VU Research Portal

(1)

VU Research Portal

Targeted chemoprevention of head and neck cancer

de Boer, D.V.

2021

document version

Publisher's PDF, also known as Version of record

Link to publication in VU Research Portal

citation for published version (APA)

de Boer, D. V. (2021). Targeted chemoprevention of head and neck cancer.

General rights

Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain

• You may freely distribute the URL identifying the publication in the public portal ?

Take down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

E-mail address:

vuresearchportal.ub@vu.nl

(2)

Chapter 1

(3)

General introduction

9

1

Head and neck squamous cell carcinomas

$QQXDOO\DURXQGSDWLHQWVDUHGLDJQRVHGZLWKKHDGDQGQHFNVTXDPRXVFHOO FDUFLQRPD +16&& ZRUOGZLGH_{,Q WKH 1HWKHUODQGV DSSUR[LPDWHO\ +16&&}

SDWLHQWVDUHGLDJQRVHGHYHU\\HDU2_{7KHVHWXPRUVDULVHLQWKHPXFRVDOOLQLQJRIWKH}

XSSHUDHURGLJHVWLYHWUDFWLHWKHRUDOFDYLW\RURSKDU\Q[ODU\Q[DQGK\SRSKDU\Q[ 0RVW LPSRUWDQW ULVN IDFWRUV DUH VPRNLQJ DQG H[FHVVLYH DOFRKRO FRQVXPSWLRQ 5LVN LV UHODWHG WR SDFN \HDUV RI VPRNLQJ DQG XQLW \HDUV RI DOFRKRO FRQVXPSWLRQ ZLWK D V\QHUJLVWLFDO HͿHFW XSRQ FRPELQDWLRQ RI ERWK_{%HVLGHV FKHPLFDO FDUFLQRJHQV}

LQIHFWLRQ ZLWK D KLJKULVN W\SH RI WKH KXPDQ SDSLOORPDYLUXV KU+39 FDQ FDXVH +16&& DV ZHOO PRVW SDUWLFXODUO\ LQ WKH RURSKDU\Q[_{)XUWKHUPRUH VRPH JHQHWLF}

GLVRUGHUVVXFKDVWKHJHQRPHLQVWDELOLW\V\QGURPH)DQFRQLDQHPLD)$SUHGLVSRVH WKHDͿHFWHGLQGLYLGXDOVIRUWXPRUVLQWKHKHDGDQGQHFNUHJLRQ_.

$IWHULQLWLDOSUHVHQWDWLRQWKHGLDJQRVWLFZRUNXSIRU+16&&FRQVLVWVRIFRPSXWHG WRPRJUDSK\ &7 PDJQHWLF UHVRQDQFH LPDJLQJ 05, DQG SRVLWURQ HPLVVLRQ WRPRJUDSK\ 3(7 RQ LQGLFDWLRQ VXSSOHPHQWHG ZLWK H[DPLQDWLRQ XQGHU JHQHUDO DQHVWKHVLD 5HJLRQDO PHWDVWDVHV LQ WKH O\PSK QRGHV RI WKH QHFN DUH GLDJQRVHG E\ SDOSDWLRQLPDJLQJXOWUDVRXQGJXLGHGÀQHQHHGOHDVSLUDWLRQF\WRORJ\DQGVHQWLQHO QRGHELRSV\:KHQVXUJHU\LVDSSOLHGDVXSIURQWWUHDWPHQWPLFURVFRSLFH[DPLQDWLRQ RI WKH VXUJLFDO VSHFLPHQ LV DGGHG WR WKH GLDJQRVWLF SURFHGXUHV 'HSHQGLQJ RQ WKH GLDJQRVWLFÀQGLQJVWXPRUVDUHVWDJHGDFFRUGLQJWRWKHHLJKWKHGLWLRQRIWKH7XPRU 1RGH0HWDVWDVLV710VWDJLQJV\VWHPDQGFODVVLÀHGLQGLVHDVHVWDJHV,WR,97_.

7KH ÀUVW OLQH WUHDWPHQW VWUDWHJ\ GHSHQGV RQ WKH VWDJH RI WKH WXPRU DW WLPH RI GLDJQRVLVWKHVLWHRIWKHWXPRUDQGRWKHUUHOHYDQWGLDJQRVWLFÀQGLQJV,QDGGLWLRQ SDWLHQWUHODWHG IDFWRUV VXFK DV DJH DQG FRPRUELGLWLHV DUH FRQVLGHUHG LQ WUHDWPHQW SODQQLQJ :KHQ VXUJHU\ LV DSSOLHG WKH KLVWRORJLFDO ÀQGLQJV RI WKH VSHFLPHQ DUH KLJKO\ UHOHYDQW DV ZHOO (DUO\ VWDJH WXPRUV GLDJQRVHG LQ RI WKH SDWLHQWV DUH SULPDULO\WUHDWHGZLWKUDGLRWKHUDS\57RUVXUJLFDOUHVHFWLRQDORQHZKLOHIRUPRUH DGYDQFHG VWDJH WXPRUV VXUJHU\ LV FRPELQHG ZLWK SRVWRSHUDWLYH UDGLRWKHUDS\ RU FKHPRUDGLRWKHUDS\RUXSIURQWFKHPRUDGLRWKHUDS\LVDSSOLHGZLWKVXUJLFDOVDOYDJH IRUUHVLGXDOGLVHDVH2UDOFDQFHUVDUHW\SLFDOO\WUHDWHGE\XSIURQWVXUJHU\,QJHQHUDO FLVSODWLQLVWKHSULPDU\FKRLFHIRUFRQFRPLWDQWFKHPRWKHUDS\7–9_.

)RU +39QHJDWLYH WXPRUV WKH ÀYH\HDUV VXUYLYDO UDWH UHPDLQHG DURXQG RYHU WKHODVWGHFDGHV_{. Despite innovation and improvements in diagnostic and treatment}

VWUDWHJLHVVXFKDVVHQWLQHOQRGHELRSVLHVDQGWUDQVRUDOURERWLFUHVHFWLRQV_{, the rate}

RIORFRUHJLRQDOUHFXUUHQFHVGLVWDQWPHWDVWDVHVDQGVHFRQGSULPDU\WXPRUVUHPDLQV KLJK$ORFDOUHFXUUHQFHSUHVHQWVZLWKLQDGLVWDQFHRIWZRFPIURPWKHRULJLQDOWXPRU DQGZLWKLQDWLPHVSDQRIOHVVWKDQWKUHH\HDUV7_{6HFRQGSULPDU\WXPRUVGHYHORSIXUWKHU}

WKDQWZRFPIURPWKHRULJLQDOWXPRURUODWHUWKDQWKUHH\HDUVDIWHUWKHÀUVWWXPRUDQG RFFXULQDSSUR[LPDWHO\RIWKHSDWLHQWVSHU\HDU_{,QSDWLHQWVSUHVHQWLQJZLWK}

(4)

General introduction Chapter 1

1

LPSURYHWUHDWPHQWUHODWHWRSDWLHQWVZLWK+39QHJDWLYHWXPRUVLQWKLVWKHVLVZHZLOO IRFXVRQWKLVSDUWLFXODUVXEJURXS Carcinogenesis

&DQFHU DULVHV E\ WKH DFFXPXODWLRQ RI VRPDWLF JHQHWLF DQG HSLJHQHWLF FKDQJHV FDXVLQJ WKH PDOLJQDQW WUDQVIRUPDWLRQ RI D QRUPDO FHOO LQWR D WXPRU FHOO D SURFHVV GHÀQHGDVPXOWLVWHSFDUFLQRJHQHVLV%2;_{)RUWXQDWHO\WKHUHDUHVHYHUDOEDUULHUV}

WKDWQHHGWREHWDNHQEHIRUHDQRUPDOFHOOEHFRPHVDFDQFHUFHOO7KHVHSKHQRW\SLF FDSDELOLWLHVWKDWPXVWEHRYHUFRPHNQRZQDVWKH¶KDOOPDUNVRIFDQFHU·KDYHEHHQ SRVWXODWHG E\ +DQDKDQ DQG :HLQEHUJ27_{0XOWLSOH PXWDWLRQV LQ UHOHYDQW VLJQDOLQJ}

SDWKZD\VQHHGWREHDFFXPXODWHGWRDOORZWKHWUDQVIRUPDWLRQWRDPDOLJQDQWFHOO7KH PROHFXODUFKDQJHVWRRYHUFRPHWKHVHEDUULHUVLQ+16&&VKRZDODUJHYDULHW\7KH QXPEHURIGULYHUJHQHVOLQNHGWR+16&&GHYHORSPHQWLVFRQVHTXHQWO\HVWLPDWHG EHWZHHQDQGFDQGLGDWHV7KHODUJHPDMRULW\RIWKHVHJHQHVDUHPXWDWHGLQRQO\ DORZSHUFHQWDJHRIWXPRUVDQGWKHIXQFWLRQDOFRQVHTXHQFHVDUHPRVWO\XQNQRZQ 7KHUHIRUHPDQ\RIWKHVHJHQHVUHPDLQFDQGLGDWHGULYHUJHQHV7KHPRVWIUHTXHQWO\ FKDQJHGDQGFRQVHTXHQWO\ZHOOHVWDEOLVKHGGULYHUVLQ+16&&DUHTP53, CDKN2A,

FAT1 and NOTCH1_.

*HQHUDO PXOWLVWHS PRGHOV RI D WXPRU W\SH DUH GHGXFHG IURP GHVFULSWLYH JHQHWLF DQDO\VLVRIWXPRUDQGSUHFXUVRUFKDQJHVIROORZHGE\IXQFWLRQDOH[SHULPHQWV$OVR +16&&LVSUHFHGHGE\SUHFDQFHURXVFKDQJHV7KHSUHFXUVRUVWDJHVRI+16&&ZHUH ÀUVWGHVFULEHGE\6ODXJKWHUet al.DOUHDG\LQDQGH[SODLQHGDVDSURFHVVRI¶ÀHOG FDQFHUL]DWLRQ·LQWKHXSSHUDHURGLJHVWLYHWUDFW28_{7KLVÀHOGFDQFHUL]DWLRQFRQFHSWZDV}

EDVHGRQWKHIUHTXHQWRFFXUUHQFHRIPRUSKRORJLFDOO\DEQRUPDOWLVVXHVXUURXQGLQJ +16&&V WKH H[LVWHQFH RI PXOWLSOH WXPRUV LQ SDWLHQWV DQG WKH SHUVLVWHQFH RI WKH DͿHFWHGWLVVXHDIWHUWUHDWPHQW29_.

7KH ÀUVW JHQHWLF SURJUHVVLRQ PRGHO IRU +16&& GHYHORSPHQW ZDV SURSRVHG E\ &DOLIDQRet al.DQGEDVHGRQ3&5EDVHGPLFURVDWHOOLWHPDUNHUDQDO\VLVRIDVXEVHWRI FKURPRVRPDOORFL_{7KH\VWXGLHGPLFURVFRSLFDEQRUPDOLWLHVGHWHFWHGLQWKHPXFRVDO}

HSLWKHOLXPLQGLFDWHGDVG\VSODVLDDQGJUDGHGDVPLOGPRGHUDWHVHYHUHG\VSODVLDRU carcinoma in situ &,6%\VWXG\LQJWKHVHJHQHWLFPDUNHUVDVWDUWRIXQUDYHOLQJWKH PROHFXODUEDVLVRIPXFRVDOFDUFLQRJHQHVLVZDVPDGHDQGHDUO\JHQHWLFHYHQWVZHUH LGHQWLÀHGVXFKDVORVVRIKHWHUR]\JRVLW\/2+DWFKURPRVRPHDUPVSSDQGS 7KHVHHDUO\FKDQJHVZHUHGHWHFWHGLQEHQLJQVTXDPRXVK\SHUSODVWLFDQGG\VSODVWLF OHVLRQVZKLOHODWHUHYHQWVVXFKDV/2+DWFKURPRVRPHVTTSTTTDQG SZHUHGHWHFWHGLQWLVVXHVHFWLRQVGHÀQHGDV&,6DQGLQYDVLYHOHVLRQV_*HQHUDOO\ WKHVHYHULW\RIWKHPRUSKRORJLFDOFKDQJHVFRUUHODWHVZLWKDQLQFUHDVLQJQXPEHURI FKURPRVRPDODOWHUDWLRQV_. UHODSVHGGLVHDVHWUHDWPHQWIRUFXUHLVGL΀FXOWDQGRIWHQWKHVHSDWLHQWVRQO\TXDOLI\ IRUSDOOLDWLYHFDUHWRH[WHQGWKHLUOLIHH[SHFWDQF\DQGRULPSURYHTXDOLW\RIOLIHDV PXFKDVSRVVLEOH_. 1HZWKHUDSHXWLFRSWLRQVDUHXUJHQWO\DZDLWHGWRHUDGLFDWHWKHWXPRUFHOOVDQGSUHYHQW UHODSVHV+RZHYHULGHQWLÀFDWLRQRIWKHUDSHXWLFWDUJHWVLQ+16&&LVKDPSHUHGE\ WKH IDFW WKDW PXWDWLRQV LQ RQFRJHQHV UDUHO\ RFFXU DQG RQ WKH RWKHU KDQG EHFDXVH +16&&WXPRUVDUHYHU\KHWHURJHQHRXVDWWKHPROHFXODUOHYHO7_{7KH&DQFHU*HQRPH}

$WODV 7&*$ FRQVRUWLXP SXEOLVKHG D FRPSUHKHQVLYH JHQRPLF RYHUYLHZ RI WKH PXWDWLRQVDQGQXPHURXVFKURPRVRPDOJDLQVDQGORVVHVLQDODUJH+16&&FRKRUW ZKLFK GHPRQVWUDWHG WKH ODUJH YDULHW\ RI PROHFXODU FKDQJHV GHWHFWHG LQ +16&& $WOHDVWFDQGLGDWHFDQFHUJHQHVKDYHEHHQUHSRUWHGWKDWPLJKWSOD\DUROHLQ +16&&0RVW+39QHJDWLYHWXPRUVVKDUHDORVVRIIXQFWLRQRITP53 and inactivation of CDKN2A,Q+39SRVLWLYHWXPRUVPIK3CAPXWDWLRQVDUHPRVWIUHTXHQW_{. Besides} WKHVHVRPDWLFPXWDWLRQVVHYHUDOFRS\QXPEHUDOWHUDWLRQV&1$VDUHDFTXLUHGLQWKH PDMRULW\RI+16&&V+39SRVLWLYHDQG+39QHJDWLYHPRVWRIWHQORVVRISSDQG TDQGJDLQRITSDQGSLVVHHQ6RPH&1$VDUHDVVRFLDWHGZLWK+39VWDWXV OLNHORVVRISLQ+39QHJDWLYHWXPRUVDQGJDLQRITLQ+39SRVLWLYHWXPRUV_{. In} DGGLWLRQDVPDOOVXEVHWRIWKH+39QHJDWLYHWXPRUVGLVSOD\DFRS\QXPEHUVLOHQW SURÀOHWKHVHWXPRUVDUHDOVRRIWHQTP53ZLOGW\SHDQGW\SLFDOO\FRQWDLQHRAS and CASP8PXWDWLRQV_. 7KHFRPSUHKHQVLYHRYHUYLHZRIWKH7&*$FDQEHH[SORLWHGIRUWKHLGHQWLÀFDWLRQRI GUXJJDEOHWDUJHWVIRUWKHUDS\EXWFXUUHQWO\WKHDYDLODEOHRSWLRQVIRU+16&&DUHYHU\ OLPLWHG 2QH )'$DSSURYHG VWUDWHJ\ LV EORFNLQJ WKH ELQGLQJ RI HSLWKHOLDO JURZWK IDFWRU (*) WR LWV UHFHSWRU (*)5 E\ FHWX[LPDE W\SLFDOO\ DSSOLHG IRU SDWLHQWV ZKR DUH XQÀW WR UHFHLYH FLVSODWLQ +RZHYHU UHFRUGHG UHVSRQVHV WR FHWX[LPDE DUH JHQHUDOO\PRGHVWDQGUHFHQWO\LWZDVVKRZQWKDWFHWX[LPDELVLQIHULRUWRFLVSODWLQ ZKHQFRPELQHGZLWKUDGLRWKHUDS\LQ+39SRVLWLYHWXPRUV²_{,PPXQHFKHFNSRLQW}

LQKLELWLRQ VHHPV DW SUHVHQW D PRUH SURPLVLQJ VWUDWHJ\ IRU +16&& WUHDWPHQW $ YDULHW\RILPPXQHFKHFNSRLQWLQKLELWLQJDQWLERGLHVKDYHEHHQDSSURYHGIRUYDULRXV WXPRUW\SHVLQFOXGLQJ+16&&DOWKRXJKH΀FDF\LQ+16&&LVVWLOOOLPLWHGZLWKOHVV WKDQRIWKHSDWLHQWVUHVSRQGLQJ_. ,QJHQHUDO+39SRVLWLYHWXPRUVKDYHDPRUHIDYRUDEOHSURJQRVLVFRPSDUHGWRWKH +39QHJDWLYHWXPRUV,QDUHFHQW'XWFKFRKRUWVWXG\LWZDVVKRZQWKDWWKHÀYH\HDUV RYHUDOOVXUYLYDORISDWLHQWVZLWKDQ+39SRVLWLYHWXPRULVYHUVXVLQSDWLHQWV ZLWKDQ+39QHJDWLYHWXPRU22_{7KHUHIRUHWKHGLUHFWLRQRIFOLQLFDOUHVHDUFKIRUWKHVH}

WXPRUV LV FXUUHQWO\ PRUH IRFXVHG RQ WUHDWPHQW GHHVFDODWLRQ²_{$OWKRXJK VRPH}

+39SRVLWLYHWXPRUVVWLOOUHFXUDQGVRPHSDWLHQWVVWLOOGLHRIWKHGLVHDVHWKHULVNLV UHODWLYHO\VPDOOZKHQDSSO\LQJFXUUHQWWUHDWPHQWSURWRFROV7KHPDMRUFKDOOHQJHVWR

(5)

1

SURJUHVVLQWRRUDOFDUFLQRPD_{$OOVXEW\SHVWRJHWKHUKDYHDSUHYDOHQFHRILQ} WKHJHQHUDOSRSXODWLRQ_{7UDQVIRUPDWLRQRIDOHXNRSODNLDOHVLRQLQWRDVTXDPRXVFHOO} FDUFLQRPDRFFXUVLQDSSUR[LPDWHO\RIWKHSDWLHQWVLQFDVHRIDKRPRJHQHRXV OHVLRQWKLVSHUFHQWDJHLVKLJKHUIRUSDWLHQWVZLWKDQRQKRPRJHQHRXVOHVLRQ_{. In}

D UHFHQW UHWURVSHFWLYH VWXG\ DW WKH 98 0HGLFDO &HQWHU WKH DQQXDO WUDQVIRUPDWLRQ UDWH ZDV DV KLJK DV (YUHQ et al VXEPLWWHG ,Q JHQHUDO WKH PRVW LPSRUWDQW ULVNIDFWRUIRUSURJUHVVLRQRIDOHXNRSODNLDOHVLRQLQWRDFDUFLQRPDLVWKHSUHVHQFH RI JHQHWLF DOWHUDWLRQV (VSHFLDOO\ DOWHUDWLRQV RQ WKH FKURPRVRPDO DUPV S S S DQG T FRQWULEXWH WR D KLJKULVN SURÀOH /RVVHV RI WKHVH UHJLRQV DUH LQGLFDWRUV IRU D KLJK ULVN RI WXPRU SURJUHVVLRQ DFFRUGLQJ WR DQ HVWDEOLVKHG ULVN PRGHO_{. Other}

IDFWRUVFRQWULEXWLQJWRDKLJKHUWUDQVIRUPDWLRQULVNRIDOHXNRSODNLDOHVLRQLQFOXGH IHPDOH JHQGHU SHUVLVWHQFH RI WKH OHVLRQ WKH SUHVHQFH RI OHXNRSODNLD LQ D QRQ VPRNHUORFDWLRQRIWKHOHVLRQWKHVL]HWKHSUHVHQFHRI&DOELFDQVDQGWKHSUHVHQFH RI G\VSODVLD_{+RZHYHU WKHVH ODWWHU ULVN IDFWRUV GR QRW HPHUJH LQ DOO VWXGLHV DQG}

YDULDWLRQEHWZHHQVWXGLHVLVODUJH$VVHVVPHQWDQGGHWHUPLQDWLRQRIWKHVHYHULW\RI G\VSODVLD LV HVWLPDWHG DFFRUGLQJ WR WKH JXLGHOLQHV RI WKH :+2_{, and changes are}

JUDGHG DV PLOG PRGHUDWH RU VHYHUH G\VSODVLD 8VLQJ WKLV FODVVLÀFDWLRQ RI WKHOHXNRSODNLDOHVLRQVDUHGHVLJQDWHGDVG\VSODVWLF²_{5HFHQWO\LWZDVVKRZQWKDW}

DQRYHOIRUPRIG\VSODVLDQDPHGGLͿHUHQWLDWHGG\VSODVLDDGGVWRDPRUHDFFXUDWH SUHGLFWLRQRIPDOLJQDQWWUDQVIRUPDWLRQE\PLFURVFRSLFH[DPLQDWLRQ7KLVQHZIRUP ZDV SUHVHQW LQ RI WKH UHYLHZHG VDPSOHV DQG WRJHWKHU ZLWK FODVVLFDO G\VSODVLD DGGVXSWRDVFRUHRISRVLWLYHIRUDQ\IRUPRIG\VSODVLD52_.

(U\WKURSODNLDOHVLRQVDUHOHVVSUHYDOHQWWKDQOHXNRSODNLDOHVLRQVEXWKDYHDYHU\KLJK WUDQVIRUPDWLRQ UDWH RI DURXQG 7KHVH OHVLRQV PRVWO\ FRQWDLQ VHYHUH G\VSODVLD &,6RUHYHQVKRZLQYDVLYHFDUFLQRPDDOUHDG\XSRQGLDJQRVLV(U\WKURSODNLDOHVLRQV UDQJHLQVL]HIURPFPDQGDUHYLVLEOHDVHU\WKHPDWRXVDUHDVRIWKHPXFRVD_{. In}

FDVHRIDPL[RIZKLWHDQGUHGSDWFKHVLWVKRXOGEHGLDJQRVHGDVHU\WKUROHXNRSODNLD_.

1H[WWRWKHVHYLVLEOHOHVLRQVLWKDVEHHQGHGXFHGWKDWPRVWSUHFDQFHURXVFKDQJHVDUH PDFURVFRSLFDOO\QRWYLVLEOH7KLVSHUFHSWLRQLVEDVHGRQJHQHWLFVWXGLHVRIVXUJLFDO PDUJLQV DQG PXFRVDO ELRSVLHV VXUURXQGLQJ WXPRUV²_{3UHVHQFH RI WKHVH PXFRVDO}

FKDQJHVFDQEHHVWDEOLVKHGE\XVHRIDPLFURVFRSHLQFDVHRIWKHSUHVHQFHRIG\VSODVLD +RZHYHUHYHQEH\RQGPLFURVFRSLFFKDQJHVWKHPXFRVDOHSLWKHOLDOFHOOVPD\DOUHDG\ FRQWDLQ FDQFHUDVVRFLDWHG FKDQJHV ZLWKRXW DQ\ PRUSKRORJLFDO FKDQJHV DOEHLW WKLV PD\ FKDQJH ZLWK WKH UHFRJQLWLRQ RI WKH QHZO\ GHÀQHG GLͿHUHQWLDWHG G\VSODVLD ,Q WKHVHFDVHVPXFRVDODEQRUPDOLWLHVDUHRQO\GHWHFWDEOHE\WDNLQJDELRSV\IURPWKH VXVSLFLRXV DUHD DQG SHUIRUPLQJ JHQHWLF DQDO\VLV IRU LQVWDQFH RQ WLVVXH IURP WKH VXUJLFDO PDUJLQV VXUURXQGLQJ D WXPRU RU E\ EUXVKLQJ VXVSLFLRXV WLVVXH DUHDV LQ KLJKULVN SDWLHQWV_{$ SUHYLRXV VWXG\ ZLWK HQUROOHG SDWLHQWV VKRZHG FDQFHU}

DVVRFLDWHGJHQHWLFFKDQJHVLQELRSVLHVRIWHQSDWLHQWV,QVHYHQSDWLHQWV 7KHGULYHUJHQHORFDWHGDWFKURPRVRPHSRIZKLFKWKHORVVRIIXQFWLRQLVLGHQWLÀHG

DVDQHDUO\HYHQWLQWXPRULJHQHVLVLVTP530XWDWLRQVLQTP53 PLJKWEHFRQVLGHUHGDV DJDWHNHHSLQJPXWDWLRQLQ+16&&7_{3UHYLRXVO\9DQ+RXWHQet al.GHWHFWHGVPDOOIRFDO}

patches of TP53PXWDWHGFHOOVLQVXUJLFDOPDUJLQVRI+16&&SDWLHQWVDVWKHDSSDUHQW YHU\ÀUVWVLJQRIPDOLJQDQWWUDQVIRUPDWLRQ_{7KHVHIRFDOSDWFKHVPD\SURJUHVVLQWR} ODUJHUÀHOGVRIPXWDWHGFHOOVDQGÀQDOO\JLYHULVHWRFDQFHU_{/RVVRIIXQFWLRQDOTP53} WRJHWKHUZLWKORVVRICDKN2A ORFDWHGDWFKURPRVRPHDUPSLVPRVWSUHGRPLQDQWO\ SUHVHQWLQDERXWDQGRI+39QHJDWLYHWXPRUVUHVSHFWLYHO\_{. In addition} WRWKHVHWZRHDUO\HYHQWV+39QHJDWLYHWXPRUVFRQWDLQPDQ\RWKHUPXWDWLRQVDQG FKURPRVRPDOORVVHVDQGJDLQV

BOX 1: Cancer progression. &DQFHUGHYHORSPHQWVWDUWVZKHQDQRUPDOSULPDU\PXFRVDOFHOO

DFTXLUHVWKHÀUVWJDWHNHHSLQJPXWDWLRQ7KLVPXWDWLRQDOORZVWKHFHOOWRREWDLQDJURZWKDGYDQ WDJH UHODWLYH WR LWV QHLJKERULQJ FHOOV WKH VRFDOOHG EUHDNWKURXJK SKDVH_{. Over time these}

FHOOV DFFXPXODWH DQG IRUP D SUHFDQFHURXV OHVLRQ 0XWDWLRQV SURYLGLQJ D JURZWK DGYDQWDJH DUHNQRZQDVGULYHUPXWDWLRQV1RZDGD\VWKHUHDUHDERXWLGHQWLÀHGGULYHUJHQHVIRUWKH FRPPRQFDQFHUW\SHV_{$IWHUDFTXLULQJWKHLQLWLDOGULYHUPXWDWLRQDGGLWLRQDOKLWVFDQJLYH}

ULVHWRWKHLQYDVLYHSURSHUWLHVRIWKHDͿHFWHGFHOO2QFHJDLQHGWKHFHOOLVDEOHWRLQYDGHLQWRWKH QRUPDOVXUURXQGLQJWLVVXH_.

%HVLGHV PXWDWLRQV LQ WKH GULYHU JHQHV HYHU\ FHOO DFTXLUHV H[WUD PXWDWLRQV DV D UHVXOW RI FHOO GLYLVLRQH[RJHQRXVIDFWRUVRURWKHUFHOOXODUSURFHVVHV0RVWRIWKHVHPXWDWLRQVGRQRWFRQWULEXWH WRWKHGHYHORSPHQWRIFDQFHUDQGDUHFDOOHGSDVVHQJHUPXWDWLRQV_{$OWKRXJKWKHVHSDVVHQJHU}

PXWDWLRQVPD\LPSDFWWKHÀWQHVVRIWKHFHOOVDQGSURYLGHQHRDQWLJHQVWKDWFDQEHUHFRJQL]HG E\LPPXQHFHOOV_{1H[WWRWKHSRLQWPXWDWLRQVDSSHDULQJLQHYHU\FHOOVHYHUDOFKURPRVRPDO}

FKDQJHVDUHSUHVHQWDVZHOO0RVWVROLGWXPRUVFRQWDLQVHYHUDOFKURPRVRPDODEHUUDWLRQVORVVHV DQG JDLQV RI SDUWV RU ZKROH FKURPRVRPHV FDXVLQJ DQHXSORLG\ $ WKLUG W\SH RI DOWHUDWLRQV DFFRXQWDEOHIRURUDWOHDVWLQYROYHGLQWXPRUSURJUHVVLRQDUHWKHHSLJHQHWLFFKDQJHV$OWHUDWLRQV LQPHWK\ODWLRQVWDWXVRIFRGLQJJHQHVFDQFDXVHDQDGYDQWDJHDQGDQLQFUHDVHGJURZWKUDWHDV ZHOO_.

Precancerous lesions in the mucosal lining

$V GHVFULEHG DERYH SUHFDQFHURXV ÀHOGV FRQVLVW RI D JURXS RI FHOOV LQ WKH PXFRVDO HSLWKHOLXP FRQWDLQLQJ FDQFHUDVVRFLDWHG DOWHUDWLRQV 7\SLFDOO\ WKHVH FHOOV VWLOO ODFN LQYDVLYHJURZWKDELOLWLHVDQGFRQVHTXHQWO\GRQRWVKRZPHWDVWDWLFEHKDYLRU_{. Some}

SUHFDQFHURXVÀHOGVPDQLIHVWDVYLVLEOHOHVLRQVLQWKHPXFRVDOOLQLQJHLWKHUDVZKLWH SDWFKHVOHXNRSODNLDRUUHGSDWFKHVHU\WKURSODNLD

/HXNRSODNLD LV GHÀQHG DV D SUHGRPLQDQWO\ ZKLWH SODTXH RI TXHVWLRQDEOH ULVN KDYLQJH[FOXGHGRWKHUNQRZQGLVHDVHVRUGLVRUGHUVWKDWFDUU\QRLQFUHDVHGULVNIRU cancer_{7KHUHDUHWZRPDLQVXEW\SHVRIOHXNRSODNLDGHVLJQDWHGDVKRPRJHQHRXV}

RU QRQKRPRJHQHRXV (VSHFLDOO\ WKH QRQKRPRJHQHRXV OHVLRQV WHQG WR KDYH D KLJKHU ULVN RI PDOLJQDQW WUDQVIRUPDWLRQ²_{$ WKLUG VXEW\SH WKH SUROLIHUDWLYH}

YHUUXFRXV OHXNRSODNLD 39/ LV GHÀQHG DV PXOWLIRFDO OHXNRSODNLD 7KLV W\SH RI OHVLRQ SUHVHQWV DV D QRQKRPRJHQHRXV SODTXH ZLWK D YHUUXFRXV NHUDWRWLF VXUIDFH ,W LV PRVW FRPPRQ LQ ZRPHQ DQG TXLWH DJJUHVVLYH VLQFH RI WKHVH OHVLRQV

(6)

1

)RU ODUJHU OHVLRQV DQG SUHFDQFHURXV FKDQJHV WKDW DUH QRW YLVLEOH WR WKH QDNHG H\H WUHDWPHQWLVFXUUHQWO\QRWDQRSWLRQDQGFOLQLFDOPDQDJHPHQWFRQVLVWVRIVXUYHLOODQFH DQGZDWFKIXOZDLWLQJ+HQFHDWUHDWPHQWLQWHUYHQWLRQLVXUJHQWO\DZDLWHG+RZHYHU WKHVHPXFRVDOSUHFDQFHURXVFKDQJHVDUHQRWLQYDVLYHWXPRUVDQGDOWKRXJKWUHDWPHQW VKRXOG EH HͿHFWLYH WR[LFLW\ KDV WR EH YHU\ OLPLWHG IRU WKLV SUHYHQWLYH VWUDWHJ\ 5DGLRWKHUDS\DQGF\WRWR[LFFKHPRWKHUDS\DUHWKHUHIRUHQRWLQGLFDWHGDOWHUQDWLYHO\ VHYHUDOFKHPRSUHYHQWLRQVWUDWHJLHV%2;KDYHEHHQDSSOLHG

BOX 2: Chemoprevention. 7KHXVHRIV\QWKHWLFELRORJLFDORUQDWXUDODJHQWVFRXOGEHGHSOR\HG

WR SUHYHQW FDQFHU GHYHORSPHQW ,Q JHQHUDO FKHPRSUHYHQWLRQ FRXOG EH GLYLGHG LQWR WKUHH FDWHJRULHV 3ULPDU\ SUHYHQWLRQ WR DYRLG WKH GHYHORSPHQW RI DQ\ SUHFDQFHU RU FDQFHU FHOOV LQ D KHDOWK\ LQGLYLGXDO RU WKRVH ZLWK SDUWLFXODU ULVN IDFWRUV 6HFRQGDU\ SUHYHQWLRQ WR DYRLG WKH WUDQVIRUPDWLRQRISUHFDQFHUFHOOVLQWRFDQFHUFHOOVDQGWHUWLDU\FKHPRSUHYHQWLRQWRSUHYHQWD UHFXUUHQFHRUDVHFRQGSULPDU\WXPRUDIWHUGLDJQRVLVDQGWUHDWPHQWRIWKHLQLWLDOWXPRU_.

&KHPRSUHYHQWLRQDFWVYLDVHYHUDOPHFKDQLVPVIRULQVWDQFHE\LQGXFLQJ'1$UHSDLUPRGXODWLRQ RI VLJQDO WUDQVGXFWLRQ RU LQGXFWLRQ RI DSRSWRVLV LQ SUHPDOLJQDQW FHOOV_{. Since prevention}

VWUDWHJLHVDUHLQWHQGHGWRSUHYHQWDQGQRWWRFXUHWKHUHDUHVHYHUDOHOLJLEOHFKDUDFWHULVWLFVIRUWKH DJHQWRIFKRLFH7R[LFLW\QHHGVWREHPDQDJHDEOHLWVKRXOGEHHͿHFWLYHDWDORZGRVHDQGHDV\ WRDGPLQLVWHU,QFDVHRI+16&&VHYHUDOVWUDWHJLHVKDYHEHHQH[SORUHGDVFDQFHUSUHYHQWLRQ VWUDWHJ\LQWKHSDVW

2QH RI WKH PRVW VWXGLHG FKHPRSUHYHQWLYH DJHQWV DUH WKH UHWLQRLGV QDWXUDO DQG V\QWKHWLFYLWDPLQ$DQDORJV7KDQNVWRWKHLUUROHLQFHOOSUROLIHUDWLRQDQGGLͿHUHQWLDWLRQ WKH\JDLQHGLQWHUHVWDVDJHQWVIRUFDQFHUSUHYHQWLRQ,QVHYHUDOFDQFHUW\SHVLWZDV GHPRQVWUDWHGWKDWWKHVHUHWLQRLGVDUHDEOHWRVXSSUHVVFDQFHUSURJUHVVLRQUHYLHZHG in_{6HYHUDOFOLQLFDOWULDOVZHUHFRQGXFWHGLQSDWLHQWVZLWKRUDOOHXNRSODNLDOHVLRQV}

WRUHGXFHDQGUHPRYHWKHOHVLRQVDQGLQVRPHFDVHVDVSUHYHQWLRQIRUVHFRQGSULPDU\ WXPRUV²_{,QWHUHVWLQJO\+RQJet al.VKRZHGSRVLWLYHHͿHFWVRIWKHUHWLQRLGVRQWKH}

SUHYHQWLRQRIVHFRQGSULPDU\WXPRUVEXWWKH\DOVRVKRZHGWKDWLQWKHVDPHSDWLHQWV WKHVH YLWDPLQ $ GHULYDWLYHV ZHUH QRW DEOH WR SUHYHQW ORFDO UHJLRQDO DQG GLVWDQW UHODSVHV_{$OWKRXJKLQLWLDOUHVXOWVZHUHSURPLVLQJODWHUWULDOVGLGQRWFRQÀUPDQ\} RIWKHEHQHÀFLDOHͿHFWV_{$QRWKHUWHVWHGFKHPRSUHYHQWLYHDJHQWLVEOHRP\FLQDQ} DQWLELRWLFPRUHRIWHQXVHGIRUWKHWUHDWPHQWRIFDQFHUZLWKLQLWLDOSURPLVLQJUHVXOWVRI FKHPRSUHYHQWLYHDFWLYLWLHVLQRUDOFDQFHU_{+RZHYHUWKHQXPEHURIVWXGLHGSDWLHQWV} ZDVOLPLWHGDQGEHFDXVHRIWR[LFLW\EOHRP\FLQLVQRORQJHUVWXGLHG$QRWKHUSURPLVLQJ VWXG\WUHDWHGSDWLHQWVZLWKKLJKULVNSUHFDQFHURXVOHVLRQVZLWKFHWX[LPDE7KLV VWXG\ VKRZHG SURPLVLQJ UHVXOWV ZLWK FOLQLFDO DQG KLVWRORJLFDO UHVSRQVHV LQ WKH WUHDWHG JURXS +RZHYHU D ORZ QXPEHU RI SDWLHQWV ZDV HQUROOHG LQ WKH VWXG\_{. A}

VHULRXVOLPLWDWLRQRIFHWX[LPDELVWKDWLVKDVWREHDGPLQLVWHUHGLQWUDYHQRXVO\DWDQ RXWSDWLHQWFHQWHUZKLFKLVXQGHVLUDEOHIRUDFKHPRSUHYHQWLYHVWUDWHJ\2WKHUDJHQWV WHVWHG ZHUH LQKLELWRUV RI F\FORR[\JHQDVH &2; DGPLQLVWUDWLRQ RI 1DFHW\OO F\VWHLQHDQDQWLR[LGDQWDQGO\FRSHQH72_{,QDGGLWLRQVHYHUDORWKHUQDWXUDOFRPSRXQGV}

WKH JHQHWLFDOO\ GHWHFWHG ÀHOGV H[WHQGHG LQWR WKH VXUJLFDO PDUJLQV LQGLFDWLQJ WKDW WKHVH ÀHOGV SDUWLDOO\ VWD\HG EHKLQG LQ WKH SDWLHQWV XQQRWLFHG ,QWHUHVWLQJO\ WKH LGHQWLÀHGÀHOGVZHUHLQSDUWJHQHWLFDOO\UHODWHGWRWKHWXPRUVDQGLQSDUWJHQHWLFDOO\ XQUHODWHG_{6XEVHTXHQWUHWURVSHFWLYHVWXGLHVUHYHDOHGWKDWWKHVHÀHOGVFKDUDFWHUL]HG}

E\FDQFHUDVVRFLDWHGJHQHWLFFKDQJHVH[SODLQDSSUR[LPDWHO\KDOIRIWKHORFDOUHODSVHV LQ+16&&SDWLHQWVDQGDSSUR[LPDWHO\KDOIRIWKHVHFRQGSULPDU\WXPRUVLQGLFDWLQJ WKH PDMRU FOLQLFDO LPSDFW RI WKHVH SUHFDQFHURXV FKDQJHV RQ WKH SURJQRVLV RI WKH patients59_.

100% 7%

13%

85%

Figure 1: Schematic overview of the various precancerous mucosal changes.$OOSUHFDQFHURXVFKDQ

JHVFRQWDLQ+16&&DVVRFLDWHGJHQHWLFDOWHUDWLRQVSHUGHÀQLWLRQEOXHFLUFOH0RVWRIWKHVH SUHFDQFHURXVFKDQJHVDUHG\VSODVWLFHVWLPDWHGDVJUHHQFLUFOHDQGFDQEHGHWHFWHGXVLQJWKH PLFURVFRSH$ERXWRIWKHSUHFDQFHURXVFKDQJHVDUHYLVLEOHDVOHVLRQVUHGFLUFOHRIZKLFK DERXWWZRWKLUGVDUHG\VSODVWLF

+HQFH DV LQGLFDWHG LQ )LJXUH DOO PXFRVDO SUHFDQFHURXV FKDQJHV FRQWDLQ FDQFHU DVVRFLDWHGJHQHWLFFKDQJHVSHUGHÀQLWLRQDVXEJURXSLVYLVLEOHDVG\VSODVLDXQGHU WKHPLFURVFRSHDQGDVPDOOHUVXEJURXSLVPDFURVFRSLFDOO\YLVLEOH2QHRIWKHFXUUHQW FKDOOHQJHVLVWRGHWHFWDOOPXFRVDOSUHFDQFHUVLQSDWLHQWV6HYHUDOLPDJLQJDSSURDFKHV DUHEHLQJXVHGVLQFHFHOOVXQGHUJRLQJSUHPDOLJQDQWFKDQJHVDUHNQRZQWRFRQWDLQ FKDQJHVLQWKHLUVWUXFWXUHDQGPHWDEROLFDFWLYLW\7KHVHPD\OHDGWRDGLͿHUHQFHLQ DEVRUSWLRQRIÁXRUHVFHQWOLJKWZKLFKFDQEHH[SORLWHGWRGHWHFWWKHVHSUHFDQFHURXV FKDQJHVEHFDXVHRIWKHUHGXFWLRQRIDXWRÁXRUHVFHQFHXSRQÁXRUHVFHQWVSHFWURVFRS\_. )XUWKHUDQDO\VLVGHPRQVWUDWHGWKHDVVRFLDWLRQRIORVVRIDXWRÁXRUHVFHQFHZLWKJHQHWLF FKDQJHVDWNQRZQLQYROYHGPDUNHUVVXFKDVFKURPRVRPHDUPVSDQGS_.

Treatment of precancerous lesions

9LVLEOHOHVLRQVZLWKOLPLWHGGLPHQVLRQVFDQEHUHPRYHGE\VXUJLFDOH[FLVLRQODVHU HYDSRUDWLRQRUFU\REDVHGVXUJHU\_{(΀FDF\RIWKHVHPRGDOLWLHVWRSUHYHQWFDQFHU}

(7)

1

ÀUVWDWWHPSWLQSDWLHQWVZLWK+16&&ELRSVLHVRIQRUPDODSSHDULQJPXFRVDWLVVXH VXUURXQGLQJ SULPDU\ +16&&V ZHUH FROOHFWHG DQG FXOWXUHG_{,Q WRWDO RI WKH}

FROOHFWHGELRSVLHVZHUHVXFFHVVIXOO\FXOWXUHGKRZHYHURQO\RQHFXOWXUHFRQWDLQHG +16&&DVVRFLDWHG JHQHWLF DOWHUDWLRQV ZKHQ DQDO\]HG IRU WKH SUHVHQFH RI DOOHOLF ORVVHVXVLQJPLFURVDWHOOLWHPDUNHUV7KHDOOHOLFORVVHVRIWKLVSUHFDQFHUFXOWXUH98 SUH6&&0ZHUHGHWHFWHGDWFKURPRVRPHDUPVSDQGSRIZKLFKWKHS/2+ZDV VKDUHGZLWKWKHFRUUHVSRQGLQJWXPRUPDWHULDO,QFRQWUDVWWKH/2+DWFKURPRVRPH DUPSZDVQRWGHWHFWHGLQWKHWXPRUFHOOV,QDGGLWLRQWKHSUHFDQFHUFHOOVFRQWDLQHG DPXWDWLRQLQTP53 ZKLFKZDVQRWSUHVHQWLQWKHWXPRU7KHVHUHVXOWVLQGLFDWHGWKDW 98SUH6&&0RULJLQDWHGIURPDQLQGHSHQGHQWÀHOGLQWKHPDUJLQZKLFKGLGQRW JLYHULVHWRWKHSULPDU\WXPRURXWJURZWK_.

1RZDGD\V WKHUH DUH QR ZHOOHVWDEOLVKHG SUHFDQFHU PRXVH RU RWKHU in vivo PRGHOV IRU WKH SUHFXUVRU VWDJHV RI +16&& ,Q VRPH UHSRUWV QLWURTXLQROLQHR[LGH 142ZDVXVHGDVDFDUFLQRJHQIRULQGXFLQJRUDOFDUFLQRPDVLQURGHQWVDQGWKHUHE\ PLPLFNLQJKXPDQFDQFHUSURJUHVVLRQin vivo_{+RZHYHUWKHJHQHWLFYDOLGDWLRQRI}

WKLVPRGHOLVYHU\OLPLWHGWKXVIDUZLWKMXVWVRPHDQDO\VHVRQWKHLQDFWLYDWLRQRIS XVLQJLPPXQRKLVWRFKHPLVWU\_.

'HYHORSPHQWRIWDUJHWHGWKHUDS\WDUJHWLGHQWLÀFDWLRQ

,QDGGLWLRQWRWKHDERYHGHVFULEHGFHOOPRGHOVDODUJHUSDQHORISUHFDQFHUFHOOOLQHV IURPOHXNRSODNLDVDQGHVSHFLDOO\IURPQRQYLVLEOHSUHFDQFHURXVFKDQJHVKDVWREH HVWDEOLVKHG DQG VWXGLHG WR UHÁHFW WKH KHWHURJHQHRXV QDWXUH RI WKH SUHFDQFHURXV FKDQJHVOHDGLQJWR+16&&7KHVHPRGHOVFDQVXEVHTXHQWO\EHXVHGWRGHYHORSDQG HYDOXDWH QHZ WKHUDSHXWLF VWUDWHJLHV $V GHVFULEHG IRU FKHPRSUHYHQWLYH VWUDWHJLHV RQO\PLQLPDOWR[LFLW\LVDOORZHGDQGWKHXVHRIWDUJHWHGDSSURDFKHVVHHPVWKHUHIRUH DSURPLVLQJVWUDWHJ\

$ ÀUVW VWHS LQ WKH GHYHORSPHQW RI VXFK WDUJHWHG WUHDWPHQW LV WKH LGHQWLÀFDWLRQ RI GUXJJDEOHJHQHWDUJHWVLQSUHFDQFHUFHOOV*HQRPLFGDWDIRUSUHFDQFHURXVOHVLRQVLV KDUGO\ DYDLODEOH EXW DV GLVFXVVHG DERYH WKH LQYDVLYH WXPRUV FRQWDLQ PDLQO\ JHQH PXWDWLRQVLQWXPRUVXSSUHVVRUVDQGWKHSUHFDQFHUVSUHFHGLQJWKHPZLOOH[SHFWDQWO\ VKRZ WKH VDPH WUHQG DOWKRXJK ZLWK OHVV JHQHWLF FKDQJHV SHU OHVLRQ +HQFH ZH VKRXOGH[SORLWIXQFWLRQDOJHQRPLFDSSURDFKHVWRLGHQWLI\YXOQHUDELOLWLHVRISUHFDQFHU FHOOV7KHUHDUHVHYHUDOWHFKQLTXHVDYDLODEOHWKDWDOORZWKHLGHQWLÀFDWLRQRIJHQHVRU SDWKZD\VFUXFLDOIRUFDQFHUDQGRUSUHFDQFHUFHOOVXUYLYDO1RZDGD\V&5,635&DV V\VWHPV DUH D ZLGHO\ XVHG WRRO IRU JHQRPH HGLWLQJ E\ LQWURGXFLQJ GRXEOH VWUDQG EUHDNV'6%DWVSHFLÀF'1$VLWHV_{+RZHYHUZKHQWKHSURMHFWVGHVFULEHGLQWKLV}

WKHVLV ZHUH LQLWLDWHG 51$ LQWHUIHUHQFH 51$L ZDV PRVW FRPPRQO\ XVHG DQG &5,635ZDVVWLOODWLWVLQIDQF\85_.

ZHUH DOVR GHVFULEHG WR KDYH FKHPRSUHYHQWLYH SURSHUWLHV )RU LQVWDQFH FXUFXPLQ ZDVVKRZQWRKDYHDQHͿHFWRQVHYHUDOPROHFXODUSDWKZD\VNQRZQWREHLQYROYHG LQFDQFHUGHYHORSPHQW_{3KDVH,DQG,,FOLQLFDOWULDOVZLWKFXUFXPLQGHPRQVWUDWHG}

VRPHHͿHFWVLQFDQFHUSUHYHQWLRQKRZHYHUDOVRVKRZHGORZDEVRUSWLRQUDWHVDIWHU administration_.

3UHYLRXV VWXGLHV DOVR LQWHQGHG WR XVH RQFRO\WLF DGHQRYLUXVHV DV VWUDWHJ\ E\ DGPLQLVWUDWLQJ WKHVH YLUXVHV WRSLFDOO\ DV PRXWKZDVK LQ SDWLHQWV ZLWK YLVLEOH OHVLRQV7KHVHYLUXVHVDUHJHQHWLFDOO\PRGLÀHGDQGUHSOLFDWHVSHFLÀFDOO\LQFHOOVZLWK D S PXWDWLRQ_{5HVXOWV RI WKH DSSOLFDWLRQ RI 21<; VKRZHG VRPH FRPSOHWH}

UHVSRQGHUVDOWKRXJKDIWHUWHUPLQDWLRQRIWKHWKHUDS\VRPHSDWLHQWVVXͿHUHGIURP UHODSVHVRIWKHOHVLRQV77_{0HDQZKLOHWKHYLUXVKDVEHHQVROGWRD&KLQHVHFRPSDQ\}

DQG FRQYLQFLQJ FOLQLFDO UHVXOWV KDYH QR ORQJHU EHHQ UHSRUWHG 7KH OHJLVODWLRQ RI WKHVHJHQHWLFDOO\PRGLÀHGRUJDQLVPVLVREYLRXVO\FRPSOLFDWHGDQGKDPSHUVUDSLG LQWURGXFWLRQLQWKHFOLQLF

,Q FRQFOXVLRQ GHVSLWH DOO WKDW KDV EHHQ WULHG PRVW SDUWLFXODUO\ IRU YLVLEOH OHVLRQV FXUUHQWO\ WKHUH LV QR VWDQGDUG WUHDWPHQW SURWRFRO &RQVLGHULQJ WKH FOLQLFDO LPSDFW RQ GHYHORSPHQW RI FDQFHU DQG ORFDO UHODSVHV LQ SDWLHQWV WUHDWPHQW RSWLRQV IRU SUHFDQFHURXVFKDQJHVDUHXUJHQWO\DZDLWHG*LYHQWKHORZWR[LFLW\SURÀOHRIWDUJHWHG DJHQWV DV D UHVXOW RI WKHLU VSHFLÀF PHFKDQLVP RI DFWLRQ WKHVH DJHQWV VHHP PRVW interesting at present.

Development of targeted therapy – precancer cell models

$Q LPSRUWDQW SUHUHTXLVLWH IRU WKHUDS\ GHYHORSPHQW LV WKH DYDLODELOLW\ RI FHOOXODU PRGHOV WKDW UHSUHVHQW WKH SUHFXUVRU VWDJH RI +16&& in vitro and in vivo. In vitro, RQO\DIHZH[DPSOHVRIVXFFHVVIXOFXOWXUHGPRGHOVDUHNQRZQWRGD\0RVWRIWKHVH FXOWXUHV ZHUH JHQHUDWHG IURP PDFURVFRSLFDOO\ YLVLEOH OHVLRQV &ROOHFWLRQ RI ELRSV\ PDWHULDORIVXFKYLVLEOHOHVLRQVZLWKGLͿHUHQWVWDJHVRIG\VSODVLDUHVXOWHGIRULQVWDQFH LQDSDQHORIDWOHDVWFHOOXODUPRGHOV²_{3URFHVVLQJDQGSURORQJHGFXOWXULQJRI}

WKHVHFHOOVUHVXOWHGLQLPPRUWDOLW\RIRXWRIFXOWXUHVZKHQNHSWRQ7IHHGHU OD\HUV7KHVHVHYHQFXOWXUHVDOOFRQWDLQHGDPXWDWLRQLQTP530RUHRYHUGLͿHUHQFHVLQ H[SUHVVLRQRISDQGSZHUHREVHUYHGEHWZHHQWKHVHYHQLPPRUWDODQGWKHWZHOYH PRUWDO FXOWXUHV 7KLV FRUUHVSRQGHG WR WKH RULJLQDO ELRSV\ PDWHULDO DQG FRQÀUPV WKHLPSRUWDQFHRIDOWHUDWLRQVLQWKHH[SUHVVLRQRIWKHVHWZRSURWHLQV)XUWKHUPRUH XSUHJXODWLRQRIFHOOF\FOHUHJXODWRUVDQGGRZQUHJXODWLRQRIGLͿHUHQWLDWLRQPDUNHUV ZDV UHSRUWHG ,Q DGGLWLRQ ORVV RI 5$5շ ZDV REVHUYHG WRJHWKHU ZLWK LQFUHDVHG H[SUHVVLRQOHYHOVRIWHORPHUDVH_.

&XOWXULQJFHOOVIURPLQYLVLEOHSUHFDQFHURXVFKDQJHVLVH[SHFWHGWREHPRUHFRPSOH[ VLQFH¶UDQGRP·ELRSVLHVVKRXOGEHWDNHQIURPWKHRUDOWLVVXHDQGFXOWXUHG7KHEHVW VRXUFHIRUWKHVHSUHFDQFHURXVFKDQJHVLVWKHPXFRVDVXUURXQGLQJRUDOFDQFHUV,QD

(8)

1

Aim and outline of this thesis

/DFN RI HͿHFWLYH WKHUDSHXWLF RSWLRQV IRU WKH HUDGLFDWLRQ RI SUHFDQFHURXV FKDQJHV UHVXOWVLQPDOLJQDQWWUDQVIRUPDWLRQWRSULPDU\WXPRUVRUORFDOUHODSVHVLQSDWLHQWV WUHDWHG IRU +16&& 6PDOO YLVLEOH OHVLRQV FDQ EH H[FLVHG EXW IUHTXHQWO\ UHFXU )RU ODUJHUOHVLRQVDQGSUHFDQFHURXVFKDQJHVWKDWDUHQRWYLVLEOHWRWKHQDNHGH\HWKHUH DUHQRWUHDWPHQW RSWLRQVDW SUHVHQW,QWKLVWKHVLVZHGHVFULEH WKH LGHQWLÀFDWLRQ RI GUXJJDEOHWDUJHWVWKDWFRXOGEHH[SORLWHGWRGHYHORSWUHDWPHQWPRGDOLWLHVDSSOLFDEOH IRUSUHFDQFHURXVFKDQJHVDVZHOODV+16&&FHOOV$VDÀUVWVWHSZHJHQHUDWHGFHOOXODU PRGHOVRISUHFDQFHURXVÀHOGV,QChapter 2ZHGHVFULEHWKHFROOHFWLRQDQGFXOWXULQJ RIPXOWLSOHELRSVLHVIURPWKHPXFRVDVXUURXQGLQJ+16&&V3UROLIHUDWLQJFXOWXUHV ZHUH JHQHWLFDOO\ DQDO\]HG 1H[W ZH XVHG WKH REWDLQHG SUHFDQFHU FHOO PRGHOV IRU WDUJHWLGHQWLÀFDWLRQE\SHUIRUPLQJDQ51$LVFUHHQZLWKSUHYLRXVO\LGHQWLÀHGWXPRU OHWKDOVL51$VIn vitro and in vivo YDOLGDWLRQZLWKERWKWKHVL51$VDQGVXEVHTXHQW VPDOO PROHFXOH LQKLELWRUV VKRZHG SURPLVLQJ UHVXOWV IRU RQH RI WKH LGHQWLÀHG KLWV Chapter 3 ,Q Chapters 4 and 5 ZH LQYHVWLJDWHG RWKHU SRWHQWLDO KLWV XVLQJ ERWK 51$LDQGDYDLODEOHVPDOOPROHFXOHLQKLELWRUV,QDGGLWLRQZHSHUIRUPHGDQDUUD\ EDVHGJHQRPHZLGHVFUHHQZLWKPL51$PLPLFVWRLGHQWLI\PL51$VWKDWFRXOGEH HPSOR\HGIRUWKHUDSHXWLFVWUDWHJLHVRIHLWKHUSUHFDQFHURU+16&&FHOOVChapter 6 )LQDOO\WKHREWDLQHGUHVXOWVDUHGLVFXVVHGLQChapter 7 DQGDIXWXUHSHUVSHFWLYHLV provided.

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·RYHUKDQJ2QFHH[SRUWHGLQWRWKHF\WRSODVP WKHSUHPL51$LVWKHQSURFHVVHGE\'LFHUUHVXOWLQJLQDQ51$PROHFXOHZLWKGRXEOH VWUDQGHG51$RIDERXWQXFOHRWLGHVWKHPDWXUHPL51$2QHRIWKHVWUDQGVWKH JXLGH VWUDQG ELQGV WR 5,6& ZKLFK LV WKHQ DEOH WR ELQG WR WKH P51$ DQG WKHUHE\ UHJXODWHVJHQHH[SUHVVLRQRIWKHJHQHVFRQWDLQLQJWKHPL51$ELQGLQJVLWHLQWKH· XQWUDQVODWHGUHJLRQ875_.

,Q DGGLWLRQ WR WKH QDWXUDOO\ RFFXUULQJ PL51$V V\QWKHWLFDOO\ GHYHORSHG VPDOO LQWHUIHULQJ51$VVL51$DVZHOODVPL51$PLPLFVPDNHXVHRIWKHVDPHPDFKLQHU\ WRPRGXODWHJHQHH[SUHVVLRQ87_{7KH'1$HTXLYDOHQWRIVL51$VDUHVKRUWKDLUSLQ51$V} VK51$VV\QWKHWLFJHQHVZKLFKDUHLQWURGXFHGLQWRWKHFHOOVE\SODVPLGVRUYLUDO YHFWRUV2QFHH[SUHVVHGWKHVHPROHFXOHVDUHDOVRIXUWKHUSURFHVVHGE\WKHLQWHUQDO PL51$PDFKLQHU\88_{$VWKH\OHQGPRUHIURPWKHPL51$SURFHVVLQJPDFKLQHU\WKH\} FRQVHTXHQWO\RIWHQEHKDYHDVPL51$VZLWKPDQ\RͿWDUJHWHͿHFWV7KLVFRXOGRFFXU ZLWKV\QWKHWLFVL51$PROHFXOHVDVZHOOEXWLQSUDFWLFHLVOHVVIUHTXHQWO\VHHQ 7KHVSHFLÀFLW\RIV\QWKHWLFVL51$VVXJJHVWWKDWWKH\DUHPRVWLQWHUHVWLQJIRUWDUJHWLQJ JHQHVDOVRDVFOLQLFDODSSOLFDWLRQ+RZHYHUWKHXVHRI51$Lin vivoKDVEHHQSURYHQWR EHH[WUHPHO\GL΀FXOWEHFDXVHRIWKHPDMRUREVWDFOHRIGHOLYHULQJWKH51$PROHFXOHV LQWRWKHFHOOV87_{. In vitro,LWLVDUHODWLYHO\VLPSOHWHFKQLTXHWRXVHIRULQVWDQFHLQDUUD\} EDVHGJHQRPHZLGHVFUHHQVWRLGHQWLI\SRWHQWLDOWDUJHWJHQHV7KHVHORVVRIIXQFWLRQ VFUHHQVDUHZLGHO\XVHGWRLGHQWLI\JHQHVWKDWPRGXODWHGUXJUHVSRQVHVRUWRÀQG QHZWDUJHWVWKDWFRXOGEHH[SORLWHGIRUGUXJGHYHORSPHQW_{7RGRWKHLUZRUNLQVLGH} WKHFHOOVVL51$VQHHGWREHWUDQVIHFWHGXVXDOO\E\HQFDSVXODWLQJWKHPLQOLSRSK\OLF DJHQWV0RVWFHOOOLQHVDOORZWKHVHWUDQVIHFWLRQVEXWVRPHDUHUHODWLYHO\UHVLVWDQWRU HLWKHUVHQVLWLYHGHPDQGLQJFRQVLGHUDEOHRSWLPL]DWLRQV7KHVHWUDQVIHFWLRQVUHTXLUH VRPHPDQLSXODWLRQVWHSVZKLFKKDPSHUWKHXSVFDOLQJWRDUUD\EDVHGJHQRPHZLGH VL51$VFUHHQVWKDWZRXOGHQFRPSDVVDOOKXPDQJHQHV)RUWKLVUHDVRQURERWLF SODWIRUPV KDYH EHHQ GHYHORSHG WR DOORZ DQG VWDQGDUGL]H WKHVH KLJKWKURXJKSXW VFUHHQVWKDWDUHDOPRVWLPSRVVLEOHWREHFDUULHGRXWPDQXDOO\

(9)

1

 0HKDQQD+5RELQVRQ0+DUWOH\$.RQJ$)RUDQ%)XOWRQ/LHXZ7et al.5DGLRWKHUDS\SOXVFLVSODWLQ RUFHWX[LPDELQORZULVNKXPDQSDSLOORPDYLUXVSRVLWLYHRURSKDU\QJHDOFDQFHU'H(6&$/D7(+39 DQRSHQODEHOUDQGRPLVHGFRQWUROOHGSKDVHWULDOLancet 393²

 *LOOLVRQ 0/ 7URWWL $0 +DUULV - (LVEUXFK $ +DUDUL 30 $GHOVWHLQ '- et al. 5DGLRWKHUDS\ SOXV FHWX[LPDERUFLVSODWLQLQKXPDQSDSLOORPDYLUXVSRVLWLYHRURSKDU\QJHDOFDQFHU15*2QFRORJ\572* DUDQGRPLVHGPXOWLFHQWUHQRQLQIHULRULW\WULDOLancet393²

 )HUULV5//HQ]+-7URWWD$0*DUFtD)RQFLOODV-6FKXOWHQ-$XGKX\)et al.5DWLRQDOHIRUFRPELQDWLRQ RIWKHUDSHXWLFDQWLERGLHVWDUJHWLQJWXPRUFHOOVDQGLPPXQHFKHFNSRLQWUHFHSWRUV+DUQHVVLQJLQQDWH DQGDGDSWLYHLPPXQLW\WKURXJK,J*LVRW\SHLPPXQHHͿHFWRUVWLPXODWLRQCancer Treat Rev63 ²

 YRQGHU*UQ-5|GHO)%UDQGWV&)RNDV(*XFNHQEHUJHU05|GHO&et al. Targeted Therapies and ,PPXQH&KHFNSRLQW ,QKLELWLRQ LQ +HDG DQG 1HFN 6TXDPRXV &HOO &DUFLQRPD :KHUH 'R :H 6WDQG 7RGD\DQG:KHUHWR*R"Cancers (Basel)11

1DXWD,+5LHWEHUJHQ00YDQ%RNKRYHQ$$-'%ORHPHQD(/LVVHQEHUJ:LWWH%,+HLGHPDQ'$0

et al. (YDOXDWLRQ RI WKH HLJKWK 710 FODVVLÀFDWLRQ RQ SSRVLWLYH RURSKDU\QJHDO VTXDPRXV FHOO

FDUFLQRPDVLQWKH1HWKHUODQGVDQGWKHLPSRUWDQFHRIDGGLWLRQDO+39'1$WHVWLQJ$QQ2QFRO2Ϳ-(XU

Soc Med Oncol29²

0F'HUPRWW-'%RZOHV':(SLGHPLRORJ\RI+HDGDQG1HFN6TXDPRXV&HOO&DUFLQRPDV,PSDFWRQ Staging and Prevention Strategies. &XUU7UHDW2SWLRQV2QFRO20

 +HJGH-96KDYHUGLDQ1'DO\0()HOL[&:RQJ'/5RVRYH0+et al.3DWLHQWUHSRUWHGTXDOLW\RI OLIH RXWFRPHV DIWHU GHHVFDODWHG FKHPRUDGLDWLRQ IRU KXPDQ SDSLOORPDYLUXVSRVLWLYH RURSKDU\QJHDO FDUFLQRPD)LQGLQJVIURPDSKDVHWULDOCancer124²  &KHQ$0)HOL[&:DQJ3&+VX6%DVHKDUW9*DUVW-et al.5HGXFHGGRVHUDGLRWKHUDS\IRUKXPDQ SDSLOORPDYLUXVDVVRFLDWHGVTXDPRXVFHOOFDUFLQRPDRIWKHRURSKDU\Q[DVLQJOHDUPSKDVHVWXG\ Lancet Oncol18²  )HDURQ(59RJHOVWHLQ%$JHQHWLFPRGHOIRUFRORUHFWDOWXPRULJHQHVLVCell61²  +DQDKDQ':HLQEHUJ5$+DOOPDUNVRIFDQFHU7KHQH[WJHQHUDWLRQCell 144² 6ODXJKWHU ' 6RXWKZLFN + 6PHMNDO : )LHOG FDQFHUL]DWLRQ LQ RUDO VWUDWLÀHG VTXDPRXV HSLWKHOLXP

FOLQLFDOLPSOLFDWLRQVRIPXOWLFHQWULFRULJLQCancer6²

%UDDNKXLV%-7DERU03.XPPHU-$/HHPDQV&5%UDNHQKRͿ5+$JHQHWLFH[SODQDWLRQRI6ODXJKWHU·V FRQFHSWRIÀHOGFDQFHUL]DWLRQHYLGHQFHDQGFOLQLFDOLPSOLFDWLRQVCancer Res63²  &DOLIDQR-YDQGHU5LHW3:HVWUD:1DZUR]+&OD\PDQ*3LDQWDGRVL6et al. *HQHWLFSURJUHVVLRQ

PRGHOIRUKHDGDQGQHFNFDQFHULPSOLFDWLRQVIRUÀHOGFDQFHUL]DWLRQCancer Res56²  $OPDGRUL*%XVVX)&DGRQL**DOOL-5LJDQWH0$UWXVR$et al.0XOWLVWHSODU\QJHDOFDUFLQRJHQHVLV KHOSVRXUXQGHUVWDQGLQJRIWKHÀHOGFDQFHULVDWLRQSKHQRPHQRQDUHYLHZ(XU-&DQFHU40²  YDQ+RXWHQ9007DERU03YDQGHQ%UHNHO0:0.XPPHU-$'HQNHUV)'LMNVWUD-et al. 0XWDWHG SDVDPROHFXODUPDUNHUIRUWKHGLDJQRVLVRIKHDGDQGQHFNFDQFHU-3DWKRO198² 9RJHOVWHLQ % 3DSDGRSRXORV 1 9HOFXOHVFX 9( =KRX 6 'LD] /$ .LQ]OHU .: &DQFHU *HQRPH

Landscapes. Science 339²

 9RJHOVWHLQ%.LQ]OHU.:7KH3DWKWR&DQFHU³7KUHH6WULNHVDQG<RX·UH2XW1(QJO-0HG373 ²

 7KLDJDOLQJDP6/HQJDXHU&/HDFK)66FKXWWH0+DKQ6$2YHUKDXVHU-et al.(YDOXDWLRQRIFDQGLGDWH WXPRXUVXSSUHVVRUJHQHVRQFKURPRVRPHLQFRORUHFWDOFDQFHUVNat Genet13²  0F)DUODQG &'<DJORP -$ :RMWNRZLDN -: 6FRWW -* 0RUVH '/ 6KHUPDQ 0< et al. The Damaging

(ͿHFWRI3DVVHQJHU0XWDWLRQVRQ&DQFHU3URJUHVVLRQCancer Res77²

REFERENCES

%UD\ ) )HUOD\ - 6RHUMRPDWDUDP , 6LHJHO 5/ 7RUUH /$ -HPDO $ *OREDO FDQFHU VWDWLVWLFV */2%2&$1 HVWLPDWHV RI LQFLGHQFH DQG PRUWDOLW\ ZRUOGZLGH IRU FDQFHUV LQ FRXQWULHV CA

&DQFHU-&OLQ68² ,.1/ZZZLNQOQO7KH1HWKHUODQGV&RPSUHKHQVLYH&DQFHU2UJDQLVDWLRQ,.1/ZZZLNQOQO 2VD]XZD3HWHUV1%RDN\H($&KHQ%<7RER%%9DUYDUHV0$$VVRFLDWLRQEHWZHHQKHDGDQGQHFN VTXDPRXVFHOOFDUFLQRPDVXUYLYDOVPRNLQJDWGLDJQRVLVDQGPDULWDOVWDWXV-$0$2WRODU\QJRO+HDG Neck Surg144² /HHPDQV&5%UDDNKXLV%-0%UDNHQKRͿ5+7KHPROHFXODUELRORJ\RIKHDGDQGQHFNFDQFHUNat Rev Cancer11²

.XWOHU',$XHUEDFK$'6DWDJRSDQ-*LDPSLHWUR3)%DWLVK6'+XYRV$*et al. High incidence of KHDGDQGQHFNVTXDPRXVFHOOFDUFLQRPDLQSDWLHQWVZLWK)DQFRQLDQHPLD$UFK2WRODU\QJRO+HDG1HFN Surg129² 0HKWD37RODU-Fanconi Anemia8QLYHUVLW\RI:DVKLQJWRQ6HDWWOH /HHPDQV&56QLMGHUV3-)%UDNHQKRͿ5+7KHPROHFXODUODQGVFDSHRIKHDGDQGQHFNFDQFHUNat Rev Cancer18² .R\IPDQ6$,VPDLOD1+ROVLQJHU)&0DQDJHPHQWRIWKH1HFNLQ6TXDPRXV&HOO&DUFLQRPDRIWKH 2UDO&DYLW\DQG2URSKDU\Q[$6&2&OLQLFDO3UDFWLFH*XLGHOLQH6XPPDU\-2QFRO3UDFW15² 278.

6]WXU] 3 :RXWHUV . .L\RWD 1 7DKDUD 0 3UDEKDVK . 1RURQKD 9 et al. :HHNO\ /RZ'RVH 9HUVXV 7KUHH:HHNO\+LJK'RVH&LVSODWLQIRU&RQFXUUHQW&KHPRUDGLDWLRQLQ/RFRUHJLRQDOO\$GYDQFHG1RQ 1DVRSKDU\QJHDO+HDGDQG1HFN&DQFHU$6\VWHPDWLF5HYLHZDQG0HWD$QDO\VLVRI$JJUHJDWH'DWD

Oncologist22²

 6FKLOOLQJ&6WRHFNOL6-+DHUOH6.%URJOLH0$+XEHU*)6RUHQVHQ-$et al. 6HQWLQHO(XURSHDQ1RGH 7ULDO6(17\HDUUHVXOWVRIVHQWLQHOQRGHELRSV\LQRUDOFDQFHU(XU-&DQFHU51²  *DUDV*$URUD$5RERWLF+HDGDQG1HFN6XUJHU\+LVWRU\7HFKQLFDO(YROXWLRQDQGWKH)XWXUHORL

80²

 *DQ 6- 'DKOVWURP .5 3HFN %: &D\ZRRG : /L * :HL 4 et al. Incidence and pattern of second SULPDU\PDOLJQDQFLHVLQSDWLHQWVZLWKLQGH[RURSKDU\QJHDOFDQFHUVYHUVXVLQGH[QRQRURSKDU\QJHDO head and neck cancers. Cancer119²

6SHFHQLHU39HUPRUNHQ-%2SWLPL]LQJWUHDWPHQWVIRUUHFXUUHQWRUPHWDVWDWLFKHDGDQGQHFNVTXDPRXV FHOOFDUFLQRPD([SHUW5HY$QWLFDQFHU7KHU18²

 /DZUHQFH06RXJQH]&/LFKWHQVWHLQ/&LEXOVNLV./DQGHU(*DEULHO6et al. Comprehensive genomic FKDUDFWHUL]DWLRQRIKHDGDQGQHFNVTXDPRXVFHOOFDUFLQRPDVNature517²

 =KDQJ<.RQHYD/$9LUDQL6$UWKXU$(9LUDQL$+DOO3%et al.6XEW\SHVRI+39SRVLWLYHKHDGDQG QHFNFDQFHUVDUHDVVRFLDWHGZLWK+39FKDUDFWHULVWLFVFRS\QXPEHUDOWHUDWLRQV3,.&$PXWDWLRQDQG SDWKZD\VLJQDWXUHVClin Cancer Res22²

 %DVHOJD-7ULJR-0%RXUKLV-7RUWRFKDX[-&RUWpV)XQHV++LWW5et al.3KDVH,,0XOWLFHQWHU6WXG\ RIWKH$QWLHSLGHUPDO*URZWK)DFWRU5HFHSWRU0RQRFORQDO$QWLERG\&HWX[LPDELQ&RPELQDWLRQ:LWK 3ODWLQXP%DVHG &KHPRWKHUDS\ LQ 3DWLHQWV :LWK 3ODWLQXP5HIUDFWRU\ 0HWDVWDWLF DQGRU 5HFXUUHQW 6TXDPRXV&HOO&DUFLQRPDRIWKH+HDGDQG1HFN-&OLQ2QFRO23²

 +HUEVW56$UTXHWWH06KLQ'0'LFNH.9RNHV(($]DUQLD1et al.3KDVH,,PXOWLFHQWHUVWXG\RI WKHHSLGHUPDOJURZWKIDFWRUUHFHSWRUDQWLERG\FHWX[LPDEDQGFLVSODWLQIRUUHFXUUHQWDQGUHIUDFWRU\ VTXDPRXVFHOOFDUFLQRPDRIWKHKHDGDQGQHFN-&OLQ2QFRO23²

(10)

22

1

 YDQ=HHEXUJ+-7*UDYHODQG$3%ULQN$1JX\HQ0/HHPDQV&5%ORHPHQD(et al. *HQHUDWLRQRI SUHFXUVRUFHOOOLQHVIURPSUHQHRSODVWLFÀHOGVVXUURXQGLQJKHDGDQGQHFNFDQFHUVHead Neck35 ²

 GH%RHU'9%ULQN$%XLM]H06WLJWHUYDQ:DOVXP0+XQWHU.'<OVWUD%et al. (VWDEOLVKPHQWDQG *HQHWLF/DQGVFDSHRI3UHFDQFHU&HOO0RGHO6\VWHPVIURPWKH+HDGDQG1HFN0XFRVDO/LQLQJMol

Cancer Res17²

 %UHPPHU-)*UDYHODQG$3%ULQN$%UDDNKXLV%-0.XLN'-/HHPDQV&5et al.6FUHHQLQJIRU2UDO 3UHFDQFHUZLWK1RQLQYDVLYH*HQHWLF&\WRORJ\Cancer Prev Res (Phila) 2²

 7DERU03%UDNHQKRͿ5+5XLMWHU6FKLSSHUV+-YDQGHU:DO-(6QRZ*%/HHPDQV&5et al. 0XOWLSOH KHDGDQGQHFNWXPRUVIUHTXHQWO\RULJLQDWHIURPDVLQJOHSUHQHRSODVWLFOHVLRQ$P-3DWKRO161 ²

 *LOOHQZDWHU$-DFRE5*DQHVKDSSD5.HPS%(O1DJJDU$.3DOPHU-/et al. Noninvasive diagnosis RI RUDO QHRSODVLD EDVHG RQ ÁXRUHVFHQFH VSHFWURVFRS\ DQG QDWLYH WLVVXH DXWRÁXRUHVFHQFH Arch

2WRODU\QJRO+HDG1HFN6XUJ124²

 3RK&)=KDQJ/$QGHUVRQ':'XUKDP-6:LOOLDPV303ULGG\5:et al.)OXRUHVFHQFH9LVXDOL]DWLRQ 'HWHFWLRQ RI )LHOG$OWHUDWLRQV LQ 7XPRU 0DUJLQV RI 2UDO &DQFHU 3DWLHQWV Clin Cancer Res 12 ²

 6WHZDUG:3%URZQ.&DQFHUFKHPRSUHYHQWLRQDUDSLGO\HYROYLQJÀHOG%U-&DQFHU109²  3HQQ\/.:DOODFH+07KHFKDOOHQJHVIRUFDQFHUFKHPRSUHYHQWLRQChem Soc Rev44²  6XQ6</RWDQ55HWLQRLGVDQGWKHLUUHFHSWRUVLQFDQFHUGHYHORSPHQWDQGFKHPRSUHYHQWLRQCrit Rev

Oncol Hematol41²

 +RQJ :. /LSSPDQ 60 ,WUL /0 .DUS '' /HH -6 %\HUV 50 et al. 3UHYHQWLRQ RI VHFRQG SULPDU\ WXPRUVZLWKLVRWUHWLQRLQLQVTXDPRXVFHOOFDUFLQRPDRIWKHKHDGDQGQHFN1(QJO-0HG323 ²

 /LSSPDQ 60 %DWVDNLV -* 7RWK %% :HEHU 56 /HH -- 0DUWLQ -: et al. &RPSDULVRQ RI ORZGRVH LVRWUHWLQRLQZLWKEHWDFDURWHQHWRSUHYHQWRUDOFDUFLQRJHQHVLV1(QJO-0HG328²  (SVWHLQ-%*RUVN\07RSLFDODSSOLFDWLRQRIYLWDPLQ$WRRUDOOHXNRSODNLD$FOLQLFDOFDVHVHULHVCancer

86²

YDQ =DQGZLMN 1 'DOHVLR 2 3DVWRULQR 8 GH 9ULHV 1 YDQ 7LQWHUHQ + (8526&$1 D UDQGRPL]HG WULDO RI YLWDPLQ $ DQG 1DFHW\OF\VWHLQH LQ SDWLHQWV ZLWK KHDG DQG QHFN FDQFHU RU OXQJ FDQFHU )RU WKH(8URSHDQ2UJDQL]DWLRQIRU5HVHDUFKDQG7UHDWPHQWRI&DQFHU+HDGDQG1HFNDQG/XQJ&DQFHU &RRSHUDWLYH*URXSV-1DWO&DQFHU,QVW92²

 .KXUL )5 /HH -- /LSSPDQ 60 .LP (6 &RRSHU -6 %HQQHU 6( et al. 5DQGRPL]HG SKDVH ,,, WULDO RI ORZGRVHLVRWUHWLQRLQIRUSUHYHQWLRQRIVHFRQGSULPDU\WXPRUVLQVWDJH,DQG,,KHDGDQGQHFNFDQFHU patients. -1DWO&DQFHU,QVW98²

(SVWHLQ -% *RUVN\ 0 :RQJ )/ 0LOOQHU$ 7RSLFDO EOHRP\FLQ IRU WKH WUHDWPHQW RI G\VSODVWLF RUDO OHXNRSODNLDCancer83²

 .KDQ=(SVWHLQ-%0DUXU6*LOOHVSLH0%)HOGPDQ/7VDL+/et al. &HWX[LPDEDFWLYLW\LQG\VSODVWLF OHVLRQVRIWKHXSSHUDHURGLJHVWLYHWUDFWOral Oncol53²

 +XQWHU.'3DUNLQVRQ(.+DUULVRQ353URÀOLQJHDUO\KHDGDQGQHFNFDQFHUNat Rev Cancer5 ²

&URRNHU . $OLDQL 5 $QDQWK 0 $UQROG / $QDQW 6 7KRPDV 60 $ UHYLHZ RI SURPLVLQJ QDWXUDO chemopreventive agents for head and neck cancer. Cancer Prev Res (Phila) 11²  .XULDNRVH 0$ 5DPGDV . 'H\ % ,\HU 6 5DMDQ * (ODQJR .. et al. $ 5DQGRPL]HG 'RXEOH%OLQG

3ODFHER&RQWUROOHG3KDVH,,%7ULDORI&XUFXPLQLQ2UDO/HXNRSODNLDCancer Prev Res (Phila)9 ²

 :DUQDNXODVXUL\D 6 &OLQLFDO IHDWXUHV DQG SUHVHQWDWLRQ RI RUDO SRWHQWLDOO\ PDOLJQDQW GLVRUGHUV Oral

Surg Oral Med Oral Pathol Oral Radiol125²

:DUQDNXODVXUL\D 6 -RKQVRQ 1: YDQ GHU :DDO , 1RPHQFODWXUH DQG FODVVLÀFDWLRQ RI SRWHQWLDOO\ PDOLJQDQWGLVRUGHUVRIWKHRUDOPXFRVD-2UDO3DWKRO0HG36²

:DUQDNXODVXUL\D 6 $UL\DZDUGDQD $ 0DOLJQDQW WUDQVIRUPDWLRQ RI RUDO OHXNRSODNLD $ V\VWHPDWLF UHYLHZRIREVHUYDWLRQDOVWXGLHV-2UDO3DWKRO0HG45²

 9LOOD$&HOHQWDQR$*OXULFK,%RUJQDNNH:6-HQVHQ6%3HWHUVRQ'(et al.:RUOG:RUNVKRSRQ2UDO 0HGLFLQH9,,3URJQRVWLFELRPDUNHUVLQRUDOOHXNRSODNLD$V\VWHPDWLFUHYLHZRIORQJLWXGLQDOVWXGLHV

Oral Dis25²

&DED\ 5- 0RUWRQ 7+ (SVWHLQ -% 3UROLIHUDWLYH YHUUXFRXV OHXNRSODNLD DQG LWV SURJUHVVLRQ WR RUDO FDUFLQRPDDUHYLHZRIWKHOLWHUDWXUH-2UDO3DWKRO0HG36²

&DSHOOD'/*RQoDOYHV-0$EUDQWHV$$$*UDQGR/-'DQLHO),3UROLIHUDWLYHYHUUXFRXVOHXNRSODNLD GLDJQRVLVPDQDJHPHQWDQGFXUUHQWDGYDQFHV%UD]-2WRUKLQRODU\QJRO83²

 /RGL*)UDQFKLQL5:DUQDNXODVXUL\D69DURQL(06DUGHOOD$.HUU$5et al. Interventions for treating RUDOOHXNRSODNLDWRSUHYHQWRUDOFDQFHU&RFKUDQH'DWDEDVH6\VW5HY7&' YDQGHU:DDO,3RWHQWLDOO\PDOLJQDQWGLVRUGHUVRIWKHRUDODQGRURSKDU\QJHDOPXFRVDWHUPLQRORJ\ FODVVLÀFDWLRQDQGSUHVHQWFRQFHSWVRIPDQDJHPHQWOral Oncol45²  +R0:5LVN-0:RROJDU-$)LHOG($)LHOG-.6WHHOH-&et al.7KHFOLQLFDOGHWHUPLQDQWVRIPDOLJQDQW WUDQVIRUPDWLRQLQRUDOHSLWKHOLDOG\VSODVLDOral Oncol48²  =KDQJ/3RK&):LOOLDPV0/DURQGH'0%HUHDQ.*DUGQHU3-et al./RVVRIKHWHUR]\JRVLW\/2+ SURÀOHVYDOLGDWHGULVNSUHGLFWRUVIRUSURJUHVVLRQWRRUDOFDQFHUCancer Prev Res (Phila)5²

(O1DJJDU$.&KDQ-.&*UDQGLV-57DNDWD76ORRWZHJ3-:+2&ODVVLÀFDWLRQRI+HDGDQG1HFN 7XPRXUVIARC Press 

 *UDYHODQG$3%UHPPHU-)GH0DDNHU0%ULQN$&REXVVHQ3=ZDUW0et al.0ROHFXODUVFUHHQLQJRI RUDOSUHFDQFHUOral Oncol49²

%URXQV ( %DDUW -$ .DUDJR]RJOX .+ $DUWPDQ ,+$ %ORHPHQD ( YDQ GHU :DDO , 0DOLJQDQW WUDQVIRUPDWLRQRIRUDOOHXNRSODNLDLQDZHOOGHÀQHGFRKRUWRISDWLHQWVOral Dis20(²( :RR6%*UDPPHU5//HUPDQ0$.HUDWRVLVRIXQNQRZQVLJQLÀFDQFHDQGOHXNRSODNLDDSUHOLPLQDU\

VWXG\Oral Surg Oral Med Oral Pathol Oral Radiol118²

 /HH -- +XQJ +& &KHQJ 6- &KHQ <- &KLDQJ &3 /LX %< et al. &DUFLQRPD DQG G\VSODVLD LQ RUDO OHXNRSODNLDVLQ7DLZDQSUHYDOHQFHDQGULVNIDFWRUV2UDO6XUJ2UDO0HG2UDO3DWKRO2UDO5DGLRO(QGRG 101²

 :LOV /- 3RHOO -% (YUHQ , .RRSPDQ 06 %URXQV (5($ GH 9LVVFKHU -*$0 et al. Incorporation of GLͿHUHQWLDWHG G\VSODVLD LPSURYHV SUHGLFWLRQ RI RUDO OHXNRSODNLD DW LQFUHDVHG ULVN RI PDOLJQDQW progression. Mod Pathol33

 0D\PRQH 0%& *UHHU 52 .HVHFNHU - 6DKLW\D 3& %XUGLQH /. &KHQJ$' et al. 3UHPDOLJQDQW DQG PDOLJQDQWRUDOPXFRVDOOHVLRQV&OLQLFDODQGSDWKRORJLFDOÀQGLQJV-$P$FDG'HUPDWRO81²  7DERU 03 %UDNHQKRͿ 5+ YDQ +RXWHQ 90 .XPPHU -$ 6QHO 0+ 6QLMGHUV 3- et al. Persistence of

JHQHWLFDOO\DOWHUHGÀHOGVLQKHDGDQGQHFNFDQFHUSDWLHQWVELRORJLFDODQGFOLQLFDOLPSOLFDWLRQVClin

Cancer Res7²

 *UDYHODQG$3*ROXVLQVNL3-%XLM]H0'RXPD56RQV1.XLN'-et al./RVVRIKHWHUR]\JRVLW\DWS DQGSLPPXQRSRVLWLYLW\LQVXUJLFDOPDUJLQVSUHGLFWORFDOUHODSVHLQKHDGDQGQHFNVTXDPRXVFHOO carcinoma. ,QW-&DQFHU128²

(11)

Chapter 1

 &KHQ $/ +VX &+ /LQ -. +VX 00 +R <) 6KH 76 et al. 3KDVH , FOLQLFDO WULDO RI FXUFXPLQ D FKHPRSUHYHQWLYHDJHQWLQSDWLHQWVZLWKKLJKULVNRUSUHPDOLJQDQWOHVLRQVAnticancer Res21 ²

+HLVH & 6DPSVRQ-RKDQQHV $ :LOOLDPV $ 0F&RUPLFN ) 9RQ +RͿ '' .LUQ '+ 21<; DQ (EJHQHDWWHQXDWHGDGHQRYLUXVFDXVHVWXPRUVSHFLÀFF\WRO\VLVDQGDQWLWXPRUDOH΀FDF\WKDWFDQEH DXJPHQWHGE\VWDQGDUGFKHPRWKHUDSHXWLFDJHQWVNat Med3²

 5XGLQ &0 &RKHQ ((: 3DSDGLPLWUDNRSRXORX 9$ 6LOYHUPDQ 6 5HFDQW : (O1DJJDU$. et al. An $WWHQXDWHG$GHQRYLUXV21<;$V0RXWKZDVK7KHUDS\IRU3UHPDOLJQDQW2UDO'\VSODVLD-&OLQ

Oncol21²

0F*UHJRU ) :DJQHU ( )HOL[ ' 6RXWDU ' 3DUNLQVRQ . +DUULVRQ 3 ,QDSSURSULDWH 5HWLQRLF $FLG 5HFHSWRUD([SUHVVLRQLQ2UDO'\VSODVLDV&RUUHODWLRQZLWK$FTXLVLWLRQRIWKH,PPRUWDO3KHQRW\SH

Cancer Res57²

 0F*UHJRU ) 0XQWRQL $ )OHPLQJ - %URZQ - )HOL[ '+ 0DF'RQDOG '* et al. 0ROHFXODU FKDQJHV DVVRFLDWHGZLWKRUDOG\VSODVLDSURJUHVVLRQDQGDFTXLVLWLRQRILPPRUWDOLW\SRWHQWLDOIRULWVUHYHUVDOE\ D]DF\WLGLQHCancer Res62²

 +XQWHU.'7KXUORZ-.)OHPLQJ-'UDNH3-+9DVV-..DOQD*et al. 'LYHUJHQW5RXWHVWR2UDO&DQFHU

Cancer Res66²

7DQJ;+8UYDOHN$02VHL6DUIR.=KDQJ76FRJQDPLJOLR7*XGDV/-*HQHH[SUHVVLRQSURÀOLQJ VLJQDWXUHVIRUWKHGLDJQRVLVDQGSUHYHQWLRQRIRUDOFDYLW\FDUFLQRJHQHVLVJHQRPHZLGHDQDO\VLVXVLQJ 51$VHTWHFKQRORJ\Oncotarget6²

 3H\VHU1':DQJ/=HQJ<$FTXDIRQGDWD0)UHLOLQR0/L+et al.67$7DVD&KHPRSUHYHQWLRQ 7DUJHWLQ&DUFLQRJHQ,QGXFHG+HDGDQG1HFN6TXDPRXV&HOO&DUFLQRPDCancer Prev Res (Phila)

9²

7DQJ ;+ .QXGVHQ % %HPLV ' 7LFNRR 6 *XGDV /- 2UDO &DYLW\ DQG (VRSKDJHDO &DUFLQRJHQHVLV 0RGHOHGLQ&DUFLQRJHQ7UHDWHG0LFHClin Cancer Res10²

)HOOPDQQ&*RZHQ%*/LQ3&'RXGQD-$&RUQ-(&RUQHUVWRQHVRI&5,635&DVLQGUXJGLVFRYHU\ DQGWKHUDS\Nat Rev Drug Discov16²

 &RQJ/5DQ)$&R['/LQ6%DUUHWWR5+DELE1et al.0XOWLSOH[JHQRPHHQJLQHHULQJXVLQJ&5,635 &DVV\VWHPVScience339²

 <DWHV/$1RUEXU\&-*LOEHUW5-&7KHORQJDQGVKRUWRIPLFUR51$Cell153² ;LQ< +XDQJ 0 *XR :: +XDQJ 4 =KDQJ /= -LDQJ * 1DQREDVHG GHOLYHU\ RI 51$L LQ FDQFHU

WKHUDS\Mol Cancer16

 5DR''9RUKLHV-66HQ]HU11HPXQDLWLV-VL51$YVVK51$6LPLODULWLHVDQGGLͿHUHQFHVAdv Drug

Deliv Rev61²

 %DXHU -$ <H ) 0DUVKDOO &% /HKPDQQ %' 3HQGOHWRQ &6 6K\U < et al. 51$ LQWHUIHUHQFH 51$L VFUHHQLQJDSSURDFKLGHQWLÀHVDJHQWVWKDWHQKDQFHSDFOLWD[HODFWLYLW\LQEUHDVWFDQFHUFHOOVBreast Cancer

Res125

 &ROH.$+XJJLQV-/DTXDJOLD0+XOGHUPDQ&(5XVVHOO05%RVVH.et al. RNAi screen of the protein NLQRPHLGHQWLÀHVFKHFNSRLQWNLQDVH&+.DVDWKHUDSHXWLFWDUJHWLQQHXUREODVWRPDProc Natl Acad