NG DNG SINH HC PHN T TRONG BNH L HUYT HC

TS.BS Phan Th Xinh i Hc Y Dc TP HCM B Mn Huyt Hc 09/03/2013

NI DUNG
Bt thng NST v gen trong bnh l huyt hc:
Bch cu cp dng ty Bch cu cp dng lympho Nhng ri lon tng sinh ty Lymphoma

a u ty

TM QUAN TRNG CA SHPT TRONG HUYT HC

TRIU CHNG LM SNG
Ty , sinh thit ty, V du n t bo

CHN ON
Phn tch NST, FISH, PCR V DNA sequencing Bt thng NST, t bin gen,...

NH GI TIN LNG, LA CHN PHC IU TR THCH HP V THEO DI IU TR

3

BT THNG NST V GEN TRONG BCH CU CP DNG TY

(ACUTE MYELOID LEUKEMIA: AML)

BNG PHN NHM NGUY C THEO BT THNG NST

Nhm nguy c Tin lng tt Bt thng nhim sc th t(8;21)(q22;q22) t(15;17)(q22;q12-21)

Grimwade D: 42% Byrd JC: 48%

Tin lng trung bnh

inv(16)(p13q22)/t(16;16)((p13;q22) Nhim sc th bnh thng -Y, +8, +11, +13, +21, +22 del(7q), del(9q), del(11q), del(20q), t(9;11)(p22;q23) Bt thng 11q23, bt thng NST khc

inv(3)(q21q26)/t(3;3)(q21;q26), -5, -7, del(5q)

Tin lng xu t(6;9)(p23;q34), t(6;11)(q27;q23), t(11;19)(q23;p13.1)

Bt thng Nhim sc th phc tp (3)

(American Society of Hematology 2006)

T L SNG CN SAU 5 NM KHI IU TR HA TR LIU

< 60 tui

> 60 tui
Ngun: Alan K Burnett, School of Medicine, Cardiff University, Cardiff, UK

Nhm tin lng xu phi c iu tr vi phc ha tr liu mnh v ghp ty sau khi t lui bnh hon ton

46,XY,t(8;21)(q22;q22) TRONG AML

t(8;21)(q22;q22) TO RA T HP GEN AML1/ETO

No.8

t(8;21)(q22;q22)

No.21

q22 q22 Gen ETO

Gen AML1

der(8)

der(21)

AML1/ETO
8

t(8;21)(q22;q22) TO RA T HP GEN AML1/ETO

AML1 (21q22)
5

BN Neg Pos

ETO (8q22)
395 bp

1 2 503 542

3 796

5 963 1057 275

2 413

3 655

4 745

M: Thang chun BN: Bnh nhn Neg: Chng m (nc) Pos: Chng dng (KASUMI-1)

AML1

ETO

T hp gen AML1/ETO hng t u tn n tm trn der(8)

inv(16)(p13;q22)/CBFB/MYH11

type A

4 379 4

5 474 5 1708 11 1921

12 2134 12

13 2296 13

14 14

88 %

B
C D E F G

1 case
1 case 5% 5% 1 case

4
4 4 4 378 4 4

5
1528 5 1201 5 994 7 8 8 1306 9 9 1459

10
10 10

11
11 11

12
12 12 12

13
13 13 13 13 13 13

14
14 14 14 14 14 14 14

8 7 1099 8

9 9

10 10

11 11

12 12

1 case 1 case 1 case

1 case

H I
J

4 4 5 9 10 11 12

13

CBFB-A

MYH11-B1

MYH11-B2

inv(16) to ra 10 kiu bn sao ca t hp gen CBFB/MYH11

46,XY,t(15;17)(q22;q12) TRONG AML-M3

t(15;17)(q22;q12) TO RA T HP GEN PML/RARA

PML (15q22)
BN

Pos Neg

RARA (17q21)
688 bp (type bcr1) type bcr2 289 bp (type bcr3)

bcr1

4 1264 1335

5 1479 5

6 1737 405 6

~55%

bcr2

~5%

M: Thang chun BN: Bnh nhn Neg: Chng m (nc) Pos: Chng dng (NB4)

bcr3

3 1263

~40%

PML

RARA

TIN B TRONG IU TR AML-M3

PML l promyelocytic leukemia RARA l retinoic acid receptor alpha All-trans retinoic acid (ATRA) tc ng vo ch RARA ca t hp gen PML/RARA lm t bo i vo qu trnh bit ha.

Arsenic trioxide (ATO) tc ng vo

phn PML ca t hp gen PML/RARA lm t bo i vo chu trnh cht t nhin (apoptosis)

Phc phi hp ATRA hoc

ATO vi ha tr liu chun

Gim t l t vong do DIC Tng t l lui bnh (75% c cha lnh bnh)

nh Lng PML/RARA Bng RQ-PCR Theo Di Tn Lu T Bo c Tnh Trong AML

Hematology 2006;2006:505-516

14

BNG PHN NHM NGUY C THEO T BIN GEN

40 50% trng hp c NST bnh thng:
Nhm nguy c Bt thng
t bin NPM1 Tin lng tt

Bnh cnh lm sng

46-62% N, blast ty cao, CD33+ cao nhng CD34-

t bin CEBPA
FLT3-TKD FLT3-ITD

15- 20% AML vi NST bnh thng v del(9q)

10-14% 28-34% (Exon 14, 15) 5-10% (Exon 5-11) T ghp c th ci thin tin lng ~ 10% Thng kt hp vi tht bi giai on tn cng

Tin lng xu

MLL-PTD t bin WT1

(American Society of Hematology 2007)

Nghin cu trn 550 BN NST bnh thng, thi gian theo di trung bnh 5 nm FLT3-ITD(+)/NPM1(-)
FLT3-ITD(+)/NPM1(+) FLT3-ITD (-)/NPM1 (-) FLT3-ITD(-)/NPM1(+)

FLT3-ITD(-)/NPM1(+) FLT3-ITD (-)/NPM1 (-) FLT3-ITD(+)/NPM1(+)

FLT3-ITD(+)/NPM1(-) Gale RE, 2008;111:2776-278416

T BIN FLT3-ITD TRONG AML

Bnh nhn nam, 10 tui Ty : t bo non 35% Chn on AML-M1 Kho st t bin FLT3 t exon 14-exon 20

t bin p.E611_F612ins13 trn exon 14 ca gen FLT3

Tin lng xu

T BIN c-KIT TRONG AML C t(8;21)

Bnh nhn n, 37 tui Chn on AML-M2 C t(8;21) v t bin c-KIT Hon tt iu tr tn cng v 2 t tng cng Ti pht ngay sau ngng ha tr liu

t bin im M541L, D816Y, N822K v L862L

Tin lng tt

t bin c-KIT

Tin lng trung bnh

BNG PHN NHM NGUY C THEO BT THNG NST V GEN

Nhm nguy c
t(8;21) inv(16) /t(16;16) t(15;17) NST bnh thng Tin lng trung bnh -Y, +8, +11, +13, +21, +22, t(9;11) Bt thng khc khng thuc nhm tin lng tt va xu inv(3)(q21q26)/t(3;3)(q21;q26), -5, -7, del(5q), Tin lng xu t(6;9)(p23;q34), t(6;11)(q27;q23), t(11;19)(q23;p13.1),

Bt thng NST

Bt thng v gen
NST bnh thng c t bin CEBPA hoc t bin NPM1 khng kem vi FLT3-ITD

Tin lng tt

t(8;21), inv16, t(16;16) c kem t bin cKIT

NST bnh thng c t bin FLT3-ITD m khng c s hin din ca t bin NPM1, CEBPA

Bt thng NST phc tp (3)

NCCN 2010

19

TN SUT CA NHNG NHM TIN LNG

Xu: 34%

Tt: 45%

Trung bnh: 21%

Grimwade, D. et al. Hematology 2009;2009:385-395

20

QUY TRNH XT NGHIM DTPT TRONG AML

Chn on AML

- Mu: 1-2 mL BM / 10 mL PB (blast 10%) trong heparin/ACD - Chuyn n phng XN trong vng 1-2 ting ng h - Nhim sc th - FISH v RT-PCR
PML/RARA, AML1/ETO, CBFB/MYH11 NST bnh thng Gii trnh t chui DNA RQ PCR Bt thng NST khc Lp li NST /FISH theo di iu tr

Lp li RQ-PCR theo di iu tr

t bin gien: Khng c t bin gien FLT3, NPM1, c-KIT CEBPA v WT1 RQ-PCR hoc Flow cytometry 21 theo di iu tr

BT THNG NST V GEN TRONG BCH CU CP DNG LYMPHO

(ACUTE LYMPHOBLASTIC LEUKEMIA: ALL)

Bt Thng NST Trong ALL

Nhm nguy c
Tin lng tt

Bt thng
Hyperdiploidy (>50) Bt thng 12p nh t(12;21)(p13;q22) (TEL/AML1) Near hyperdiploidy (47-50) Near tetraploidy (82-94) S lng NST bnh thng

Bnh cnh lm sng

Trisomy NST 4, 10 v/hoc 17: Tin lng rt tt 10% ca childhood ALL 25% ca B-precusor ALL Ci thin vi phc iu tr mnh T l tht bi vi iu tr cao ~ Near hyperdiploidy

t(9;22)(q32;q11) (BCR/ABL)
Tin lng xu t(4;11)(q21;q23) (MLL/AF4) t(1;19)(q23;p13) (E2A/PBX1) Chuyn v lin quan n c-myc ti 8q24 vi mt Ig ti 14q32, 2p12 hoc 22q11 Bt thng 9p

S lng BC cao, ln tui, thng l nam, v ALL-L2

S lng BC cao v thng < 1 tui Pre-B (cIg+) B-cell ALL hoc Burkitt leukemia. S dng ha tr liu tn cng liu cao dng cho Burkitt lymphoma s ci thin c tin lng xu ca B-cell ALL ~ 10% ca childhood ALL. S lng BC cao, T-cell immunophenotype, v tng kh nng ti pht

A BI TRONG ALL

53,XX,+3,+4,+7,+9,+11,+18,+21

A BI TRONG ALL

72,XXY,+1,+2,+3,+3,+4,+5,+6,+6,+7,+8,+9,+10,+11,+11, +12,+13,+14,+15,+16,+18,+19,+20,+21,+21,+22

Cc ri lon gen trong a bi NST/ALL

Gim biu hin gen m ha asparagine synthetase (men tng hp L-asparagine) Tng biu hin gen SLC19A1 (m ha cht vn chuyn MTX vo tb) trn NST 21 tng tch t ca MTX polyglutamates trong t bo (thng c 3, 4 NST 21/a bi) Tng biu hin ca gen proapoptotic CAS-P8AP2 ti NST 6q15 t bo blast a bi c xu hng tri qua qu trnh cht theo chng trnh t bo mt cch t pht kh r

Gii thch
Vn nhy vi ha tr liu: corticosteroids, mercaptopurine, thioguanine, cytarabine, L-asparaginase (ASP) v methotrexate (MTX)

t(12;21)(p13;q22) TO RA T HP GEN TEL/AML1

a
TEL(12p13) 1a tel 2 3 4 5 6 7 8 cen

Breakpoint region 15 kb 2 3

AML 1(21q22) 1 tel

7a

7b

8 cen

Breakpoint region 150 kb

TIN LNG TT

der(19)t(1;19)(q23;p13) TO RA T HP GEN E2A/PBX1

t(1;19)(q23;p13) TO RA T HP GEN E2A/PBX1

E2A (19p13)
M BN1 BN2 Neg Pos

PBX1 (1q23)
400 bp 373 bp

M: Thang chun BN1, BN2: Bnh nhn Neg: Chng m (nc) Pos: Chng dng

TIN LNG XU E2A PBX1

QUY TRNH XT NGHIM DTPT TRONG ALL

Chn on ALL
- Ly 1-2 mL BM / 10 mL PB (blast 10%) trong heparin/ACD - Chuyn n phng XN trong vng 1-2 ting ng h - Nhim sc th - FISH v RT-PCR

Cy NST khng mc BCR-ABL, MLL-AF4, E2A-PBX1 v TEL-AML1 - NST bnh thng - Bt thng s lng RQ PCR PCR tm IG-TCR

Lp li RQ-PCR theo di iu tr

RQ-PCR: IG-TCR v Flow cytometry theo di iu tr

30

BT THNG NST V GEN TRONG NHNG RI LON TNG SINH TY

(MYELOPROLIFERATIVE NEOPLASMS: MPN)

Phn Loi MPN in Hnh Theo t Bin Gen

BCR/ABL(+) Bch cu mn dng ty (CML)
MPN BCR/ABL(-) a hng cu (95 ~ 97% JAK2V617F) Tng tiu cu nguyn pht (~ 60% JAK2V617F) X ty nguyn pht (~ 60% JAK2V617F)

32

BCH CU MN DNG TY IN HNH C t(9;22)(q34;q11)

CHUYN V NST PHILADELPHIA

No.9

No.22

der(9)

der(22)

BCR gen

Ph chromosome
BCR/ABL gen

t(9;22)(q34;q11)
6.0 kb 7.0 kb

8.5 kb BCR/ABL mRNA P210

BCR/ABL

ABL gen
ABL

ABL/BCR gen ABL/BCR mRNA

P145

Hat tnh tyrosine kinase (-)

Hat tnh tyrosine kinase (+++) 9q34: 22q11:

ABL gen BCR gen

Pht hin bng: - Nhim sc th - FISH

34

t(9;22)(q34;q11)

9q+

22qNST Ph

35

FISH S Dng Vysis BCR/ABL Probe

300 kb

BCR probe

ASS gene-56kb

650 kb

ABL probe 36

CC KIU BN SAO BCR/ABL TRONG CML

Cent minor BCR major BCR
b1
1

Tel

micro BCR
b5 c1 c2 c3
4

BCR gen (22q11)

e1

b2
2

b3
3

b4

c4

c5

c6

c7

e12---13-----14------15----16

e17---18---19----

20----21----22---23

ABL gen (9q34)

#
1b intron 1 1a intron 2

##
a2 a3 a1 1

# splicing exons

Minor BCR/ABL mRNA

2 e1

e1 a2 b2 a2

e1a2 : 198bp

b2a2 : 212bp
b3a2 : 287bp e19a2

Major BCR/ABL mRNA

3 4

e1 b5

b2 b3 a2 e19 a2

e1

b1 b2

Pht hin c bng k thut RT-PCR hoc RQ-PCR

37

BCR/ABL HOT HA CC CON NG TN HIU TRONG CML

Bt thng s kt dch ca t bo
BCR/ABL

c ch s cht theo chng trnh

T bo tng sinh

(Ngun: Lichman MA., 2006 )

IMATINIB C CH C HIU BCR/ABL

Imatinib Mesylate (gleevec, glivec): Vin: 100 mg

Liu: 4 vin/ngy
(Ung/g mn) Theo di: ngoi tr,

BN khng phi
nm vin.

Nhng XN Trong Chn on v Theo Di iu Tr CML

Thm khm v XN
- Khm LS

Ch nh
Lc chn on/TD iu tr Mi 3 thng cho n khi khng cn lch to Nghi ng bnh tin trin hoc khng thuc Lc chn on/TD iu tr Mi 1-2 tun cho n khi H n nh, sau mi 6 tun Nghi ng bnh tin trin hoc khng thuc Lc chn on/TD iu tr 6, 12, 18 thng hoc ti khi lui bnh hon ton (CCR) Nghi ng bnh tin trin hoc khng thuc

-Huyt (H)

- Phn tch NST (BM)

-RQ-PCR (BCR/ABL) -FISH (BCR/ABL)(PB)

Mi 3 thng t lc CCR
Chn on khng chc chn (Ph- hoc cy NST khng mc) Mi 3 thng nu khng c RQ-PCR theo di Chn on khng chc chn (LS in hnh nhng Ph- hoc cy NST khng mc) Nghi ng bnh tin trin hoc khng thuc

-PCR (BCR/ABL) - t bin BCR/ABL kinase

THEO DI P NG IU TR CML
S lng t bo ung th

100 10 1 0,1 0,01 0,001 0,0001

1012
p ng huyt hc Huyt , PMNB

1010 109
p ng DTTB
p ng SHPT

m t l tb NST Ph+ o s copy BCR-ABL

106

Khng pht hin c bn sao BCR-ABL

T l BCR-ABL (International scale)

Khuyn co s dng p ng SHPT s tng tnh chnh xc hn trong nh gi p ng iu tr

THEO DI P NG IU TR BNG K THUT RQ-PCR

Nhng t Bin Ca BCR/ABL kinase domain Lin Quan n Khng Imatinib Trn Lm Sng 30 v tr v 44 loi t bin c pht hin
90 v tr v 112 loi t bin

Gy chuyn cp?
244-255
276-290 294-301 331-352

P loop

C-helix

SH3

SH2
359 Catalytic

379-398

Kinase domain

Activ. loop

Contact residues (Azam M v cng s. Cell 112:831, 2003)

Pht hin c bng k thut gii trnh t chui DNA hoc ASO-PCR

43

T BIN BCR/ABL TRN BN CML KHNG IM

Bnh nhn nam, 39 tui Chn on CML C t(9;22)(q34;q11) v BCR/ABL: b3a2 Imatinib: -400 mg/ngy x 1 thng -800 mg/ngy x 2 thng

Chuyn cp

Ti pht 3 thng sau ghp ty, c iu tr gim bch cu v nng , BN t vong 2 thng sau ti pht

D ghp ty

44

Tng liu imatinib

iu tr nilotinib

Khng matinib

iu tr dasatinib

Khng nilotinib

Th nghim LS vi MK-0457

Khng dasatinib

(Borrow J, West Midlands Regional Genetics Laboratory 2007)

T BIN GEN JAK2V617F

a hng cu (95 ~ 97% JAK2V617F)

Tng tiu cu nguyn pht (~ 60% JAK2V617F)

X ty nguyn pht (~ 60% JAK2V617F)

CU TRC V CHC NNG JAK2 (1)

JAK2: tyrosine kinase, khng phi th th. C vng JH1 (Janus homology domain) v JH2 (2 mt).

Janus

the two faced Roman God of doorways

JH1 c hot tnh kinase. JH2 khng c hot tnh kinase nhng c chc nng iu ha ngc JH1. JAK2 rt quan trng cho s to hng cu.
47

CU TRC V CHC NNG JAK2 (2)

JAK2 lun gn vi th th ca erythropoietin (EPOR) Nu EPO gn vi EPOR, phn ng bt cp ca th th xy ra v JAK2 c hot ha. t bin JAK2V617F (nm trn JH2) lm mt chc nng iu ha ngc ca JH2 i vi JH1 v gia tng tnh p ng vi EPO. Khi c t bin JAK2V617F, tn hiu c truyn i sinh hng cu m khng cn hin din EPO.
Tng sinh Bit ha Sinh ung th

EPOR

EPO

48

CC KT PHT HIN T BIN JAK2V617F

T l pht hin JAK2V617F ty thuc vo k thut khc

nhau:
- Gii trnh t DNA (direct sequencing): Thp hn - Allele Specific Oligonucleotide-PCR (ASO-PCR): Cao hn

Ti Vit Nam, s dng k thut ASO-PCR:

- a hng cu nguyn pht: 98% (kho st 51 BN)
- Tng tiu cu nguyn pht: 59% (kho st 122 BN)

49

KT QU KHO ST T BIN JAK2V617F

ASO-PCR Gii trnh t DNA

T BIN GEN TRONG MPN VI BCR/ABL(-)

PV: a hng cu nguyn pht ET: Tng tiu cu nguyn pht MF: X ty nguyn pht

Cross NCP, American Society of Hematology 2011

51

BT THNG NST V GEN TRONG LYMPHOMA

BT THNG NST CA NON-HODGKIN LYMPHOMA

Tng sinh t bo B (chim khong 88%):

- Diffuse large B cell lymphomas (30%)

- Follicular lymphoma (25%) - Mucosa-associated lymphatic tissue (MALT) lymphoma (7.5%) - Mantle cell lymphoma (6.0%)

Bt thng NST thng gp lin quan 14q32 (IGH), 3q27 (BCL6),

11q13 (BCL1), 18q21.3 (BCL2)

Tng sinh t bo T (khong 12% )

Bt thng NST thng gp lin quan 14q11.2 (T cell receptor chain:TCRA), 7q34 (TCRB), 7p14 (TCRG), 2q23 (ALK)
53

BT THNG NST CA NON-HODGKIN LYMPHOMA

Bt thng NST
t(14;18)(q32;q21) t(3;22)(q27;q11.2), t(3;14)(q27;q32) t(2;3)(p12;q27) 3q27 c gien BCL6

Non-Hodgkin Lymphoma
70-90% follicular small cleaved cell 40% trng hp DLBCLs 10% follicular lymphoma

Small noncleaved cell

t(8;14)(q24;q32) v diffuse large B cell (DLBCLs) Hu ht cc trng hp mantle cell 54

t(11;14)(q13;q32)

BT THNG NST V GEN TRONG A U TY

BT THNG NST CA A U TY LC CHN ON

a u ty: ri lon tng sinh c tnh dng tng bo Bt thng NST gp trong khong 85% lc chn on:

Phn nhm nguy c

Nguy c chun

Nguy c chun

Ngoi ra: - del(17)(p13): tin lng xu - del(13q), del(1p)

American Society of Hematology 2012

56

HNG NGHIN CU IU TR TRNG CH PHN T TRONG A U TY

American Society of Hematology 2012

57

CC IM CN LU
1. 2. 3. 4. 5. 6. Tm quan trng ca SHPT trong huyt hc Phn nhm nguy c trong bnh bch cu cp dng ty (AML) Tin b trong iu tr AML-M3 Phn nhm nguy c trong bch cu cp dng lympho (ALL) t bin gen JAK2V617F: gp trong bnh no? K thut pht hin? Nhim sc th Philadelphia, cc kiu bn sao BCR/ABL v k thut pht hin. 7. 8. 9. Vn theo di p ng iu tr trong CML c im t bin gen BCR/ABL gy khng imatinib, k thut pht hin. Bt thng NST trong lymphoma

10. Bt thng nhim sc th trong a u ty