DTIC ADA424123: Development of COMET-FISH Method for Rapid Identification of Persons at Risk for Breast Cancer Development. Addendum

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by  Defense Technical Information Center

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Auard Number: 2AND17-91-1-0526

opment of CONBT-FIEH Method for Rapid Tenzification

ersons ac aisk for Sxeast Cancer Development

POINCIPAL ISVESTIGMTOR: ROvert Bristow, M.D., Ph.o.

CONTRASTING ORG:

University Health Henso-k
Torsato, Cntario WSC 20 Canada

REPORT Da’

Type

ORT: Final Addendun

U.S. Anny Medical Rexcurch und Macerie! Cerne
Fort Betrick, Werylané 21702-5012

DIS/S.MUTLON Siai'amsv1: Approved fox P.blie Reseane;
Distriiutioa Unlind-ed

che views, opiniona and/or finéings contained in thie
Unswe of the asthor{e} end ahou.d nor 6 construed aa
Bepartuenl uf Lhe Arny poritior, policy or decision unless so
Sesigrated by other docurentation

por are

REST AVAILABLE COPY

20040713 042
Fo Approved.
‘OND Wo. 5740185

‘dene obra camary 2008 [Pinar Addorun 11? Gog 2822 - 16 Bee 2003)

Pe ROTHSRIsr .
poherl BeLetow, H.3., PED.
G.ivecedzy Health setwor's
Torosts, OuTare MG 2Ce Condy
Shot soo. ordet anor uhn.on.ca
U.S. iemy Medics! Researck and storie] comand

origiual conta:ns color places: ALL OTIC repssd:etioae will be i= black and whize

approved for eublis Foleage, bistvib:tion Unlinitee

A. proportion of broase cancers have: genetic alterations ot VR UU2-q12 (HER-2ineu), 2013.2 (ZNEZLT gene}
ard L7PI3.1 (FH. We have documented global gocetic instability withm malignant breast cancer eell lines
| (MCE: MDA-MB-468, amd CRL:-2336) by coupling the DNA-damage Curaeé wssny to fluorescence in situ
hybridivation (Comet FISI1). Gene-sposfic: instability al pS and HERZ Loci was also observed, Following
genotoxic insull OR and HO»), we found that p53 locus seas capable of rapid and preferential repaic wher
‘compared to the ITER-2 locos, Comtet-FISIT is a rapid and easy assay which iv amcuable tor the study of gene-

spovifw instability or DNA repairin tissues relating to cancer risk or pitngession,

Taclaceitind Unetnsai fina eetonn: fied 2admibed
TN TR SO = ‘Sinaia rp IA ew 27
OF CONTENTS

rom: Cover

Standard Forres $1203

Table of Contents

U. Intredvction

UL. Body
a) Methods and Mavesiels-Technalogy Development
bb) Rosus-Aims 1 and 2 addvessing Stalemen! nf Wock

TV. Rey Resourch Accomplishments
\. Repomable Outcomes

WI Conclusions

YI Reforecces

VAL Appetilices¥ are

12

xu

18
‘Mon: chan one ia eight secnnen sill bs tivgnosedl with brosstcaneor inthe. Lime
‘when compared to the geaeral population, ch Fire: deyros female relives of brcast cancer

piliomts have atwo to thuoefold inocease in breast

canoer visk( 1). Tew biomarker assays ae

allele to identity nds isk for brea! er niente e-seogzneky in sated
oust andlor micro enviconenente! lactes(2-4). Genel: natahility oa coromon finding cag
the prozens ef iumprigeaceis nnd the prcscnes af non-<adoe c'ncsncnosme shemale in breast
cancer cells may eect an underlying prelisposition for chramosonta instabiity et pecific gene
Joci(S). 8 aumbor of gsoes bave beea impticaol in fain! an sparc bron cancer
carciaogecesisinluding he estrogen al pongcste cto ceceptns rene wid cep
cpidsemal prose actor cept Tueily members Gvluding HER oct), BRCA, BRCA2,
ATM and 581), Altered expression af te above genes can be nsosiated witn an mcressed

‘reas cancer ssk und aller rales of cast eancer progression, @, 7)

Chromosome instabitity within brent cancer cells may arise asa consequence a
‘discordant cell eysle checkpoint contol and the repair of specific genetic loci following DNA

dtaeange¢2, 8). The BRCAL ord BRCA2 genes are preter

18 forthe association hetween

‘chromosomal :nstabifity, decteased Lideity ef DNA repuir al sn increased tisk for breast cancer
(7). Mutations in BRCAI and BRCA2 can prevent ayynoreiale homelogius recombination

{rough altered binding co the FANCDD?2 und RADSI proteins and iit the $ er G2 eel] eye

checkpoints fullowing DNA darnage(6), Otaer common genetic alleations wssociated with breast
cancers include auipliticatioas of HER-2iveu or 7NF247 (loca on 17qH1.2-q12 and 2013.2,

respective'y) and deletions er mutation ia p$3 (locared on 1713.1). The TIFR-2inev oncogons
encodes 2 185 KD tamennbrane tyrosine kinase sccoptor with axtensive homaloay to the
epidermal grows factor seerptor. Amplification of ITER-2inen is asiousaled with inorensed cell
proliferation, lace geale and chrorcprul-ameuploidy in ssveciation with an aggressive

Tinie eourvo(9), Broast tuners whi

ot express mutations inthe p53 gene acquite siilar

haracevistics, prasurnubly due Io allexco GL and G2 cell eyele checkpoint vente]

and decroasss

DNA repsis (10, 11). The oncogene ZNFIL7 encodes a member of the Knugpet

Fumify of canscr’priem Factors, ‘Ts gone Is amplified early ir breast cimuer coremogenesis i

one fifth oF reas! uemazs and is associated wit poor prngnasis (12).

In this study we hypothesized thut penele instability af specie gent Tosi, such as pS
aud HER-2/neu, night be a key chareteistic of brcasc cancer. Indeed, using & combsned Comet
FISK protocol, we observed insreased instabiily at hath these Tac in boast cancer vells, Our

aaa su

thatthe case and rapidity ofthe Comet-TISTT assay could make ituscful ia che

alin

Setting for ansesxing (he relative stab.lity of specie gers laci associated vith either

Dbrews cancer risk or progression.
HI. BODY OF REPORT
Original AIMS in Scatement of Work:
1) Vedovelop an validate the COWMT-FISH any to dovect genetic imetaiity ot howe

fencer ef Wah
2) Twapply COMMT-TISTT to shaw hetevigeneity im genetic instaieg at these Toe

‘We have completed both AIMS a3 summarized below in the Methods and Results section,

A) MATERIALS AND METHODS-1TECHNOLOGY DEVELOPMENT
oll Lines and Comel Assay

The breasl concer cell Jina use this sty wens tained Fm ATOC (VA: USA) and
included: MCF-7 {also denoted ITTB22: BRCAI** and p33*""7); MDA-MI468 (also denoted
HYB122: BRCAL* and p53"), and CRL22AG (BRCAT™ and pS", The GHISIOB
Gymmphoblastoid) and AGHILM {normal mammary epithelial} cll lines were obtained fem
Coriell (NI: TSA), All cel Snes ware colued as per the supplier eeommendations.

“The alkaline Comet assay was performed essensally as previously described (13, 14),

relly, ells were atmisoa

75 ol of 0.5% iow melting agarose at 37°C and spxead 09 4.1%

agarose ple-conted slide. Slides were chen placed in ioe-cald lysis butler @2.5 MINaCl, 1D0reia
FIDTA, 10 mM ‘1uizma base, 10% DMSO, 16 Triton X) and lysed overnight, After lysis, the
slides wate placed in horizontal electeaphinesis tanks filled seth: electrophoresis ler (300 mM

N20H ! 1miM EDTA: pH 13.0) for 20 minuoss, and den subjected electrophoresis 1.250220

iA for x further 20 minutes. After cleeteophovesis, the slides were washed (044 Ml Tyis HCA, p}1
7.5; hue dimes 5 micumes each) and ueied. Pur the neutral comet ansay, initial eps of the
rouacall were similar save dor additions] incubstion with Protelnas>-K for 60 minutes at 37°C,

Addidoually, after ysis, slides wore placed in hucizouta electeypharesis tas Fife with
elevoaphcresis buffer (Ix TRI, pET 8.0) and after 20 saluntes, ciectcophovesis was calried aut ut

25 ¥P80-45mA for 20 rainates. Ta both Cen

wasup lies Were as-dvied ad stained with

ethidium bromide (2yelre) prior ts scaring. The relative amavet of fagmceted DNA contained

‘ithe the Comets ell, compaced to the won-iragmentad TINA withic the Comer heed, 22

doterminca by fluorescent ing

analysis (Northern Felipso vofiwace) (4, 15} 9 datermiae the

parameters: relative DNA ie tall; comet tall ength end Olive ull rmonren. Tn
experiments, GMISIOB and HTH22 cells exe expased to 2 of 10.Gy (using a Cs sume al
dose rate of 1.22 Gy/in as doseribedi(12) ar 100. uM TLO: Zor S minutes to induce DNA

ouble-svanw (DNA-db) und single-strand (DIVA. set, breaks. respectively. Came were thon

charactorived m0, 18 and 60 minutes post-teatinent fuiTowing addition of free aod.

Comet-FISH

The Comet assay car be combined with fluorescence in situ hybrization (HSH)
methodology to investigate the lnculalion of specific gone lock an individual cell. Whether the

FISIT signa les within inthe comic head or tlt indicates whether the weence of meres lies

‘within, arin the vicinity of, » damaged region of DNA. TISH probes ware abtained trom Vysis
{nc (Spectrum Orange: I incis; USA ard included HER-2 (190K; |7yl | 2-q12}, ZNP2I7 (320
‘es 20q13,

‘an p53 (14518; I7p13.1). A 17-Alpha centromeric probe (Spectrum Green) wa

ss aseferenes, Exposuie ta igh atkali dng the alkaline Comet assay donates DNA
allowing for direct FIST ae 3er tho manufeeae’ recornmendiions aul previnaly

descrbed(15). FISH probes were denatured at 80°C and applied onto the detydcated and died
Side and allowed wy hybridise ovemight ia hunted atephere a 17°C. This was followed

Iny gentle washes in 2x SSC at 37°C fare LO avinules befare drying al room tempececne. The
lddes wore coumlorstainedl with

iunmediately using » Mibum

Touatesvent microscope fit

wat sppropaiat tlers, Hor nenteal Camel-T7SH, ke DNA wes

first

ically desatuved (03M NuOIT for 2 minntos at om temperature) prior ta paube

hybridization

Initial KISH of ce'Tulae cystospins (400 eels por cell lin) determined tha: AGLI24,
GOIISIOB, MCE 7 and MDA.MB4AR cells bad bi-llelic p53 gone signals, wheteas CRT-2336

‘wus mmorulllie for p53 (data uot shown). AGI 134, CMI310B snd MCH-7 oeils bad bi-alletio

TIER-2 signals. Only 20% cells withis die MDA-MB4BR und CRI-2336 call lies had biallelic
HIER-2 signals, the romeioing cells inthe population with groeter than 2 HEIR-2 signals, AT. ive
cell lines were consistency hi-"llic for ZNE217, Subsequene soaring of Comel-FISH signals

‘eas carried cal by viual:sing the position ofthe Mlusrescent hybridisation signal within the

comet “bead and Lit" profile. The relative distribution of FISH sigesls im thr Comet head versus

tke comer til,

interpreted ax stale, aul anstabie, genetic loi respectively, TT aegale wore

‘both Tosutions,thogo were classified as labile genet foe,

RESULTS (SUE APPENDIX IFOR TABLES 1,2 AND ALT FIGURES PERTAINING TO

AIMS 1 AND 2 IN STATEMENT OF WORK)

Tuite alkaline and neutre! Comel usseys asses

od basctine BNA nvigrutios po

sma for
cndogenous DNA bres eating o ongoing getetcinstaitty within ormal (GAILS10B and
AG134) ond malignent (MICR-7; MDA-VTBAGS snd CHULA336) cell nes( 14,16, 17)
Following saline lysis, all the breast cancer al Sues howe evadence of fnoveased
cndogeuous DNA breaks Gi. nereased Olive tall utoments and til enn) in comspacison to

anormal uel (ce ‘able 1). This was oso true immsditely following 2Gy of ionizing reciation,
Tae

ata ave camsatent wth mevensed alkali ube sites (Le, DNA sicgle-stcand rai de
ova in che fraginented chrornuti> that mcgcnted outside the Ireas:caneer ecll vel. A sitar
tend was lwerveal in the endogenous level of DNA, deuble-stand brecks using Ihe neat

Come a85

x. When combine togsther, both endayencoas DNAckby and induced DNA-dshe
‘were gree in the tee breest cancer sel ics, wen coeipund Wy normal cells (pst; Mann
‘Woimey tn). The increased numberof nie DNA 39 or DKA-ds renics amongst the breast
‘cancer cal fine pane was not reoted to difeenta! BRCAL a 953 stuunlgenetype, These

results ace causinien! with opereased genetic insuiliy and ongoing DNA. dima w:thit

-malignent breast oells (18, 19)

‘We subsequently used Comet SH to specifically interrogate the eadogen us sability of
{ZNHII?. p53. and TIN loc. "Ihese data are presented in Table 2 with representative images
shown in Figure 1, We optinized cbs hybridization conditions following, cial or neat
lysis withthe ZNP217 probe. Hydization of highly detectable rahe (240Ka) occuszed within
6-705 of alkaline comets and BF-90 5 of Roser! comets. For the ZVE217 locus, we didnot
detect aiterences in hybridization effcieney between normal an emer cel ines (sea not
sown), Addiionally, les hun Seh of ZNE217-FISH signals were observe bein ths tail of
the const in uny af tho five cell ns, eamusten with chromosomal tabLity at this ns (ata
ol shown}, HER? ar p53 TSH sigrals were observed m the comet head in greater than 98% of
cell in AGII3 of GM113100 cells, consistent wilh genetic nbiity wt both Let in nora cols

To MCE-7 cells however, ory 6U to 73 per

FR-2.a9d p53 loci, Thi

abnormal patter increased in the MDA-MB468 and CRI.2336 cel nes, in which only 17 1023,

pomcont of cells bud stable HER-2Teci and 43 to 50 pessent of wets had stable p53 loc
respectively. These dave are consistent with

ased genetic instability al WHK-2 und p53

vrtbir

cast catcer cels and thet increased inwtzility van he associa in cells whicaexpresn
matamt p53 protein and au sbcogated G1 checkpoim
Is anather series of Comel-HISH oxporimionts se acsessed dhe location nf p58 and HER-

2 signals over time following IR or 1Op-treatments, as a moasnce of ropa. af dhe ate

‘ortaining Seeman damn (soe sepresenttive images ia Figu7e 2) FISH signa were
entered within the cil of he comet in the majosity of AGL1M4 and MDA-VIB468 cells
immediatly following adi or THO, exposune, Cells were chen ineuhate. fot 15 60
annutes (elowing fo potential eps of DNA-anbs andl DNA-debe within 1 hou following
teeatment a tine when reusimal reper Kinstics cous). ACO and 19 minutes following either

‘aextment,antiple p53 and TIER.

yw were observed in AGLI34 and MDA-MP.A68 cells,

After 60 minutes, both gonetic lei were ful

paired in mn

8! cot with na evidence of signal

seattor in th

eet ail, At the sane time gol

‘he scared 5 signal solved to solely 2
cieverabe signal nthe til he MIDA-M4GS cells, whereas he seatorng of the HER 2
signals pomistod (c% Fire 2A and 2B). Those dita suggest that despite both lc being located
«9 chromasome 17; there is prsfesential psi of die 153 las (or t's sssocored etvoaatin

domain) in both nermal and malignant vet

be

Further raw huckground data celating to e-Mye, Cyclin D, 2NF2H? snd RB loci 2
found a Tables 3 to 15 ia Appendix Hand essentially supports the results of HER-2 and p53

tetling above,