCLUSIONS:
Ar DOWNREGULATION OF [ILA-A AND BWS, BUT NOL BW4,
ALLOSPECIFICITIES IN LEUKEMIC CELLS. AK ESCAPE MECHANISM
IROM CTL AND IK ATTACK? (Appondis #
HLA class antigen defocss may hive a acgative rypucl on the yruseiny application oF T cell
tase immunetherapeuticsmategies for Ingulmen’ «lek. Theretore in the present shady taking
advantage of a large pune of THA clas allele-specific huenan ronoclona antibodies we have compared
K-LA class [antigen expression an leakemie cells witk chat on autologous and allogeccis normal 22s
Doweregulation ot HLA-A andior -B allospectficiciss was presorin the majority al de patients stuled,
Eowevet. dowuregnlation did not affect sll HLA cless [alles unitonly, ul wes alrut exclusively
testristed 7o HLA A allespecificities and to HLA-B allospeviliviics whieh belong ta the IILA-Bwé
qroup, The kiter aJaspecif.ites, at variance fron thuse which belong the TILA Bae gremp, do not
adslate she irteraegions oP buktenir vells with NK ells. Therefire bur ests sige tha she selet:ive
downregulation oF HLA-A ard HT.A-PowS allasnocifciies associclad with HLA-Dved preservation
Provides feukernic cells wth an eseape mechanism not only fem CTL, bur also from NK cells, cts &
Fesult T eell-hased imnmanathecapautissratepies far leckemis should ullizs HLABw4 alloantigens as
resirctiag clements since a selective HLA-#4 allote loss would provide leukemic ells with am sscaps
mechanism
B: HLA ANTIGEN EXPRESSION IN BREAST CANCIR: A MULTICENTER STLDY

ATTILIZING FORMALIN-FIXED PARAFFINIZED TISSUES (Appendis # 3).

Despite the posstaleolinical significance andl po‘entil fy T-tell sel irnmotherspy, evaluation
‘02 malignant lesions for HLA class [mligen expression isnt perleried soatitely, even for parents who
are candidates for such the=apy. ‘This reflec, al legs in part #eluetanse by pathologie 10 utlize frozen
tssmue scotions in IHC assays. Litle information is available shaw: the selulness oF foetalinfixed
pparufftc-embedded theues (FFPT) as subsleatas in TTC ass evaale tissue expression of HLA,
antigens We therefore usderinek a ruivenfer shuly lw level and standeccize aa ILC protoce! using
FEPTs und anti-HLA mAs, To dete if loss ol expression af MIC Class 1 molecules atthe protein
lovel reflect altertions at (re gone level, DNA rim miceodiascoted normal ane. tumor tissue were
eeeled wits microsulellitas al tae MDIC class [6p21.3 focus (HLA. A, B, C dekacxinamts) an atthe

5421 beta 2 m.croglobulin locus tor carcerdance of expression, HI.A glass Tunligen dovat-regulotion ia

conjunction with cellular hetorogencity of cxprossion in Ihrae sree! carcinoma cases was cancardantly
reported by tke foct participating laboratories wit the aniTTL.A class] aribody 14C-10 and veth che
anti-beta 2 mieroplobulin T368. Punbu-mione, 1s suing 0° nos ane metigngae mammary cells as
deel hy: the fr lear fa he lesions saved with the arci-HLA class IL LG. 19 contrast
indltraing Iynyhoestes were strongly sta.ned by LGM. Downregulation of class was reflected by LOW
in cases U and 3 for the 15q21 locus and in ense | at the 6p21 loons, The revulls indicate that PEPTs
eprcicnt a uscfil substrate upon whish to movilor HT. antigen eepressior in malignant lesions,
capevially when appropriate marksrs gre uss lo liMerentinte wealigramt celle érom lymphocytes and
dendeile el,

‘The mannseript has boon submited co "Breast Cancer Reve

le" (Appendis 3).

C: L088 OF HETEROZYGOSITY AT HLA LOCI 6P21 AND 15Q21 (Appendix # 4)

Todletermine if loss af expecssion of MHC Class { malecules at che protein level rellyl allem
athe gcae level. the HLA and Cancer component ofthe 13" International Hisloegmpauihility Workshop
uncertaak a small prot makicenter study, ‘The goal was to optimi and assess the epwotucibilicy of tae
assays to measure the expression ef HLA slase Tnligens znd ia detect LOTT al the TTLA Tous (6p21] and
atthe B2m locos in formalin fixed, pura Tn ernbeddsl wesaual breast epithelinm and malignant breast
Tesiem

[DNA ftom mictodisseared normal and tumor tissue were
AIH! class Tp? 143 Incus4HT.A-A, B, C deternsinants) ala the L5q2l ela? miexeglaulin locas Gu
eswentince express (Table 1), TTT ets Eantigen davwn-regulation in conjunction with celtular
heteruyenety of expresso in thee bras carcinoma eases was concordant reported by che four
participating laocatories sith the antlILA eiass € antibody HC-10 and with the auti-bota 2
ricroglobul: L38s, Enmhermare, the four lasoratories detected ce staining. of moral yr maliygant
brosst sus forthe lesions stained with the anti-HA class THLGIT, Tn enntraa, infirating lymphocytes
were stiongly stained by LGIT, Downregulation of clase was reflected ay LOH in caacs and 3 for tke
15q21 Jocus and in ease 1 st the 6p21 locus, The ess indizace that FEPTs represent uselil subse I
monitor HLA antigen expression in maTignemnt lesions, especially whem appruprin e markers are used te
differentiate malignant cells fran Lymphoeytes ani denudiie cells,

Lose af helesorygatitslex-markees oprosentative of tae HLA loci ar Gp2 and 1S92agresd with,
dovin regucation. of expression af e!ass 1 gone expression, LOH assoc:ated with loss of the HT.A loos a
chromosome 6p supports aa extoxécd enechanism taatacey contribute to HLA haplolyc luss previously
described mn differant Fustolagical ter types, Contscninating stoma ean mask TOF resis und

jatel with eaierosateites atthe
icroscopic or ser microdissection, ta separate storna and tumo: weuld facilitate an interpretation of
LOM in cases with unclear results

REFERENCES,
1. Maiincola, EM. Jafis, b, Hicklin, DJL Bsvape of human sofid turnans torru 7 eell cecagnition:
sc mccbanisins and funstional significance. Arty tmmanol. Im press, 1958.

molecu

Restifo NP, Marincola FM, Kassakan: V, Taabenberger J, Yarnell JR, Rosenberg 88. Loss of
funelonnl blu 2-microglebalin in metastacie melacottas from five pationts receiving iamamulheszy.
Nal Cancer Inet, BH: 100-118, E86,

Joger by Ringhotfer M, Kezbach J, Aran Mf, Oesch T, Jager &, Kath A. Imnnunesslection in vive:
Indspeadent loss of MHC class Td eelanocy-« lTeeutiation actigex: expression in metastatic
anstonama medicaced by antiger-specitic CTI. ul. J. Cancer 71-142-147, 1997

4, Moller P, Mattfelat T, Grass Geta, Pxpressiim of HLA-A, -B, €, LI -DP, -DQ, and of HLA-D-
asseciated invariant chv'n (i i non-neoptastie miemmary epithelium. fib-oadenoma, adenoma. and
fonreingmal the breasl, Amn | Patkol 125:

5, Moievaua AM, Cestaara AM, Fano RA, Calling G, Expression nf MTTC eles Tand clans tC antigers
iu primary breast carcinomas ane synchremus risa satsstases. Clin Exp Metastasis 13: 43.48, 1995

APPENTIX

1: Stady Instromtents
HILA Tmmumohistoctemisiry Rlevirore Date Teleforea

2: Manuscripts Publishet
Thosenreg sation oF HLA-A and Bw, but not Bx, aLoepecifieftes in lencemie cells. An escape
‘mechan foes CTL and NK artack’! Copyright (2) 2093 American Society oF Tlematclagy:
Blood Kirst Edition Paper, prepuilished oalie December 4, 200%; DOI 10.1 182cploo-2003-07.
2500

3: Manuserips Submited:

son in bresst cancex: a multicenter study utilizing formalin-Fixed paratiia.72d
din 82)

TILA antigen expres
tissuoe, suamitred "Breast Cancer Nosearoh” iy

Repan tn LATS,
Long a helerorygonity al ILA loci 6p21 ané 15q2t. submitted 924/03
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Blood First Edition Paper, prepublished online December &, 2003; DO! 10.118zibiosd+2003.07.2500

Article title: Downregulation of HLA-A arid Bw, but not Bw4,
allospectiicties in leukeric eol's, An escape mechanism from CTL and
NK siltack?
Authors: Demanet C’, Mulder A”, Deneys V*, Worsham Mf, Claas F *,
Ferrane 5 ®,

Affiliations: HA Isboraton:. Acade"12 Hesptal VUE, B-ussels &

i, ‘Dep ef
lreruncher-atsegy ard Blacctrans‘usen, Leicen Universy Medicat Carler. Len, be
Neherlarde, "Dept. af Immcanheme-sony, Universié Grttlioue de Louvain, Sussee,
Belg, Dest of Patlogy, Hanry Ford Hosp sl, Data, Ml, USA. "Dart. of Ir-unctogy,
Roswal Park Cancer rete, Bualo, KY, USA.

umntag thor evecricens eacage fim Cl ae NK eee

Abbreviations: ALL: acute Werphoblacic leukemia, ALL: acuta myeoid loukomia, CLL:
cfvorie lymphatic laukemia HLA human .eukocsta anger, NK: Metaal Xie call, CDC:
cole ront tesordenleylelotety, Mt moan Tuoreseen25 index.

Keywords: ALA viass ., buke~a, Ueiruugulatio, Bad, Bu6, Natal KiLSE cul
menses ell ane

Conespandence

0r.€. Deane

cedars Heap: Yrja Unive tet 2ussel

HLA Laborleny

Larbouklo 195, 1059 Br ess Celie

Prone: 192.2477 87 08

Fox #80 2477 69 77

tra bition dear ena wane

Total toxt word counts: 3847
Abstract word Counts: 203

Scientific heading: Neoplasia
Floaneial supports This eludy ws supuertsl by & grant fom the ‘Fonds. voor

‘fotenechappotiy Onde zouk Vane * (FA

{0-Viamceon No, 1..457.99) avd by PAIS

grants Casi une,

HHS,

850 CAGTH08 and FS0 CAIGIGG awarced by the National Cancar

1

‘Copyright (} 2002 Amerlzan Soclety of Hematology