GIA TRI PHAT HIEN M O T SO BIEN DOI Dl TRUYEN 6 BENH NHAN LO XE Ml CAP DONG TUY CUA PHUONG PHAP PCR SO V 6 l NHI§M SAC THE DO
Vu Minh PhUofng, Pham Quang Vinh
• TrUdng Dai hoc Y Ha Noi
Muc tieu: so sanh ket qua ciia phUang phap PCR vdi nhiSm sac the do trong phat hien cac bien doi di truyen t(i5,i7)(q22;q2V vdi bien doi gen PML/RARa, t(8,21)(q22; q22) vdi bien doi gen AMLi/ETO va inv(i6)(pi3q22) vdi bien doi gen CBF/]/MYH17 d benh nhan la xe mi cap dong tuy. Doi tUang va pbi/ffng phap nghien curu: 87 benh nhan duac chan doan la xe mi cap ddn^ tiiy theo tieu chuan FAB duac dieu tri tai vien Huyet hoc truyen mau Trung uang va Khoa Huyet hoc truyen mau benh vien Bach Mai. Cac benh nhan duac choc hiit dich tuy lam xet nghiem nhiSm sac the do va PCR xae dinh cac bien doi gen PML/RARa, AMLI/ETO, CBFfi/MYHl. Ket qua va ket luan: trong 16 benh nhan nhom co PML/RARa chi co 9 benh nhan phat hien co chuyen doan t(i5;i7) (q21;q22) chiem ty le 56,25%. Trong i/ benh nhan nhom cd bien doi gen AMLI/ETO co 9 b$nh nhan phat hien co t(8;21)(q22;q22) chiem 52,9%. Trong 7 benh nhan cd bien doi gen CBFfi/MYHl 1 khong co benh nhan nao phat hien co inv(16)(pl3q22)
Tif khoa: t(15,17)(q22;q21), PML/RARa, t(8,21)(q22;q22), AMLI/ETO inv(16)(p13q22), CBFP/MYH11
I. DAT VAN DE
Cac bien ddi nhiem sac the t(15,17) (q22;q21) vdi bien ddi gen PML/RARa, t(8,21) (q22;q22) vdi bien ddi gen A M L I / E T O va inv(16) (p13q22) vdi bien ddi gen CBF(3/MYH11 la nhifng bien ddi di truyen quan trong co vai tro trong chan doan, tien lifong va lUa chon phac dd dieu tri benh Id xe mi cap dong tuy. O Viet Nam, tif nhCfng nam 80 da phat trien ky thuat di truyen te bao de phat hien cac bat thudng nhiem sac the. Tuy nhien ky thuat di truyen te bao trong danh gia cac bat thudng di truyen con nhieu han che. TCr nam 2006 vien Huyet hoc truyen mau T W da ap dung ky thuat di truyen phan tif (RT - PCR va nested - PCR) de nghien ciTu mot so' cac bien ddi gen co vai tro quan trong trong benh mau ac t i n h . Vi vay chung toi tien hanh nghien cifu de tai nay v6i muc tieu:
So sanh ket qua ciia phuong phap PCR vdi nhiim sac the do trong phat hien cac bien doi di truyen t(15,17)(q22;q21) vdi bi§'n ddi gen
PML/RARa, t(8,21)(q22; q22) vdi bien ddi gen AMLI/ETO va inv(16)(p13q22) vdi bien ddi gen CBFP/MYH11 d benh nhan lo xe mi cap dong tiiy.
II. DOI TliQlNG VA PHl/aNG PHAP NGHIEN cOU
I.Doi tifging nghien cu'u: 87 benh nhan dUOc chan doan xae dinh Id xe mi cap dong tuy theo tieu chuan cua FAB (nam 1986) difpc dieu tri tai khoa C8 vien Huyet hoc truyen mau T W va Khoa Huyet hoc truyen mau benh vien Bach Mai tCr 3/2007 den 3/2009 bao gdm 24 benh nhan lo xe mi ca'p the M3 va 63 Id xe mi cap the tuy khac.
2. PhUdng phap nghien ciJu , ,-, .
- Cac benh nhan diroc choc hut dich tuy lam xet nghiem nhiem sac the dd theo ky thuat nhuom bang G va phan tfch nhiem sac the tren may AXO 100 (Zeiss) va lam xet nghiem RT - PCR, nested - PCR xae dinh cac bien ddi gen PML/RARa, A M L I / E T O va C B F p / M Y H l 1
- So sanh ddi chieu ket qua cua 2 phirong phap.
I I I . K E T Q U A 1 : ; , , , ,
Bang 1. Ket qua phan tich bien ddi gen PML/RARa, AMLI/ETO va CBFfi/MYH11 d 87 benh nhan la xe mi cap dong tuy
Nhom 1 2 3 4 5
Dac diem
The M3 CO PML/RARa The M3 khong co PML/RARa Co A M L I / E T O
C6CBF(3/MYH11
Khong CO bien ddi gen (trCr the M3)
n 16 8 17 7 39
(%) (18,4) (9,2) (19,5) (8) (44,8)
Tdng 87 (100)
Nhan xet bang 2:
- Trong 16 benh nhan nhdm cd PML/RARa: cd 9 benh nhan cd chuyen doan t(15;17) chiem ty le 56,25%. Co 5 benh nhan khdng cd chuyen doan t(15;17) chiem ty le 31,25%
- Trong 8 benh nhan nhom khdng cd PML/RARa: khdng cd benh nhan nao co chuyen doan 1(15:17).
Bang 2. So sanh phan tich nhiem sac the cua la xe mi cap M3 co bien ddi gen PML/RARa
Cong thu'e nhiem sic the
Co PML/RARa N = 16
Khong CO PML/RARa N = 8
n (%) n (%)
46, XX,t(15;17)(q22;q21) 4 (25) 0 (0)
46, XY,t(15;17)(q22;q21) 5 (31,25) 0 (0)
46,XX 1 (6,25 3 (37,5)
46,XY 2 (12,5) 1 (25)
46, XY, 17q - (6,25) 0 (0)
45, XY, - 7 1 (6,25) 0 (0)
46, XY, t(14;15) 0 (0) 1 (12,5)
46, XY/50, XY 0 (0) 1 (1.2,5)
49,XX, + 1 1 , + 1 3 , + 2 1 / 44, XX, - 12, - 19
0 (0) 1 (12,5)
Khong phan bao 2 (12,5) 1 (12,5)
Nhan xet bang 3 (trang 3): . ' ..•.,•,!•• , - . . . . , . . ; . . ,-,^-, ,-,,.. ...
- Trong 17 benh nhan nhdm cd bien doi gen AMLI/ETO dUcfc lam phan tich nhiSm sSc the co 9 benh nhan cd t(8;21) chiem 52,9%. ' ' s ; ' Us^i n-.!ti ^ k ) paivt \\A<t\
- Trong 39 benh nhan nhom khdng co bien ddi gen khdng co benh nhan nao co t(8;21).
Bang 3. So sanh phan tich nhiim sac the d benh nhan co bien ddi gen AMI 1/ETO
Cong thu'e nhiem sSc the
Co A M L I / E T O Khong co bien doi gen n = 17 n = 39
45,X, - Y, t(8;21)(q22;q22) 46,XX, t(8;21)(q22;q22)
46, XX/46,XX, t(8;21) 46, XY 46, XX 45, XY, - 2 2 , 1 6 q - 46, X Y / 4 5 , X Y , - 2 2
46, XY, t(9;22) 45, XY, - 2 2 / 4 6 , XY 44, XY, 5q+, - 2 , - 1 2 42, XY, - 2 , - 3 , - 1 4 , - 1 5 ,
, 12q - , 1 0 q - 46,XX/46,XX,^6q+
46,XX,t(ll;17)(q23;q21) Khong phan bao
n 2 6 1 2 2 1 1 0 0 0 0 0 0 2 Bang 4. So sanh phan tich nhiem sac the d benh nhan (
(%) (11,8) (35,3) (5,9) (11,8) (11,8) (5,9) (5,9) (0) (0) (0) (0) (0) (0) (11,8) :d bien ddi
n 0 0 0 18 12 0 0
3 (%) (0) (0) (0) (46,2) (30,8) (0) (0) (2,6) (2,6) (2,6) (2,6) (2,6) (2,6) [7,7) gen CBF/3/MYH11
Cong thu'e nhiem sic the
C6CBFP/MYH11 n = 7
Khong CO bien doi gen n = 39
n (%) n (%)
46, XX 46, XY 46, XY, t(9;22) 45, XY, - 22 /46, XY 44, XY, 5 q + , - 2 , - 12 42, XY, - 2, - 3, - 14, - 15,
, 1 2 q - , lOq - 46,XX/46,XX,16q-^
46,XX,t(ll;17)(q23;q21)
3 4 0 0 0 0 0 0
(42,9) (57,1) (0) (0) (0) (0) . _ (0) (0)
12 18
30,8 46,2 (2,6) (2,6)' (2,6)
(2,6) _ (2,6)
(2,6)
Khong phan bao 0 (0) 3 (7,7)
Nhan xet: khdng phat hien 1 trudng hap nao cd inv(l6)(pl3q23). Ca 7 trUdng hap thudc nhdm cd cd bien doi gen CBF/3/MYH11 deu cd kieu nhiim sSc the binh thUdng tren phan tich nhiim sic the.
IV. BAN LUAN
1. Doi chieu ket qua phan tich bien doi gen PML/RARa vdfi phan tich n h i i m sSc the
Theo nhif ket qua d bang 2 tren 16 benh nhan CO bien ddi gen PML/RARa dUdc phan tich n h i i m sac the, co 5 benh nhan khong thay t(15;17) (q22;q21) chiem ty le 3 1 % . Nghien cufu cua Sucie M va cong sU tren 1 5 benh nhan Id xe mi cap the M3 chi thay 5 benh nhan co td 5;17)(q22;q21) nhUng lai co tdi 1 3 benh nhan co bien ddi gen PML/RARa bang ky thuat PCR [9]. TUdng tif Andrea Biondi tien hanh phan tich nhiem sac the tren 28 benh nhan Id xe mi ca'p the M3 co bien ddi gen PML/RARa , ket qua chi co 26 benh nhan co t (15;17)(q22;q21), 2 benh nhan con lai co kieu nhiem sic the binh thudng [2]. Nghien cUu cua D Sainty tren 90 trUdng hdp Id xe mi cap the M3 khong CO t(l 5 ;1 7)(q22 ;q21) sau dd da phat hien 4 trUdng hdp cd 1(1 5 ;1 7) d mot quan the te bao nhd
[31. Nhu vay neu nhU so' te bao cd t(1 5 ;1 7) rat it thi rat khd phat hien chuyen doan nay tren phan tfch nhiem sac the. NhU vay ro rang ky thuat PCR trong phat hien bien ddi gen PML/RARa cd do chinh xae cao hon cac ky thuat di truyen te bao.
2. Doi chieu ket qua phan tich bien doi gen A M L I / E T O vdi phan tich nhiem s i c the
Theo nhu ket qua trinh bay d bang 3, trong 1 7 benh nhan cd bien ddi gen A M L I / E T O chi cd 9 trUdng hdp phat hien t(8;21 )(q22;q22) chiem ty le 53%, 4 trUdng hdp khong cd bien ddi nhiem sac the va 2 trUdng hop cd bien ddi nhiem sac the phUc tap, Mrozek (2001) khi nghien cUu 21 trUdng hop cd bien ddi gen A M L I / E T O , chi cd 15 trUdng hop thay cd t(8;21) chiem ty le 71 %, 6 trUdng hdp cdn lai khdng phat hien t(8;21) ma la cac bien ddi n h i i m sac the phUc tap khac [7], Eun Kyung Cho tien hanh phan tfch nhiem sac the cua 20 benh nhan cd bien ddi gen A M L I / E T O dUdng tinh, chi phat hien dUpc t(8;21) d 10 benh nhan
chiem ty le 5 0 % [4]. Sarriera JE (2001) chd rang ky thuat RT - PCR cd dp nhay cad hon cac ky thuat di truyen te bao trong phat hien t(8;21) (q22;q22) vdi bien ddi gen tUdng ifng [8]. NhU vay ty le phat hien t(8;21) trong nhdm benh nhan co A M L I / E T O d nghien cufu nay cung tUdng ddng vdi cac nghien cifu khac. NhUng ket qua nay deu chifng minh tfnh ifii viet rd ret cua ky thuat PCR so vdi ky thuat di truyen te bao.
3. Doi chieu ket qua phan tich C B P p / M Y H I I vdi phan tich nhiem sac the , , j ,
Theo ket qua d bang 4 ca 7 benh nhan cd bien ddi gen C B p p / M Y H l l deu khong phat hien duoc inv(16)(pl 3q22). Ket qua nay khdng cd gi la ba't thudng, tren thifc te rat khd phat hien dao doan nay bang phUdng phap nhuom bang. Pham Quang Vinh (2003) nghien cUu tren 203 benh nhan Id xe mi cap ddng tuy khdng phat hien dUdc mdt trudng hop nao cd inv(l 6)(p13q22) [1].
Fumihiko Monma (2006) nghien cifu tren 224 benh nhan Id xe mi cap ddng tuy, phat hien 3,6%
cd inv(16)(pl3q22) trong khi dd tdi 7,6% cd bien ddi gen C B F p / M Y H l l [6]. Francesco Lo Coco (2008) nghien cufu 443 benh nhan thay inv(16) (pl3q22) chiem 7% trong khi ty le cd bien ddi gen C B F p / M Y H l l la 8,8% [5]. Ben canh nhUng khd khan trong phan tfch inv(l 6)(pl 3q22) bang cac ky thuat.nhuom bang thi hhOng benh nhan lo xe mi cap the nay thudng cd nhUng bien ddi di truyen phdi hop vi the eang lam giam ty le phat hien invd 6)(pl 3q22).
V. KET LUAN
Nghien cUu ddi chieu ket qua nhiem sac the dd vdi PCR trong xae dinh cac bien ddi gen PML/RARa, A M L I / E T O va C B F p / M Y H l l tren 87 benh nhan Id xe mi cap ddng tuy thay:
Treng 16 benh nhan nhdm cd PML/RARa chi cd 9 benh nhan phat hien cd chuyen doan t (15;17)chie'mty le 56,25%.
2. Trong 17 benh nhan nhdm cd bien ddi gen A M L I / E T O dUdc lam phan tich n h i i m sac the cd 9 benh nhan phat hien cd t(8;21) chiem 52,9%.
3. Trong 7 benh nhan cd bien ddi gen CBFp/
MYFH11 khdng cd benh nhan nao phat hien cd inv(16)(pl3q22).
TAI LIEU THAM KHAO
1. Pham Quang Vinh (2003). Nghien cUu bat thudng nhiem sac the trong cac the benh Id xe mi cap d ngUdi Idn tai Vien H H - T M T W . Luan an tien sy y hoc.
2. Andrea Biondi, Alessandro Rambaldi, Pier Paolo Pandoff et al (1992). Molecular monitoring of the myl/retinoic acid receptor alpha fusion gene in acute promyelocytic leukemia by PCR. Blood Vol 80 N°July 15; 492 - 4 9 7 .
3. Danielle Sainty, Vincenzo Liso, Angelo Cantu Rajnoldi et al (2000). A new morphologic classification system for acute promyelocytic leukemia distinguished cases with underlying PLZF/RARA gene rearrangements. Blood 15 August, Vol 96 No 4; 1 2 8 7 - 1 296.
4. Eun Kyung Cho, Soo Mee Bang, Jeong Yeal Ahn (2003). Prognostic value of A M L I / E T O fusion transcripts in patients w i t h acute myelogenous leukemia. The Korean Journal of Internal.
Medicine: 18; 13 - 20.
5. Francesco Lo - Coco, Antonio Cuneo,
Fabrizio Pane et al (2008). Prognostic impact of genetic characterization in the G I M E M A LAM99P multicenter study for newly diagnosed acute myeloid leukemia. Haematologica; 93 (7), 1 0 1 7 - 1024.
6. Fumihiko Monma, Kazuhiro Nishii, Junko Shiga et al (2006). Detection of the CBFbeta/
M Y H l l fusion gene in de novo acute myeloid leukemia ( A M D : a single institution study of 224 Japanese AML patients. Leukemia Research: LR - 2 5 5 9 - 2 5 6 4 .
7. Krzystovs Mrozek, Thomas W Prior, Colin Edwards et al (2001). Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv (16) in a prospective series of adults with de novo acute myeloid leukemia: a cancer and leukemia group B study. Journal of Clinical Oncology, Vol 19, N°9 ( M a y l ) : 2 4 8 2 - 9 2 .
8. Sarriera JE, Albitar M, Estrov Z et al (2001).
Comparison of outcome in acute myelogenous leukemia patients w i t translocation (8;21) found by standard cytogenetic analysis and patients with A M L I / E T O fusion transcript found only by PCR testing. Leukemia Jan; 1 5 (1); 57 - 6 1 .
9. Sucie M, Zadro R, Burazer B et al (2002).
A c u t e p r o m y e l o c y t i c l e u k e m i a M 3 : cytomorphologic, immunophenotypic, cytogenetic, and molecular variants. J Hematother Stem Cell Res D e c ; l l ( 6 ) ; 941 - 5 0 .
Summary
COiVlPARISON OF OUTCOME IN DETECTING SOME
ABBERANT PCR TESTING AND GENETIC BY CYTOGENETIC ANALYSIS IN ACUTE MYELOGENOUS LEUKEMIA PATIENTS
Objectives: (1). Compare the result of PCR testing and cytogenetic analysis in detecting t(15,17) (q22;q21) w i t h PML/RARa, t(8,21 )(q22;q22) with A M L I / E T O and inv(l 6)(pl 3q22) with C B F p / M Y H l l in the acute myelogenous leukemia patients. Methods: 87 patients diagnosed acute myelogenous leukemia by FAB criteria in National Institute of Hematology and Blood transfusion and Departmentof Hematdldgy and BIdod transfusion - Bachmai Hospital. All patient were taken bone marrow aspiration for cytogenetic
analysis and PCR testing to detect PML/RARa, AMLI/ETO, CBFp/MYHl fusion genes. Results and conclusions: (1) Rate the patient were detected t(l 5;1 7)(q21 ;q22)/ the patient with PML/RARa positive was 56.25% (9/16). (2) Rate the patient were detected t(8;21)(q22;q22)/ the patient w i t h AMLI/ETO positive was 5 2 . 9 % (9/17). (3) In 7 patienst w i t h C B F p / M Y H l l positive no one was detected invd6) ( p l 3 q 2 2 ) .
Keywords: t(15,17)(q22;q21), PML/RARa, t(8,21)(q22;q22), A M L I / E T O inv(16)(p13q22), C B F p / M Y H l l
CAC DOT BIEN DONG MAM TAI VUNG NONG CUA GEN APC 6 CAC BENH NHAN UNG THU DAI TRL/C TRANG
THE DA POLYP TUYEN GIA DINH
N g u y i n PhUOng A n h \ Nguyen Nghiem Luat^ Tran Van Khanh^
^Benh vien K; -TrUdng Dai iioc Y Ha Ndi
Muc tieu: cac ddt bien ddng mam d gen Uc che khdi u APC (tumor-supresor adenomatous polyposis coli) cd lien quan vdi ung thu dai trUc trang the da polyp tuyen gia dinh. Muc dich cua de tai nay ia phat hien cac dot bien ddng mam tai viJng ndng tren exon 15 cua gen APC d cac b$nh nhan ung thu dai trUc trang the da polyp tuyen gia dinh d Viet Nam. Doi tu'ang va phuang phap nghien ciiu: gen APC cua cac benh nhan ung thu dai true trang the da polyp tuyen gia dinh dUac khuech dai bang ky thuat phan Ung chudi polymerase (PCR), sau chudi DNA duac xae dinh trinh tU trUc tiep de phat hien cac dot bien ddng mam. Ket qua: da phai hien cac dot bien diem d 21 benh nhan vdi ty le phat hien dot bien la 16/21 (76,2%), bao gom dot bien dich khung (cai 1 bp, xoa 1 bp, xoa 2 bp) la 34/39 (87,2%), dot bien thay the bp (thay the 1 bp va 2 bp) la 5/39 (12,8%) va dot bien vd nghJa (stop codon) la 13/21 (61,9%>). Khdng phat hien duac cac dot bien doan Idn d cac benh nhan nay. Ket luan: (1) Da phat hien cac dot bien dich khung (cai 1 bp hoae xoa 1-2 bp), dot bien thay the bp (1-2 bp) va cac ma vd nghla d vung ndng ciia gen APC d benh nhan ung thU dai true trang the da polyp tuyen gia dinh Viet Nam. (2) Khdng phat hien duac dot bien doan Idn d vung nong ciia gen APC d cac benh nhan nay.
Tif khoa: gen APC, ung thif dai trifc trang
I. DAT VAN DE
Cac dot bien ddng mam d gen Uc che khdi u APC (tumor - supresor adenomatous polyposis coli gen: APC) la nguyen nhan cua hau het cac trUdng hop da polyp tuyen gia dinh (hereditary familial adenomatous polyposis; FAP) [5, 9]. Da polyp tuyen gia dinh la mot rd'i loan di truyen trdi cua nhiem sac the thUdng (autosomal dominant inher- ited disorder) chiem khoang 1 % cac ung thU dai trang ndi chung. Da polyp tuyen gia dinh dUdc dac trUng bdi tU hang tram den hang nghin cac
polyp tuyen trong dai trUc trang va cd kha nang tien trien thanh ung thU dai trUc trang [9]. Do dd, viee chan doan sdm da polyp tuyen gia dinh d nhUng thanh vien trong gia dinh cd nguy co la rat quan trpng de ngan ngUa sU phat trien ciia ung thu. Nhd nghien cdu sang loc cac thanh vien gia dinh bi da polyp tuyen gia dinh, ngUdi ta da phat hien hon 300 dot bie'n ddng mam khac nhau d gen APC. Khoang 9 5 % cac dot bien dich chuyen va vd nghTa gay nen sU cat ngan cac protein APC [5, 9). SU ma't chUc nang cac protein APC lam