ALA, P - THR VA V - VAL CUA EBNA - 1 6 VIRUS EPSTEIN

Summary

AUTOLOGOUS PERIPHERAL BLOOD STEM CELL CARRIED - OUT AT THE CENTRAL MILITARY HOSPITAL 108 FOR THE PATIENTS WITH NON - HODGKIN LYMPHOMA

AND MULTIPLE MYELOMA

High - dose chemotherapy with autologous peripheral blood stem eell(PBSC) transplantation is the front - line treatment for certain patients with non - Hodgkin's lymphoma (NHL) and multiple iTiyeloma(MM).

Objectives: applying the autologous PBSC transplantation in treatment of the patient with NHL and M M , evaluating its safety and effectiveness. Method: PBSCs of the NHL and M M patients being in remission were mobilized by the combination of Cyelophosphai-nide(2 - 4g/m') and G - CSF ( 5 - 1 0 mg/kg/day);

PBSCs were collected and eryopreserved in - 196°C; BEAM and high - dose Melphalan were used as the pre - transplant conditioning regimens for NHL and M M patients respectively; autologous PBSC transplan- tations were performed and followed - up. Results: the mean time of PBSC mobilization was 10.25 ± 1.7 days; the mean CD34-I- cell count collected by 3 - 4 consecutive apheresises was 6.95 ± 2.60 x lOVkg;

the cell viable rate of the PBSC eryopreserved in - 196''C was approximately 9 0 % ; PBSC transplantation was carried - out safely with the rapid engraftment and hematopoietic recovery (the mean times of granulocyte and platelet recovery were 11.0 ± 0.9 days and 11.8 ± 1.6 days respectively); the event - free survival of the NHL patients after transplantation was longer in comparison with that one of M M patients.

Conclusion: autologous PBSC transplantation was performed safely and effectively.

Keywords: peripheral bood stem cell (PBSC), autologous PBSC transplantation, non - Hodgkln's lym- phoma (NHL), multiple myeloma (MM)

XAC DjNH 3 PHAN TYP P - ALA, P - THR VA V - VAL CUA EBNA - 1 6 VIRUS EPSTEIN - BARR PHAN LAP TAI VIET NAM

Le Thanh Hoa', Vu Thi Tie'ng Hoang Thj Mlnh Chau', Le Thanh H a ^ Nguyin Dinh Phuc^

'Vien Cdng nghe sinh hoc; 'Hoc vien Quan y; ^Trddng Dai hoc Y Ha Noi EBNA - 1 lien quan mat thie't de'n ung thd vdm mui hong (NPC) va tang nhiim virus. Da hinh tai vi tri acid amin 487 cho phep xic dinh 5 phin typ: P - ala, P - thr, V - vai, V - leu, V - pro (P: prototype; V:

Variant): Can lim sing td trong miu benh pham NPC cua benh nhin Viet Nam cd bao nhieu phin typ, thudc loai nao. Muc tieu: phit hien va xac djnh phin typ EBV bang phdang phip phan tich dac diem phin td EBNA - 1. Doi tugng va phuang phap nghien ciiu: tach DNA, thdc hien PCR, thu nhin doan gen EBNA - 1 (0,76 kb); giii trlnh td, so sanh; phin tich acid amin EBNA - /, nhan dinh da hinh tai vi tri 487 de xac djnh phan typ tren 22 miu bdnh phim sinh thiet td benh nhan mic NPC nhip vien 4 vung, gom Hi Noi (6 miu), Hai Phdng (6 miu), Di Ning (3 miu) vi TP Ho Chi Minh (7 miu). Ket qua: trong sd 22 miu NPC cua Viet Nam phit hien 3 phin typ: P - ala, P - thr vi V - vai, chda phit hien V - leu va V - pro. Ha Ndi cd 4 miu V - vai; 2 mau P - thr; Hai Phdng cd 4 V - vai, IP- ala, 1 P - thr; Da Ning cd 2 V - vai, IP- thr; TP hid Chi Minh cd 4 V - vai, 2 P - ala, ? P - thr. Ket luan: ba phin typ P - ala, P - thr vi V- vai Ion tai trong sd 22 miu NPC, ngoai trd P - ala chda phit hien d Hi Ndi, 3 vung cdn lai deu chifa ca 3 phin typ. Phin typ V - vai gap d tat ca benh pham, ddac xic nhin cd the cd vai trd chinh trong tien tnen ung thd (tumorigenesis) cua NPC. Sing loc phan td xic djnh phin typ EBNA - 1 li can thiet vl cd the giup cho djnh hddng chan doin sdm va phdng chdng NPC . TO khoa: EBV, NPC, EBNA - 1, PCR, P - ala, P - thr, V - vai, V - leu, V - pro

I. D A T V A N DE

Epstein - Barr virus (EBV) gay sd n h i l m d p h i n ldn cpng ddng tren the gidi ngay tfl tudi nhd , nhifng ehi mpt sd phat trien thanh ung thfl vdm mui hpng (NPC, nasopharyngeal carcinoma), Burkitt lymphoma, benh Hodgkin T - lymphoma (Gruhne et al., 2009). Protein EBNA - 1 (Epstein - Barr Nuclear Antigen 1) la loai cd mat trong tat e l mpi dang t i l n trien khdi u eua EBV, xue t i l n qua trinh tang nhiem I n h hfldng d i n sfl nhan len cua he gen, sao chep va phan chia te bao (Aiyar et al., 1998). Chudi polypeptide cua EBNA - 1 chfla 641 acid amin (aa), chia lam 2 vung ehfle nang chinh, vung N va vung C, ndi vdi nhau bang chudi acid amin chen, gdm n h i l u motif lap glycine/alanine (GAR) tfl aa 90 den aa 327 (Mai et al., 2007). Cac acid amin vj trf tfl 459 - 607 dau tan cung C cd vai trd trung gian cho D N A b i m dinh va hinh thanh phan tfl kep (dimerization) (Wu et al., 2002). Da hinh tai vi tri acid amin 487 cho phep xac dinh 5 phan typ P - ala P - thr, V - vai, V - leu, V - pro (P: prototype; V: variant), lien quan den lifu hanh EBV trong mo, mau va dich hpng (Bhatia et al., 1996). Theo nghien eflu g i n day, EBV phan lap d vung Nam Trung Qud'c chu y l u la V - vai (Zhang et al., 2004; Mai et al., 2007), ddng vai trd quan trpng tang cUdng sao chep dd'i vdi E B N A - 1 ( M a i e t a l . , 2010).

Ben canh nghien cflu lam sang, tien trien benh, EBV phan lap tai Viet Nam cung da difpe nghien cflu ve dac diem sinh hpe phan td, trong dd cd phan tfch gen EBNA - 1 (Do et al., 2008;

Nguyen Dinh Phuc, 2006). Tuy nhien, viec x l c dinh phan typ (subtyping) dfla tren b i l n ddi acid amin 487 chfla dfldc d l cap den tai Viet Nam.

Muc tieu cua nghien cflu nay la: Xac dinh cac phan typ EBV qua phan tich dac diem cau true phan td EBNA - 1 co trong cac mau benh pham NPC cua benh nhan Viet Nam. So sanh vdi cac phan typ da dUgc biet tr€n the gidi de budc dau

xac dinh dac diem sinh hgc, dich ti bgc phan td EBV d cac benh nhan ung thU vdm miii hgng tai Viet Nam.

II. DOI TUONG VA PHUONG

PHAP

NGHIEN CL/U

Trong nghien cdu nay, chdng tdi phan tich da hinh tai vi tri acid amin 487 eua EBNA - 1 de x l e dinh eac phan typ prototype va variant d 22 mau benh pham cua benh nhan ung thfl vdm hpng (NPC) nhap vien tai Hai Phdng, H I Npi, Da Nang v a T P Hd Chf M i n h .

1 . Mau benh pham va xii ly cho nghien cOu Mau tUPi thu bang phUdng p h l p sinh t h i l t , bao quan d - 20°C, tfl 4 vung: i) mau Ha Ndi, ky hieu:

H N va EB; ii) mau Hai Phdng, ky hieu HP; iii) mau TP Hd Chi M i n h , ky hieu S; iv) m l u Da Nang ( m i l n Trung), ky hieu: MT.

DNA tdng sd difdc tach chiet sd dung bo kit cua hang Bionner (Han Qudc). Tdm tat nhfl sau:

n g h i i n m l u t h i n h huyen dich; cho proteinase K, u va cho RNase, Cho dung mdi GC, u d 70°C, rdi cho Isopropanol, ly tam de thu djch. Chuyen len cdt Ipc, ly tam, loai bd dich dfldi. Cho dung mdi W l , ly t i m , bd dich dUdi. Cho dung mdi W 2 , ly tam, bd djeh difdi. Chuyen cot sang dng Eppen- dorf mdi, eho djeh tach EL, ly tam, thu dich ben dudi la DNA tdng sd cua mau, bao quan d - 20°C.

Kiem tra D N A bang dien di tren gel agarose 1 % , nhudm ethidium bromide. D N A hien thj la vach sang dat tieu chuan de thflc hien PCR.

2. ThOc hien PCR, glal trinh to, xO ly sd^ lieu Doan gen EBNA - 1 (khoang 0,76 kb) thu dfldc bang PCR vdi khudn la D N A tdng so', sfl dung cap mdi; EBK1F: 5' GTCATCATCATCCGGGTCTC 3' va EBVR: 5' CGATTGAGGGCGTCTCCTAAC 3'.

Chuoi gen difpe thu nhan sau khi giai trinh tfl trflc tiep s i n pham, hoac sau khi tach ddng dflpc xd ly bang ehflpng trinh SeqEdl.03, sap x I p bang

AssemblyLIGN 1.9 va MacVector 8.2 (Accelrys Inc.), so sanh ddi c h i l u bang ehflpng trinh GENEDOC 2.5. Thanh p h i n acid amin dflpc thu nhan bang d c h sfl dung bp ma cua vi sinh vat bae thap (vi khuan, bacterial code) cd trong Ngan hang gen.

I I I . K E T Q U A

1. Thu nhan chuoi gen EBNA - 1

Vdi cap mdi EBK1 F - EBVR, doan gen k h o i n g 0,76 kb dflpe thu nhan va mpt sd dfldc giai trinh tfl trflc t i l p , mpt sd dflpc tach ddng, k i t q u i trinh bay d hinh 1. San pham gen EBNA - 1 phan Idn la dPn bang, chat Ifldng td't, ehdng toi giai trinh trflc tiep sfl dung mdi EBK1 F (hinh lA); mpt sd difdc tach ddng d l thu clone dung kich thfldc va giai trinh tfl bang mdi M l 3F [hinh IB).

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4.3fil kb - 2.(127 k b -

- 7 M b|i

Hinh 1. Kiem tra trUc tiep san pham PCR doan gen EBNA - 1 trUdc (A) va sau khi tach ddng (B)

Chi chu: A. S5, SI7, SI8, S20, HN9, HN11: san pham PCR cua cac miu dai dien; B. DNA cua cic ddng te bao 1 - 2: cua SI8; 3 - 4: cua HN9; 5 - 6: cua HNl 1 cit kiem tra bang EcoRI.

2. Xac dinh phan typ EBV qua phan tich vj tri aa 487 cua EBNA - 1

Vj tri acid amin 487 difdc sfl dung de so sanh ddi chieu EBNA - 1 cua 22 m l u EBV thu nhan tfl benh nhan NPC nhap vien tai Hai Phdng (HPI - 6); tai Ha Npi (HN2, 9, 1 1 , 12, 11 L, EB10); tai Da N l n g (MT7, 9, 10); va tai TP Ho Chf Minh (SI, 5, 7, 16, 17, 18, 20). Mpt sd chung qudc te dUpc so sanh, dac biet la ehung ed d i l n B95 - 8 (1) va B95 - 8 (2) dUpe sfl dung de phan biet prototype; ehung C l 8 va GDI (Trung Qudc) dai dien Nam Trung Qud'c eua Chau A va chung AG876 (Ghana) dai dien EBV typ 1 va Chau Phi.

K i t qua so sanh trinh bay d hinh 2 eho tha'y, tai vj trf 487, 22 ehung EBV cua Viet Nam chda 3 loai acid amin, dd la V (valine), A (alanine) va T (threonine), tfl dd x l c djnh thupe 3 loai p h i n typ: P - thr (Prototype threonine); P - ala (Prototype alanine) va V - vai (Variant valine).

K i t q u i xac dinh phan typ cua 27 chung, bao gdm d 22 chung eua Viet Nam va 5 ehung tham chieu cua t h i gidi, difpe liet ke d bang 1. Bang 1 cung cho tha'y ehi t i e t trong 6 m l u eua Ha Npi, cd 4 m l u thudc V - vai va 2 m l u thuoc P - thr ( H N l 1 va H N l 1 L); trong 7 m l u TP Ho Ch' M i n h , ed 4 m l u thupe V - vai, 2 m l u thudc P - ala (SI 8 va S20) va 1 m l u thupe P - thr (S7); trong 6 m l u Hai Phdng, ed 4 m l u V - vai, 1 m l u P - thr (HP4) va 1 m l u P - ala (HP6); trong 3 m l u mien Trung (Da N l n g ) , ed 2 m l u V - vai va 1 m l u P - thr (MT7).

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Hinh 2. So sanh mgt phan trinh tU acid amin EBNA - 1 cua 27 chung gom 22 chung EBV Viet Nam; 2 bien the cua chung cd dien B95 - 8; chung AG876 va 2 chung cua Trung Qudc (GDI va CIS). Cac chijng cua Viet Nam trong dau moc; vi tri aa 487 dugc chi dan bang mui ten; aa d vi tri 487 cua cac chUng EBV Viet

Nam dugc dong khung dgc.

Bang 1. Ket qua xac dinh phan typ ciia cac chung EBV qua phan tich acid amin tai vi tri 487 cua EBNA - 1

Stt 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Chung AG876 B 9 5 - 8 ( l ) B95 - 8 (2) GDI HN2 HN9 HN11 HN12 EB10 SI S5 S16 S17 S18

aa487 L A A V V V T V V V V V V A

Ke't qua xac djnh phan typ

V - leu P-ala P-ala V - v a l V - v a l V - v a l P-thr V - v a l V - v a l V - v a l V - v a l V - v a l V - v a l P-ala

Stt 15 16 17 18 19 20 21 22 23 24 25 26 27

Chung S20 HPI HP2 HP3 HP4 HP5 HP6 MT7 MT9 MT10 S7 H N l l L C18

aa487 A V V V T V A T V V T T T

Ket qua xac djnh phan typ

P -ala V - v a l V - v a l V - v a l P-thr V - v a l P-ala P-thr V - v a l V - v a l P-thr P-thr P-thr

IV. B A N LUAN

K i t qua phan tich trinh tfl acid amin cda 22 chung EBV Viet Nam cho thay cd 3 phan typ (V - vai, P - thr va P - ala): phan typ V - vai ndi trdi trong tat d d e m l u , dUpc xae nhan ed vai trd

trong t i l n t r i l n ung thu (tumorigenesis) eua NPC va phan b d dja ly vung Nam va Ddng Nam A. Sang Ipc phan tfl xac dinh phan typ EBNA - 1 de dinh hfldng phdng chdng NPC la e i n t h i l t tai Viet Nam.

Cd 14 trong sd 22 mau nghien edu thupe v l

V - vai. EBV ed dac tinh da hinh b i l n ddi gifla cae ehung phan lap d cac vung dia ly thudc Nam va Ddng Nam Chau A (Zhang et al., 2004; Wang et al., 2010) va nghien edu eua Mai et al (2007;

2010) chflng minh cac chung EBV thupe V - vai d vung Nam Trung Qud'c, ed k h i nang tang cfldng sao chep va cd vai trd quan trpng trong tien trien NPC. Tuy nhien, mpt nghien cflu k h I e v l EBV d B l e Trung Qud'c eho thay da hinh phan typ EBV la dae d i l m eua eac chung dia ly vung Nam Trung Qud'c va Ddng Nam A, khdng phai la y l u t d gan l i l n vdi d i l u hda tien trien ung thU vdm (Wang et al., 2010). Cho du ehfle nang la gi, thi viee xae djnh dflpc 3 phan typ (V - vai; P - thr va P - ala) cua EBV trong m l u benh pham ung thfl vdm mui hpng cua Viet Nam va xac djnh tinh da hinh phan typ cua EBV da cho mpt nhan xet bfldc dau la: EBV Viet Nam, ve mat djch t l hpe, ed bj anh hfldng cua vet dich te EBV tfl Quang Ddng - Nam Trung Qudc tdi Ddng Nam Chau A.

V. KET LUAN

Ba phan typ P - ala, P - thr va V - vai dflpc phat hien tdn tai trong sd 22 mau NPC cua Viet Nam, ngoai trd P - ala chfla phat hien d cac mau Ha Npi, cac mau d 3 vung cdn lai deu chda d 3 phan typ.

Phan typ V - vai da gap d trong tat d d c mau, dfldc x l c nhan ed the cd vai trd quyet dinh trong t i l n t r i l n ung thu vdm mui hpng (tumorigenesis), loai benh gap nhilu theo phan bd dia ly vung Nam va Ddng Nam A. Sang Ipe phan tfl xac djnh phan typ EBNA - 1 de cd the dinh hfldng cho chan doan sdm va phdng ehdng NPC la can t h i l t tai Viet Nam.

Chung tdi cam an tai trg kinh phi cua Bd Khoa hoc va Cdng nghe thdng qua de tai KCIO.31/06 - 10 (chu nhiem de tai - PGS.TS Nguyin Dinh Phuc) de thdc hien cdng trlnh nay.

T A I LIEU T H A M KHAO

1. Nguyin Dinh Phuc (2006). Nghien cdu ehan doan lam sang va gen virus Epstein - Barr

trong ung thfl vdm mui hpng. Luan an Tien sT Y hpe. Trfldng Dai hpe Y Ha N p i .

2. Alyar A, Tyree C and Sugden B (1998): The plasmid replicon of EBV consists of multiple eis - acting elements that facilitate D N A synthesis by the cell and a viral maintenance element.

EMBOJ 17: 6 3 9 4 - 6 4 0 3 .

3. Bhatia K, Raj A, Gultlerrez M l , Judde |G, Spangler G, Venkatesh H and Magrath IT (1996):

Variation in the sequence of Epstein Barr virus nuclear antigen 1 in normal peripheral blood lymphocytes and in Burkitt's lymphomas.

Oncogene 13: 1 7 7 - 1 8 1 .

4. Do NV, Ingemar E, Phi PT, Jenny A, Chinh TT, Zeng Y and Hu L (2008). A major EBNAl variant from Asian EBV isolates shows enhanced transcriptional activity compared to prototype B95.8. Virus Res 132 (1 - 2): 1 5 - 24.

5. Gruhne B, Sompallae R, MarescottI O, Kamranvar SA, Gastaldello S and Masucci MG (2009). The Epstein - Barr virus nuclear antigen - 1 promotes genomic instability via induction of reactive oxygen species. Proe Natl Acad Sci U S A 106 (7): 2313 - 8 .

6. Mai SJ, Ooka T, Ll DJ, Zeng MS, Jiang RC, Ju XJ, Zhang RH, Chen SP and Zeng YX (2007).

Functional advantage of NPC - related V - vai subtype of Epstein - Barr virus nuclear antigen 1 compared with prototype in epithelial cell line.

Oncology Reports, 17:141 - 146.

7. Mai SJ, Xle D, Huang YF, Wang FW, Llao YJ, Deng HX, Liu WJ, Hua WF and Zeng YX (2010). The enhanced transcriptional activity of the V - vai subtype of Epstein - Barr virus nuclear antigen 1 in epithelial cell lines. Oncol Rep 23(5):

1 4 1 7 - 2 4 .

8. Wang Y, Liu X, Xing X, Cul Y, Zhao C and Luo B (2010). Variations of Epstein - Barr virus nuclear antigen 1 gene in gastric carcinomas and

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nasopharyngeal carcinomas from Northern China. 10. Zhang XS, Wang H H , Hu LF, Ll A, Zhang Virus Res 147 (2): 2 5 8 - 6 4 . RH, Mai H Q , Xia JC, Chen LZ and Zeng YX 9. Wu H, Kapoor P and Frappler L (2002). (^004). V - vai subtype of Epstein - Barr vims Separation of the D N A replication, segregation nuclear antigen 1 preferentially exists in biopsies and transcriptional activation functions of Epstein "^ nasopharyngeal carcinoma. Cancer Lett 211 (1):

-Barr nuclear antigen I . J Virol 7 6 : 2 4 8 0 - 2 4 9 0 . 1 1 - 8 . Summary

SUBTYPING P - ALA, P - THR AND V - VAL BY ANALYSIS OF EBNA - 1 FROM EPSTEIN - BARR ISOLATED IN VIETNAM

EBNA - 1 is associated with tumorigenesis and persistent latent infection of EBV. Based on the variation at amino acid 487, EBNA - 1 is classified into five subtypes, including P - ala P - thr, V - vai, V - leu and V - pro (P: prototype; V: Variant). It should be elucidated that how many and which subtypes of EBNA - 1 in the Vietnamese EBV isolates are. Objectives: Detection and subtyping EBV in Vietnam by molecular analysis of EBNA - 1. Subjectlves: 22 biopsy isolates from the NPC patients admitted in 4 geographical regions: Ha Noi (6 samples); Hai Phong (6 samples); Da Nang (3 samples); Ho Chi Minh city (7 samples). Methods: D N A extraction, PCR amplification, EBNA - 1 fragment (0.76 kb), sequencing, comparative analysis, annotation of amino acids, polymorphism determination at the aa 487 ppsition for subtyping were used. Results: There are three subtypes detected in 22 isolates: P - ala, P - thr and V - vai;

not yet V - leu and V - pro. These include 4 V - vai, 2 P - thr in Ha Noi; 4 V - vai, 1 P - ala, 1 P - t h r in Hai Phong; 2 V - vai, 1 P - thr; and 4 V - vai, 2 P - ala, 1 P - t h r in Ho Chi Minh city isolates. Conclusion:

Three subtypes of P - ala, P - thr and V - vai detected in 22 EBV isolates; except for P - ala not in Ha Noi;

all three subtypes found in three other regions. V - vai is dominant, considered to play primary role in tumorigenesis of NPC. Molecular screening for subtyping of EBNA - 1 is essential in prevention of the NPC.

Key words: EBV, NPC, EBNA - 1 , PCR, subtype, P - ala, P - thr, V - val, V - leu, V - pro

HIEU QUA CUA GHEP TE BAO GOC TUY XUDNG LEN QUA TRINH LIEN xaONG TRONG DIEU TRI K H 6 P GIA THAN X L / O N G CHAY

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Nguyen Manh Khanh', Nguyen Thj Thu H a ^ Nguyen Tie'n Binh', Ly Tuan Khal^ Nguyin Thanh Binh'' 'Benh vien Viet Ddc, ^Benh vien Trung dang Quan doi 108, ^Hoc vien Quan y

Muc tieu: dinh gii hidu qua cua ghep khdi te bao gdc tuy xdang tdi qua trlnh lien xdang trong dieu trj chim lien xdang, khdp gia thin xdang chay. Ddi tugng va phuang phap nghien ciiu: td thing 9/2006, tai khoa Chan thdang Chinh hinh, benh vien Vidt Ddc va khoa Huye^hoc, benh vien Trung dang Quin ddi 108, 59 benh nhin chim lien xdang, khdp gia thin xdang chiy ddac ghep khdi te bao gdc tuy xdOng td than vao 6 gay, dinh gia ket qua lien xdang va mdi lien quan vdi sd Idang te bio tuy xdang, te bio gdc CD34+. Ket qua: khdng cd bien chdng tai nai lay vi ghep tuy xdang. Ty Id lien xdang 91,5%o (54/59 benh nhin) vdi thdi gian 'trung binh 24,2 tuan (9 - 44 tuan). Trung binh 13,8 ± 5,1 x W'' te bao gdc CD34-I- va