IC CONG NGH^ SINH H g C TOAN QUOC 2013
DI^U TRI N H A M T R O N G D I C H GEN K-RAS: TU" NGHieN CO'U IN SIUCO D^N T H y c NGHIEM
Lao Oi>c T h u | n \ Tnrong Kim P h u v n g \ Lieu Chi HCing^, NguySn Van Truwng\ L§ Huyen Ai Thiiy'' 'Khoa Cfing ngh$ sinh ftpc, Tn/dng D9I hgc Md Tp. Hd Chi Minh
'B$nhviSn Tdy Ninh TOM T A T
Phirong phqi di^u tri ngSm tning dich pMn tii (Molecularly targeted th«^y) la phuong p h ^ hien d^i dirpc ling dyng trong fflih vyc dieu tn ung thir bang cich sir dung khang the doa d6ng hay nhihig {dian tii nhd tin cong cic tS bto ung thu Uu diem cda phuong p h ^ niy la kh6ng anh huong d ^ cic te bio linh khic. Mgt trong nhiing "dich phSn tii" dugc hudng tdi trong phuong phip di^u trj
^ c h f a h la gen K-ras. 'nifini qua vi|c khai thac dii li?u Uen quan cac lo^ dpt bien lien gen niy t& cic cong trinh nghien cmi da dupc cong bd tnidc dSy, tSn so d$t tmng binh cd trpng so duoc thong ke nhSm jtic dinh cac dpt biln noi trfii tren gen K-ras. KS Sd, vi§c t ^ ke, dinh gii cic c§p mdi dung d^ phdt h i ^ dot bien tren gen K-ras bSng phirong phqj PCR-giii trinh ty hay phit hi§n biy dpt bi6n noi tr^i bang Allele-Specific-Rcal time-PCR (AS-Rcal tune-PCR) duoc tiln htoh. Nhung dmc nghi?m dSu dfin tu 19 mlu b?nh ph&n due paraffin dupc cung cap bdri bgnh vipn Da khoa Tiy ninh ^ nh?n m$t so dpt biln "tren gen K-ras. Ben canh dd, phuong phip AS-Real time-PCR cho ph6p phdt hi^n hai dpt bien t^ codon 12 mi phuong p h ^ PCR giii trlnh tv da bo s6L Tfi- kfioa: AS-Real limc-PCR, ^T-ras, PCR-giii trinh Ht, ung thir dai true tring.
MercAu
Dllu tt) nhim trting dfch id mOt ti^n bO mdi trong ITnh virc chuy§n sau cOa dilu tri nOi khoa d6i vol ung thif nhd i>ng dgng tdc thSnh tgu tnang nghien ci>u v l sinh hgc phSn tif. Li^u phdp mtW cho thiy c6 nhilu u-u dilm nhir tSng cirdng kha nang phyc hoi, tec dyng phg r l i it. th§m chl khi b§nh nhSn dilu t i bing hda hgc tri li$u thit bgi vln c6 t h i si> dOng li$u ph^p nSy hay kit hop li^u phdp n^y voi phuong phdp h6a hgc trj li$u s§ giup k6o ddi sv sing hon cho b^nh nhdn (Mendelsohn, 2002). Cdc lo^i ttiulc trong ligu phap ndy chi tdc dgng din t l bdo ung thif vd kh6ng gdy ra ho^c gdy rit ft tdc dOng din t l bdo Idnh, din chirc ndng binh thuerng cOa cdc co quan. Chiing can thi$p vdo cdc phdn ti> ddc hi|u trong co chl sinh ung thu- vd sg- tdng tnfdng ciia khli u. VI vdy cdc nhd nghien ct>u ggi cdc phdn h> ndy Id "cac dIch phdn tiy (molecular targets) vd ligu phdp ndy dirg'c ggi Id li|u phdp nhim tning dich phan ti> (molecularly targeted therapies) hay la lj$u phdp nham tning dfch (targeted therapies).
Mgt trong nhOng con difdng truyin tfn ht$u cua thu t h i EGFR (Epidemial Growth Factor Receptor - thg t h i ylu t l tdng trudng bilu mfl) ctS sy tham gia cCia tn.ic RAS/RAF/MAPK. Gen EGFR md hda dio thg thi nhdn to tdng tnfdng bilu bi (EGF) djnh vi tren b l m|it t l bdo. ban chit Id mOt glycoprotein cd hogt tfnh tyrosine kinase t h i hi#n vai trd dilu h6a sif tdng sinh vd bt|t hda t l bdo. Khi EGFR tifong tdc vdi EGF dan din sif hogt hda con dirdng tfn hi$u thflng qua cdc protein RAS, RAF vd MARK, trang do K-ras Id mgt pratein thuflc hg protein fRAS (Karapetis et ai, 2008). Trong trgc tmyln tfn hi0u ndy, khi K-ras d trgng thdi binh thudng, thi hogt dflng cua nfl phg thuflc vdo hogt tinh cila thg t h i EGFR;
Tuy nhifln, nlu co dflt biln trfln^gen md hfla cho K-ras thi pratein ndy se tu hoat hda khflng phg thuflc EGFR (tin hiflu v l tdng sinh vd hogt hfla t l bdo van duoc tnjyen di Iifln tgc) (Karapetis ef al., 2008). Ngodi ra, trong liflu phdp dieu trj nhim tning dich phdn ti> (EGFR) dp dgng trfln cdc bflnh nhdn ung thu dgi tryc trdng, vai tro ciia cdc khdng thi Cetuximab vd Panitumumab chi t h i hifln khi md benh nhan hodn todn khflng mang cdc dflt biln trfln K-ras, nlu khflng (ti>c Id b$nh nhdn mang dflt biln K-ras) till hiflu qud dllu trj cfln nguvc Igi (Benvenuti ef a/., 2007; Amado ef ai, 2008; Karapetis ef al., 2008; Jdnsson ef ai. 2009).
Cl ede bflnh ung thu kha p h i biln nhu ung thu dgi tn/c trang. ung thu F^li khflng t l bdo nhd, ung thu vti. ung thu thuc quan,... K-ras vin cdc dgng dgt biln gay xdo trfln chdc ndng cua gen ndy dd duvc cong b l trfln t h i gifli (Benvenuti ef al., 20;Sun etai, 2011)
Nhu vdy, ro rdng d l hudng tdi dp dgng dilu tri liflu phdp nhdm tning dich phan td cho cdc logi ung thu nfli trfln d ngudi bflnh Vlflt Nam, d n tlm hilu dflt biln trfln gen K-ras; vCing dflt biln thudng xuyen xuat hifln. kilu dflt biln, cdc phuong phdp. ky thudt dugc ap dgng d l dd tim vd phat hifln dflt bien. ... vd dfl cung chinh Id mgc tieu cua nghifln cdu budc diu ndy.
V A T L I E U V A PHU'aNG P H A P V$tli$u
Nghifln cdu sd dgng 19 mSu benh phim ung thu dgi tryc trdng dugc cung d p ti> bflnh vifln Da khoa Tdy Ninh. Mlu dupc diic trong paraffin vdi ghi chii thdi gian tiiu td thdng 1/2007 tdi thang 5/2011. Cac mlu dugc tide qudn d nhiflt dfl- 30°C trudc khi tiln hdnh tdch chilt DNA bang phuona phdp phenol/chlorofomi (Meick), proteinase K (Fennentas)...
Hfla chit sd dyng phuong phdp PCR, difln di bao gflm Master mix green (Fermentas). Mfli (IDT): TAE buffer (Bio basic)...
Phucmg phdp nghifln cdu
Khai thdc du" II#u. k^^o sdt in silico vd thiet ke mol
Cdc logi dflt biln gen K-ras dugc khai thdc du li$u trfln Pubmed vd dugc thing kfl xac djnh tin s l trung blnh cd trgng s l d l xdc djnh cdc dot biln noi trfli. Mli quan hfl gida cdc kilu dflt biln voi' ylu t l chung tflc dugc phdn tich danh gld mli
ri\Jl w u n i rv.nkJH n u i j o u i \ i 3 iNVaril;; oiNH HQ
^^
tuong quan giira chiing bdng phuang phdp phan tich phuong sai WJOVA. Dong thdi, trinh hf gen K-ras dugc thu rt^
fren NCBI bang ma s l truy c^p NC_000012.11. Viflc thilt k l moi cho phan dng PCR-gidi trinh ty vd AS-PCR dugcth(|cl hifln bing ca'c cflng cg tin sinh hgc nhu BLAST, IDT analyzer. Primer Expr^s, /\rmhyb, Batch Primer 3.
Tdch chilt DNA
Chiing tSi sd dgng xylene d l xd ly paraffin (mlu dugc dilc trong paraffin) (Lfi Thj Tnic Llnh vd cs, 2011) trudc Idii tiin hanh tdch i^ilt DNA bang phuong phap phenol/chloroform (Sacchi ef ai, 198T)
PCR-giai trinh t v vd Atlele-SpecHic Real-time PCR
Phdn dng PCR sd dgng DNA vua tach d i i l t ti> mfl diic paraffin de Idm mgch khuwi vdi cac mil giai trinh tif vd AS-Real- time PCR cho gen K-ras (Bdng 2 vd Bang 3). Phan dng dugc thyc hifln trong twig t h i tich 15 pi vdi diu trirti nhift;
95''C/5 phiit; 40 chu ky vdi 95°C/30 gidy. )CCI30 giay (X Id nhiei dfl bat cgp tuong ung cOa cac cgp mli). 72°C/30 g i ^ sau dfl kit thiic frtian dng vfli 72''C/6 phiit. Trong phuang phdp PCR^idi frinh bjr, sdn phim PCR dugc tiln hdnh ^ | frlnh ty d Marcogen (Han Qulc) vd phdn tfch kit qua gidi frinh tu bang chuang trinh Chromas Pro 2.0.4 vd rfiuongl
frinh Seaview ' KET Q U A V A T H A O LUAN
Khai thdc d u liflu
Trang qud frinh khai thdc, chiing toi thu dugc tong cgng han 80 tdi lidu tham khdo (tinh din thfli dilm thdng 1/2013) c6 lidn quan din d c phdn tidi dgt biln fr^n didi nham la K-ras cua ung thu dgi true b^ng. Trong d c kit qud nghiSn ciiu fim dugc. mau md diic paraffin dugc su dyng khd nhilu (67.27%), cdc mlu mfl tuai, mau ddng Ignh cOng dugc khfio sdt nhung vdi t^ lfl thip han. V l ky thudt phdt hidn dflt biln, phuang phdp giai binh ty Sanger dugc su dgng Id tM ylu|
(60.29%). Cdc phuang phdp khdc nhu Real-time PCR. COLD (Co-amplification at lower denahjration temp) PCR c&i^
dugc dp dyng nhung khong p h i biln. Cung qua birdc tlm hilu thflng tin ndy. chting tfli nhfln thiy, ylu t l quylt t^nh (Jf nhgy ciia cdc ky thugt sd dung chfnh Id li 1^ te bao ung thu hifln difln trang mdu (ngodi ddc tinh riflng v l dfl nhgy cOii tung kf thudt), tf Ifl nay hodn todn phg thuflc vdo ky thugt lay mlu va giai dogn bflnh ly, ky thudt lly mau t i t se d n cd4 mlu cfl fi Ifl cdc t l bdo mang dflt biln cao han cdc t l bdo binh thudng. Trong mflt sd nghidn cuu, van d l ndy dirge ^M quylt bing d d i friuc hifln thflm budc quan sdt tieu ban gidi phlu bflnh cua mau. cdc mau nao d t^ Ifl t l bdo mang 0(/i biin Idn hon 70% mfli dugc chgn cho d c budc sd dgng kT thugt Sinh hpc Phdn tu v l sau, d c mlu khflng dgt ySu diu ndy sfl bj logi.
Cdc dflt biln frdn K-ras rat da dgng. D l cd mflt d i nhin mang tinh ting quan. du liflu thu ttidp dugc mang ^ xd ly bSng phuang phdp thong kfl vdl d c h tinh tan s l trung binh d frgng so nham logi bd su khac biflt gida d c phuang ph^
nghidn cdu vd cdc kigi mau mfl sd dung trong d c nghifln ctju (dd liflu khflng frlnh bdy). Qua phdn tich thong kfl, c6 7 dflt biln noi trfli xuit hifln d nhilu loai ung thu, d tan suit cao dugc ghi nhdn bao gom G12D, G12V, G13D, G128,:
G12C, G12A. G12R (Bdng 1). Viflc phan tich phuong sai ANOVA (p=0.05) dugc thyc hifln nhim lalm fra xem cd sg' khac nhau v l tinh frgng ciJa 7 dgt biln nfli trfln giua d c chau luc hay khflng. Kit qua cho thay bay dflt biln nli trfli n6i trfln Id khflng cd sy Idiac biflt v l t^ Id d giua cdc chdu Igc vdl nhau.
Bdng 1: Ttidng k i mSu va thong s& ANOVA tren cdc kilu dflt tnin nli frpi Bay dflt blin K-ras p h i bi^n d ung Ihir d^i b y e b^ng Ting m3u d$t
bien K-ras Chiu Au Chau My Ch3u A G12D
G12V G13D G12C G12S G12A G12R
1.957671 2 845997 3.067208 4100888 4 299204 4.417707 4.858892
0.234331 0.166877 0.154773 0.112845 0.106812 0.103439 0.092211
7.708647 7.708647 7.708647 7.708647 7.708647 7.708647 7.708647
Khdc bi^t khflng cd y nghTa t h i n g k i
Khao sat in silico va ttiilt kfl mfli cho phan dng PCR-gidi frlnh t y i Gen K-ras co chilu dai 45675 bp nam frfln nhilm sac tfil s l 12. d vj tri 12p12.1 vd cfl 4 exon. Sau khi dinh \^ cdc W ,
biln noi trgtjfln frlnh tv vua ttiu nhdn dugc, diOng tfli tien hanh thilt k l d c cap mli dio phan ting PCR-gidi tinh tV , (Bang 2). Tien hanh kiem fra vj fri bat cdp, san phim khuich dgi, dg dgc hiflu vd cdc thflng so vat ly d a d c bO m6i, lA j qud thu dugc khd tiL
Trong do, L la dfl dai ciia mfli, Tm Id nhiflt dfl nflng chdy cua moi. (1) la mdc nang lugng lien kit ty do cho kha nang hli*
ttidnh d u tnic hairpin loop. (2) la mdc nang lugng Iifln kit tu do cho khd nang hinh thanh c l u biic homodimer. (3) B mdc nang lugng lien kit tu do cho khd nang hlnh thdnh d u tiiic heterodimer, P Id kich ttiudc sdn phim PCR F-nA • xufli, R-mli ngugc. - Bdng 2: Trinh tif vd cac thflng so danh gia m6l cua IDT cho gen K-ras
Gen Trinh t y moi (5'-3') F: GCCTGCTGAftAATGACTGAATA R: CTGTATCAAAGAATGGTCCTGCAC
L 22 24
Tm 53.3 55.9
(1) 1.89 0.99
(2) -3.14 -7.05
(3)
• -5.09 P(bp)
176
PC CONG NGH6 SINH HQC TOAN QUOC 2013 T4ch chiit DNA
DNAlti'tlii'!!! **' '" " ^ . I W • " " " "*• "Wiu y4u 16 may dSi (kel qui ktiong trlnh biy), diilng toi ten hanh tich chlet af mo (Juc paraffin bang phirang phdp phenol/chlarolbnn, trong d6 cac glal doan rtra mau bing ethanol u mSu vdl proieinase K phii dtfTC thi/c hl0n h6t sijc cin than.
PCR-fliai trlnh tip
Kil'n'*! " 1 ? * ' * ' •"**• '=''""9 '^ * •'="'' PCR-glai Wnh tv ving e«jn 1 gen K-ras liSn 19 miu ung thi/ dai tnre trina minh f l I ' S " " " " ^2, 13 tr«n exon 1 gen Kvas (noi t i p tnjng 7 dpt blin nil trOI) khong c6 d6t bien nio duoc ghi nhjn ininn 1) u cac v| tri khac, Chung t i l c6 ghi nh|n dirac mot s6 peak dot blin, tuy nhlin t«l cic peak dpt blin luon luon c6 S I ™ r i r..!^"'..'"'' * ° " 3 * ^ ^ ' ' " * " ' ' "'"^"9 "^ P ^ " "'"*' " ' " ' " ' a 1^ '^° ' I " " (Hl"fi 2)- D*u niy chimg t i ranq trong quin thS te b i o thu nhin to miu b$nh phim, s i t i bao blnh thirftng chiim mit 191^ r i t l i n
l\M\f\/i
BinhtiircFng GGT GGC 12 13 Giy Gly DOtbiSn
Blnhttiirdro GGT GGC 12 13 Gly Gly OCttuin
16
AAAAA/X Blnh thimng GGT GGC
12 13
< * Gly D«bi&i —
Binhthudng GGT GGC 12 13 Gly Gly D«ttiidn —
23
A A M A « BlnhUiUDngGGT GGC
12 13 tay G^
eg(bi6n — — ^ A A r > A A - BMttMKmg GGT GGC
12 13 Gly Gly OolbtSn — Hlnh 1. Kit qud gidi trinh l y codon 12 vd 13 trsn mflt sd miu
BBih thuang A C 6 ATA CAG Blnhmu'Smg ^ c G ATA
.4i\L,
Blnh ttHrfrng GAA Blnh ttHi'6Tig GAC
Hinh 2. Mflt s6 vj tri nghi ngd d ^ bien dugc ghi chu (ddu mQi tfln trfln hinh)
Tdp tmng phdn tfch kit qua gidi trinh ty t^i cdc vi fri codon 12 vd 13. chung tfli cht thiy xuit hifln mflt peak duy nhlt, du cung cfl vai cham nghi ngd, nhung r i t khd d l phan biflt giua sflng (dflt bien) hay tin hiflu nen (hlnh 1). Mflt khuyin cao da dugc dua ra doi vfli nghiSn cdu cung loai, dfl Id: viflc dp dgng phuong phap gidi trinh ty d l dfl tim dflt biln frong ung thu dfli hfli ky thudt ily mau phai th|t tit. Varelia-Garcia va cflng sy (2009) da khang <^nh phuong phdp gidi frlnh t u theo Sanger sfl khflng cd khd nang phdt hifln dflt biln neu mlu cd dufli 25% t l bdo ung thu. dli voi cdc ket q u i giai frinh ty td mlu cd s6 tugng t l t>ao ung thu frilp hon chi co t h i kit lufln d muc nghi ngd. Trong nghifln cdu budc dau ndy, c6 Ifl chting tfli cQng mac phdi van d l luong ty. Cdc mau bdnh phim diu cd tfnh chit hoi cdu, khflng kdm ghi chu noi tdp trung t l bdo ung thu hay ddnh gid so bfl % t^ ifl chilm cua t l bdo ung Ihu. Chfnh vl vdy, chung tdi chgn gidi phdp b'ln hdnh them mflt ky ttiudt dfl tlm dflt bien nda, tdp trung vao cdc vj fri dflt biln mang linh noi trfli. E)fl chfnh Id phuong phdp Allele-Specific real-time PCR.
Allele-Specific Real-time PCR
D l ttiyc hifln ky thuflt AS Real-time PCR, chOng tfli tham khao bfl moi cua tdc gia Lang vd cflng sy (2011) boi chiing dap dng hau het cdc yflu cau kilm tra in silico ngo^i trd mdc nang lugng ty do tgo heterodimer Id khflng d^t (Bang 3).
Bdng 3. Trinh ty mol/mdu do nhim phat hl$n 7 d$t biln ph£ trien trSn K-ras Dflt bi^n
G12D (c.3SG>A)
G12V
M 6 | (5'-3') F: AAACTTGTGGTAGTTGGAGCGGA R- CATATTCGTCCACAAAATGATTCTG F: AAACTTGTGGTAGTTGGAGCAGT
L 23 25 23
%GC 47 8 36.0 43.5
Tm 58.6 52.6 5 6 7
(1) -0 62 0 79 -0.62
(2) -4.88 -3,91 -4 88
(3) -10.26 -10 26
P(bp) 85 85
23
{c.35G>T) G13D (c38G>A)
G12C (c.34G>T)
G12S (c346>A)
G12A (c.35G>C)
G12R (c34G>C)
Reverse Reference
MSud6
Ft CATATTCGTCCACAAAATGATTCTG . F : GTGGTAGTTGGAGCTGGAGA
R: CATATTCGTCOVCAAAATGATTCTG F: AATATAAACTTGTGGTAGTTGGAGCCT R: CATATTCGTCCACAAAATGATTCTG F: AATATAAACTTGTGGTAGTTGGAGCGA R: CATATTCGTCCACAAAATGATTCTG F: AACTTGTGGTAGTTGGAGCTTC R: CATATTCGTCCACAAAATGATTCTG F: AATATAAACTTGTGGTAGTTGGAGCTC R; CATATTCGTCCACAAAATGATTCTG CATATTCGTCCACAAAATGATTCTG GACTGAATATAAACTTGTGGTAGTTGGA CTGTATCGTCAAGGCACT
25 20 25 27 25 27 25 22 25 27 25 25 28 18
36.0 55.0 36.0 37.0 36.0 37.0 3 6 0 45 5 36,0 37.0 36.0 36.0 35,7 50,0
S2.6 56.3 52.6 56.2 52.6 56.2 52.6 55.0 52 6 55.0 52.6 52.6 54.8 51.7
0.79 0.13 0.79 -0.62 0.79 -0.62 0.79 0.5 0 79 -0.62 0 79 0.79 -0.62 0.87
-3.91 -6.34 -3.91 -4.88 -3.91
^ . 8 8 -3.91 -6.34 -3.91 -9.49 -3.91 -3.91
^ . 8 8 -3.61
-6.6 -10.26
-10.26 -10.26
-10.26 -3.61 -10.26
^ *
90
90 84
90
Diing nhu dy doan. ttiyc nghiflm AS-Real-time PCR da ghi nhdn dugc mflt vdi kit qua duong tfnh. cg thi nhif sau:
TnsnglSmluttidnghiflm, mflt so mlu deu xuit hi|n dflt bien tgi codon 12,13: G12D (mdu s l 1.16vd37), G12V(m3u so 1) vd G12C (mau s l 16). dgc biflt mlu so 1 xuit hifln hai dflt biln G12D vd G12V. mau s l 16 xuit hifln hai ddl bi^
G12DvdG12C(Hlnh3).
i!
. / ,,—
1^
<-
1
1"-....- - ~
Hlnh 3. K4t qud AS-Real-dme PCR dirong tlnh tr6n haf mSu 1 vd 16
Lang vd cflng sy (2011) cQng da khing djnh bfl moi/mlu dfl ciia hfl cho phep phdt hifln dflt bien khi chl can Irong mSu ci 1 % DrJA dflt bien, dflng Uidi hg cQng cflng bo frong s l mau kiem fra dflt biln xuat hifln am tfnh bing PCR-giai Wnh tv, cfl hai mlu fld cho kit qua duong tinh khi Ihay bdng ky Ihuflt phdt hifln Id AS-ReaHime PCR sd dyng chinh bfl mtilmh do nhu trong nghifln cdu ndy.
K ^ LUAN
Mflt s6 kit lufln nit ra sau nghifln cdu ndy la: (1) Cdc dflt biln nli trfli frfln gen K-ras Id G12D. G12V, G13D. G1ZC.
G12S. G12A. G12R, cac kilu dflt biln trfln khong lo^i ln> lln nhau vd khflng cfl sy khac biflt dya'vdo y l u to ching K t l (2) Phuong phdp PCR-Gidi frinh ty cho phflp phdt hifln t i t ca cdc dflt bien, tuy nhifln frong cdc Irudng hgp, ntiltBl nghifln cdu hfli cdu vdi cdc mdu mfl md khflng t h i khoanh vCing xdc fljnh sy hifln difln (cfl hay khflng nhilu hay it) cila- lfl bdo ung ttiu, thi AS-Real-ttme PCR 16 ra ttiich hgp hon
TAI LI^U THAM KHAO
Amado RG. Wolf M. Pee^rs M. Van CE. Siena S (2008) Wild-type KRAS is required for panitumumab efficacy in patienis wlh metastatic colorectal cancer. JCO 26:1626-1634, J K Benvenuti S, Sartore BA. Nicotantonio FD. Zanon C, Moron M (2007) Oncogenic activation of the RAS/RAF signaling pathway impaifi the response of metastatic cotorectal cancers to anti-epidermal growth factor receptor antibody therapies. Cancer Res 67:26«-2648, Blanke CD (2005) Gefitinib in Cokirectal Cancen If Wishes Were Horees. JCO 23(24): 5446-5449.
IS^^t."^!,th J^ni°!^ri=;„"^T™^« ^'^°''J' ^"^^'^ ^ (2°°^' *^-'^^ " ^ ^ * i ° " i" ^^^ endometrium of tamoxifen-treated breast cai«t patients, with a companson of tamoxifen and toremifene Br J Cancer 92.1098-1103
Jonsson M, Ekstrand A, EdekTmg T, Ebeihard J. Grabau D (2009) Experiwices from treatment-predictive KRAS testing; high mutatwn frequency in rectal cancers from females and concunent mutafions In &ie same tumor. BMC CiinKal Pathology 9.
Karapetis CS. Kha'mbata FS. Jonker DJ, CTCallaghan CJ, Tu D (2008) K-ras mutafions and benefil from cetuximab in advanced colorectal cancer. N Engl J Med 359; 1757-1765.
Lang AH. Dreiraf HD. Geller RS, Starit N. Winder T, Geiger K, Muendlein A (2011) Optimized AUete-SpecHk: Real-THne PCR Assays for the Detetaron of Common Mutations In KRAS and BRAF. J Md Diagn. 1 3 : 1
Mendelsohn J (2002) Targeting the epWemial growth fector receptor for cancer ttierapy. JCO 20: 1-13.
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K-RAS IN MOLECULARLY TARGETED THERAPY: FROM IN SIUCO TO IN VITRO STUDY
Lao Due Thuan'', T r u o n g K i m P h u o n g \ Lfeu C h l H u ^ g ^ N g u y e n V a n T r u o n g \ Le Huyen Al Thuy'' 'HoChiMinh city Open Unhrersity, Viet Nam
^TayNinh Hospital, VietNam
Molecularly targeted therapy is a new type of cancer treatment which uses monoclonal-antibodies or small substances to identify and attack cancer cells, but not nomsal cells except normal cell. This has more advantages than classical treatments. There is well molecular-targeted agents is K-ras (Kirsten-ras).
In this study, we performed a systematic literature review in NCBI and conqnited average frequencies of K-ras mutatioiis in some common cancers. According to our data analysis, we concluded that global published inutatioiis vAuch belong to these genes neidier exclude each other nor associate with factors of race. We also successAilly designed and evaluated a set of primers for detection ofK- ras mutations by PCR-sequencing and a set of primers and probes sets are used for detecting the most seven common K-ras mutations by Allele-Specific-Real-time PCR (AS-Real-time PCR). Due to initial experimental results, an appropriate DNA extraction procedure ftom FFPE (Fomialin-Fixed, Paraffin-Embedded) samples and optimal Tm for each primer couple of airqilified mutation, containing regions on K-ras have been drawn.
Results of K-ras mutations detection on colorectal cancer patients from Hospital of Tay Ninh Pnjvmce, Viet Nam obtained by both methods: PCR-sequencing and AS-Real-time PCR, were mformed.
Key words: Allele-Specific Real-time PCR, colorectal cancer, EGFR, K-ras, PCR-sequencing.
*AiJthor for con^spondence' Tel: +84-905784471: Email: lhathuy@gmail com
