K thut xt nghim lai ti ch nhim sc th gn hunh quang (fish) pht hin

yu t HER-2/NEU trong ung th biu m

(10:36 | 19/10/2011)
1. M U
Mc d c nhiu tin b trong chn on v iu tr, song cho n nay ung
th vn l vn ln ca Y hc v t l gy t vong vn rt cao. C th iu tr ung th
bng nhiu cch nh phu thut, x tr, ha cht, tng cng min dch v nhiu khi
phi kt hp cc phng php trn vi nhau (phng php iu tr a m thc), song
nhn chung, kt qu vn cn nhiu hn ch, nht l i vi nhng trng hp ung th
giai on mun, c di cn, khng cn kh nng phu thut. Gn y, ngi ta
tm ra mt s loi thuc mi, vi bn cht l cc khng th n dng, iu tr trng ch
(target therapy) vi hiu qu rt tt nh: Gefitinib, erlotinib, lapatinib, cetuximab Mt
trong nhng loi thuc v ang c s dng kh ph bin cho nhiu loi ung th
biu m hin nay l trastuzumab (Herceptin), mt khng th n dng tc ng trc
tip trn th th Her-2/neu b mt t bo u [5], [13], [16], [29].
Tuy nhin, cc thuc iu tr trng ch, sn phm quen thuc nht v ang c
nhiu nc trn th gii s dng l trastuzumab, l loi thuc rt t tin. S l ht sc
lng ph nu Her-2/neu m tnh m vn iu tr bng trastuzumab. V th, trc ht cn
nh gi chnh xc tnh trng biu l ca yu t Her-2/neu cc t bo ung th, v l
iu tin quyt cho s thnh cng ca liu php trastuzumab m khng s lng ph tin
mt cch khng cn thit [24].
2. GII THIU V HER-2/NEU, EGF V EGFR
2.1. Yu t Her-2/neu, EGF v EGFR
Her-2/neu, cn gi l c-erbB-2; CD340 hay p185, l 1 trong 4 thnh vin ca gia
nh th th biu b ngi (Human epidermal receptor - HER). Nhm ny gm 4 loi l:
HER-1 (Th th ca yu t tng trng biu b - Epidermal growth factor receptor EGFR), HER-2, HER-3 v HER-4. Cc th th HER-3 v HER-4 t gp cc ung th
ca ngi, ngc li, HER-2 v EGFR c bit n v c nghin cu rt nhiu.
Yu t tng trng biu b (Epidermal growth factor - EGF) l nhm yu t tng
trng c bit n sm nht. Chng c Stanley Cohen v cng s (CS) pht hin
ln u tin vo nm 1962. l mt nhm gm nhiu yu t nh: Yu t tng trng
chuyn dng alpha (TGF-), amohiregulin, yu t tng trng biu b gn heparin
(heparin-binding HbEGF), -cellulin, cripto-1...
Th th ca EGF (EGFR) nm trn b mt ca mng t bo. y l
mt glycoprotein xuyn mng, phn nm bn trong mng t bo ca th th ny gn
vi men tyrosine kinaze. Men ny c hot ha khi EGF gn vi th th EGFR. Th
th EGFR do mt gene nm trong t bo quy nh, l mt tin gene sinh ung th.
Gene quy nh EGF v th th EGFR c tm thy m tuyn vbnh thng v
nhiu loi m khc.
2.2. S biu l ca HER-2/neu v gi tr tin on p ng vi iu tr

C khong 25-30% cc ung thu v c biu l qu mc ca th th Her-2/neu trn b

mt mng t bo. Ngoi ra, c th gp s khuch i gene HER-2/neu trong nhiu loi
ung th khc nh: ung th phi khng phi t bo nh (UTP KTBN), cc ung th biu
m (UTBM) tuyn ca i trc trng, d dy, tuyn nc bt, bng quang, tin lit
tuyn, bung trng, ni mc t cung[5], [11], [14], [15], [17], [18]
2.3. Tnh hnh nghin cu v Her-2/neu trong mt s bnh ung th
2.3.1. Trong UTBM tuyn v
Mc d s tng biu l v khuch i gene HER-2/neu c nhiu loi ung th khc
nhau nhng vn ny c nghin cu nhiu nht l trong UTBM tuyn v [4], [6],
[8], [19], [21], [27]... Cc s liu nghin cu thng cho thy khong 25-30% cc
trng hp UTBM tuyn v c tng biu lHER-2/neu (khong 7% i vi ung th ti
ch v 41,5% khi ung th di cn) [4]. Tuy nhin, cc s liu cng b cn cha thng
nht. C nghin cu nu t l Her-2/neu dng tnh ch l 14%, trong khi mt nghin
cu khc li a ra con s rt cao, ti 90% [4], [28]
2.3.2. Trong ung th phi loi khng phi t bo nh (UTP KTBN)
Theo Andre v CS (2004) [5], t l cc trng hp UTP KTBN c tng biu l HER2/neu khng nhiu lm v dao ng kh ln gia cc nghin cu [4], [5], [10], [12]. S
liu t cc nghin cu thng cho thy t l cc trng hp UTP KTBN c tng biu
l HER-2/neu qua xt nghim HMMD khong t 19% - 30%; Tuy nhin, cng c nghin
cu cng b t l cao hn nhiu: ti 40-80% [5].
2.3.3. Trong ung th tin lit tuyn
Nghin cu ca cc tc gi thng cho thy khong 25-30% s bnh nhn ung th tin
lit tuyn c tng biu l gene HER-2/neu [5], [20], [23].
2.3.4. Trong ung th i trc trng:
ngha ca s khuch i gene v gia tng biu l protein Her-2/neu trong UTBM i
trc trng cn t c nghin cu v cc s liu rt khc nhau gia cc tc gi (21% 59,4%)
2.3.5. Trong ung th d dy
Gn y, c kh nhiu nghin cu v s biu l ca HER-2/neu trong ung th d
dy v cc th nghim iu tr bng trastuzumab. Tuy nhin, cc s liu thu c t
nhng nghin cu khc nhau cha thc s thng nht
Ti Vit Nam, cng c mt vi cng trnh nghin cu da trn xt nghim
HMMD v tnh trng biu hin ca yu t Her-2/neu nhng ch yu l trong UTBM
tuyn v v mt vi nghin cu trong UTBM ca i trc trng, vi s lng bnh nhn
t, cc kt qu nghin cu cng cn khc nhau nhiu:

- Nghin cu ca Nguyn Vn Thnh v CS (2009) ti Bnh vin Ung bu TP.

H Ch Minh [2], trong UTBM tuyn v cho thy t l Her-2/neu dng tnh l 29,74%;
trong khi nghin cu ca T Vn T (2009) ti Bnh vin K, trn cc bnh nhn UTBM
tuyn v c tui i 35 li cho thy t l ny kh cao, ti 42,3% [3].
- Hong Kim Ngn v CS, nghin cu trong bnh UTBM i trc trng thy Her2/neu (+) 27,27% s bnh nhn; t l ny nhng khi UTBM vi c tnh cao
(high-grade) cao hn rt nhiu so vi cc trng hp c c tnh thp (low-grade)
(63,64% so vi 18,18%) [1].
Hu nh cha thy c cng trnh nghin cu no v vn ny i vi UTBM
ca phi, tuyn tin lit v cc loi ung th khc
3. CC PHNG PHP NH GI YU T HER-2/NEU
C th chia cc phng php nh gi yu t Her-2/neu thnh 3 nhm chnh
sau [24]:
- nh gi mc khuych i ca gene HER-2/neu (bng cc k thut lai ti
ch NST: FISH, CISH, SISH).
- Xc nh s biu l qu mc ca th th Her-2/neu trn b mt t bo thng
qua vic pht hin cc protein c hiu (bng xt nghim HMMD).
- nh lng protein Her-2/neu trong huyt thanh (phng php ELISA).
Trong cc phng php ni trn, hin nay c 2 phng php thng c s
dng rng ri nht l HMMD v FISH.
3.1. K thut xt nghim HMMD
HMMD l k thut c s dng ph bin nht hin nay trong vic nh gi th th
Her-2/neu. Nguyn l ca xt nghim ny l s dng cc KT c hiu bit, bn sn
trn th trng pht hin cc khng nguyn (KN) tng ng l protein Her-2/neu sn phm ca gene HER-2/neu - trn b mt t bo.
u im ca HMMD l tin li, r tin, tiu bn xt nghim d bo qun sau khi
phn tch, v ch cn s dng knh hin vi quang hc thng thng quan st. Tuy
nhin, phng php ny c nhc im l kt qu khng tht khch quan, do c th
b nh hng ca mt s yu t nh: iu kin bo qun mu trc , thi gian v k
thut c nh bnh phm, loi KT s dng (n dng hay a dng), v quan trng hn
c l kh khn khi p dng bng tnh im c kt lun chnh xc.
C mt t l kh ln trng hp kt qu biu hin l 2+ khi dng phng php HMMD
li khng thy c s khuych i gene tng ng khi dng phng php FISH. Trong
nhng trng hp ny th FISH cho nhiu thng tin v tin lng, v p ng vi iu
tr trastuzumab hn l phng php HMMD. V th, Hoa K v cc nc pht trin
quy nh khi kt qu xt nghim HMMD khng tht r rng (2+) th phi xt nghim li
bng k thut FISH.
3.2. K thut xt nghim FISH
y l phng php nh gi mc khuych i ca gene HER-2/neu trn
NST 17 ca t bo.

Cng nh cc phng php xt nghim da trn DNA khc, FISH c li im l cc

nuclieotid c kh nng bin tnh v ti t hp li. Mt c tnh rt quan trng l, dng
chui n DNA, mi axt nucleic s bt cp vi 1 axt nucleic khc theo nguyn tc b
sung ti cu trc li chui xon kp DNA. Trong k thut FISH thng quy, on
DNA ch c c nh trn b mt tiu bn s c lai vi on DNA c nh
du hunh quang (u d DNA), to thnh 1 chui xon kp DNA lai.
3.2.1. Nguyn l ca xt nghim FISH (xem s di)

Hnh 2. S v nguyn l ca k thut FISH

Ch thch: on DNA ch (mu xm nht) c c nh trn b mt tiu bn v lm
bin tnh. u d DNA (mu en) c nh du bng 1 cht nhum hunh quang
(fluorochrome) v / hoc 1 hapten khng nhum hunnh quang (nonfluorescent

hapten), bin tnh v lai trc vi DNA lp li khng nh du (mu xm sm). Sau ,
u d DNA c lai vi DNA ch. Sau khi ra loi b chui n DNA khng gn
cng nh nhng on NDA gn mt cch khng c hiu. Sau khi ra b hapten
khng gn hunh quang v cho dung dch chng phai mu vi DAPI (4,6-diamidino-2phenylindol.2HCl l), quan st tiu bn di knh hin vi hunh quang.
3.2.2. Thit b, dng c v ha cht
3.2.2.1. Thit b, dng c
Ngoi cc my mc, thit b lm tiu bn m bnh hc thng quy nh: my
x l m, my c block paraffin, my ct tiu bn vi th cn cn thm cc my mc,
thit b sau:
-

Knh hin vi hunh quang (vi cc lc hunh quang cn thit).

My lai bin tnh DNA (ThermoBrite).

My trn mu (Vortexer).

Hot plate (iu chnh c nhit ).

B n nhit (Waterbath).

My khuy t gia nhit.

My o pH.

Cn in t.

Pippet t ng.

Bung to m.

T lnh.

Coplin jar.

Tiu bn, l knh

3.2.2.2. Du d DNA gn hunh quang v ha cht

Cc u d DNA gn hunh quang cho FISH (c nhiu loi u d DNA gn hunh
quang cho FISH bn sn trn th trng bi cc hng nh Vysis, Abbott, Kreatech,
MetaSystems, ASI v Cytocell...).
Vectashield antifade.
Biotinylated antiavidin.
Biotin nick translation kit.
Bovine serum albumin (BSA).

 DAPI (4,6-diamidino-2-phenylindol.2HCl) stock solution.

dNTP: dung dch stock dATP, dCTP, dGTP, dTTP.
Cc nucleotis gn hunh quang 1 mM (FITC-12-dUTP).
Cc chui mi Oligonucleotide 50 pmol ml/1 (c hiu cho chui ch).
Dung dch PBS (phosphate buffered saline).
Xi mng cao su (rubber cement): Fixogum.
Taq DNA polymerase vi m 10 Taq.
3.2.3. Quy trnh xt nghim FISH
1. Mnh m c ct t chnh cc bnh phm s dng ct nhum, chn on
m bnh hc thng quy v xt nghim HMMD trc , vi dy lt ct 3-4m, tri
phng trn lam knh chuyn dng, kh paraffin.
2. Gi 100l dung dch m bin tnh ln tiu bn v y l knh 2450mm.
3. tiu bn trn 1 hot plate nhit 75C x 2-4 pht.
4. B l knh, cho tiu bn vo coplin jar cha cn 70% (4C) gi DNA ch dng
chui n.
5. Loi nc trn tiu bn bng cn ethylic (70%, 90%, 100%, 4C, mi chng 3 pht)
v kh trong khng kh.
6. Thm 20l dung dch u d (probe solution) ln mi tiu bn bin tnh, y 1 l knh
24 50mm ri gn cht xi mng cao su xung quanh l knh.
7. tiu bn 1-3 m 37C trong hp to m.
8. Ly tiu bn ra bung 37C v b xi mng cao su bng kp v tho l knh
bng cch ngm tiu bn trong dung dch Tween 4 SSC/0.2% ( nhit phng trong
b coplin jar 100ml).
9. Ra tiu bn 3 5 pht trong dung dch formamide (45C), sau trong dung dch
2 SSC (37C) 3 5 pht trong 1 b coplin jar 100ml.
10. Chuyn tiu bn vo dung dch Tween 4 SSC/0.2% (100ml nhit phng)
trong vi giy.
11. Thm 50l dung dch I vo mi tiu bn, y l knh 24 50mm v 37C trong
30 pht trong bung m.

12. B l knh v ra 3 ln 3 pht trong dung dch Tween 4 SSC/0.2% ( nhit

phng v lc nh nhng).
13. Thm 50l dung dch II vo mi tiu bn, y l knh 24 50mm v 37C x 45
pht trong hp to m.
14. Lm li bc 12.
15. Nhum tiu bn vi dung dch DAPI (trong b coplin jar 100ml, nhit phng) x
8 pht.
16. Ra tiu bn 3 ln trong nc vi giy v kh t nhin trong khng kh.
17. Thm 15l dung dch chng phai mu, y l knh
18. Quan st v phn tch kt qu di knh hin vi hunh quang.
3.2.4. nh gi kt qu
- c kt qu di knh hin vi hunh quang phng i 400 ln.
- Trc ht, cn xem li tiu bn m bnh hc nhum HE thng quy xc nh v tr
vng ung th xm ln.
- Sau , quan st, tm v xc nh chnh xc vng u tng ng trn tiu bn FISH.
- Tm v m trn 20 t bo u cc tn hiu mu (tn hiu ca Her-2/neu) v cc tn
hiu mu xanh lc (tn hiu tm ng ca NST 17).
- Ly trung bnh cng ca cc tn hiu mu , chia cho trung bnh cng ca cc tn
hiu mu xanh.
+ Nu kt qu > 22 l FISH (+).
+ Nu kt qu < 18 l FISH (-).
+ Nu kt qu t 18-22 th phi m li trn 60 t bo v tnh ton tng t nh
trn.
FISH l mt phng php xt nghim cho kt qu khch quan hn. N c coi l mt
xt nghim tiu chun vi nhy v c hiu cao pht hin s khuych i ca
gene HER-2/neu. Tuy nhin, nhc im ca phng php ny l gi thnh kh cao,
mt nhiu thi gian hn, phi quan st di knh hin vi hunh quang, vic c kt qu
kh khn hn do kh ghi nhn nhng du hiu hnh thi hc km theo, i khi rt kh
xc nh vng ung th xm ln trn tiu bn FISH, tiu bn khng th lu gi lu c
th xem li v tn hiu hunh quang bay mu dn theo thi gian
3.3. Mt s phng php khc nh gi yu t Her-2/neu

Ngoi 2 k thut xt nghim HMMD v FISH ni trn, cn c th nh gi yu t Her2/neu bng mt s phng php khc nh:
- K thut lai ti ch NST gn mu / hoc gn bc(Chromogenic / Silver in situ
hybridization - CISH / SISH).
- K thut khuych i gene RT-PCR (Reverse transcription - polymerase chain
reaction).
- Phng php ELISA (Enzyme-Link ImmunoSorbent Assay).
4. D KIN CCH THC TRIN KHAI XT NGHIM FISH
4.1. iu kin trin khai k thut FISH ti Khoa Gii phu bnh l
- V o to k thut: BS Trnh Tun Dng c hc v k thut ny ti Nht
Bn (nm 2007) v Singapore (nm 2009). Trong thi gian ti s mi chuyn gia
Singapore sang o to trc tip ti Bnh vin cho cc bc s v k thut vin ca
Khoa.
- V thit b, dng c, vt t v ha cht: hn ti, Khoa Gii phu bnh l c trang
b tng i y cc thit b, dng c cho k thut ny. Cc vt t tiu hao v ha
cht ban u s s dng t ngun kinh ph ca ti cp B Quc phng.
4.2. K hoch trin khai
D kin, trong khong 2 nm u s la chn khong 120 trng hp UTBM,
gm 4 nhm, mi nhm 30 BN:
+ Nhm 1: UTP KTBN.
+ Nhm 2: UTBM tuyn v.
+ Nhm 3: UTBM i trc trng.
+ Nhm 4: UTBM tin lit tuyn.
(y l nhng loi ung th hay gp cc bnh nhn cao tui ca Vit Nam v
cng l i tng hay gp ti Bnh vin TWQ 108 ni ring).
- Trc ht, cn xt nghim m bnh hc, chn on xc nh l UTBM:
+ Cc mnh bnh phm u s c c nh bng dung dch formol 10% trung
tnh, sau c x l bng my x l m t ng STP 120 (ca Hng Microm - c),
ri c trong khi paraffin bng my EC 350-2 (ca c).
+ Ct mnh vi dy 3-4m, nhum Hematoxylin - Eosin v PAS bng my
nhum tiu bn t ng HMS 70 (ca c).
+ c v phn tch kt qu di knh hin vi quang hc Axioscope 40 ca Hng
Calzeiss (c). Phn type MBH, phn c tnh v giai on ca khi u theo phn loi
ca T chc Y t th gii nm 2010.

- Sau , tin hnh xt nghim HMMD v FISH nh gi v s biu l ca

yu t Her-2/neu.
- So snh kt qu gia HMMD v FISH, rt kinh nghim v hon thin k thut,
sau s trin khai rng ri hn cho cc loi ung th khc.
5. KT LUN
Mc d c nhiu tin b trong chn on v iu tr, song cho n nay, ung
th vn l vn ln ca y hc v t l gy t vong vn rt cao.
Trn th gii, vic kho st yu t Her-2/neu bng xt nghim FISH tr thnh
thng quy. Tuy nhin, Vit Nam, cho n nay vn rt t c s Y t c th thc hin
c xt nghim ny.
Hin ti, Khoa Gii phu bnh l c trang b kh y cc thit b, my
mc cn thit, cn b c o to kh c bn, c th trin khai xt nghim FISH,
v nu lm tt tt th y s l mt trong nhng c s u tin ti Vit Nam ng dng
k thut ny tm hiu v s biu l ca yu t Her-2/neu trong ung th, gip cc Bc
s c thm hiu bit, kinh nghim v cn c khoa hc p dng liu php iu tr ch
bng trastuzumab cho cc bnh nhn ung th, ci thin cht lng v ko di thi gian
sng cho bnh nhn.
D kin, sau khi k thut ny c trin khai v hon thin, Khoa Gii phu
bnh l ca Bnh vin TWQ 108 s l ni tip nhn v xt nghim cho hu ht cc
trng hp ung th c nhu cu v loi xt nghim FISH, v hin ti cha c c s no
min Bc thc hin xt nghim ny cho cc bnh nhn UTBM. Mt khc, s c thm
iu kin tin hnh cc nghin cu khoa hc, o to v hun luyn ti Bnh vin
TWQ 108 ni ring v Vit Nam ni chung.
K thut c Thng trc Hi ng KHKT Bnh vin thng qua v nhn nh
y l k thut c nhiu li th v c tnh ng dng cao.