% BCR-ABL rearrangement

(1)

(2)

- Cytogenetics and FISH in Cancer Diagnostics -

Outline

• Chromosomal analysis

– Procedure

– Nomenclature

• FISH

– Procedure – Probes

– Multicolor-FISH – CGH

• Chromosomal abnormalities in cancer

– CML, MPD, MDS, AML, ALL, CLL, myeloma, lymphoma

• Clinical application of cytogenetics

(3)

Analysis of metaphases

Stain (G-banding)

Slide making Fixative solution

Hypotonic solution

Mitotic inhibitor

Culture

Harvest

Specimen 24-72 Hour culture

High-resolution (MTX) Mitogen-add culture

Direct

Procedure of chromosomal analysis

(4)

Karyotyping

(5)

Malignant cells + Normal BM cells Poor quality of metaphase

Metaphase cells of bone marrow

(6)

Clone

A cell population derived from a single progenitor

The same structural rearrangement : 2 metaphase cells The same chromosomal gain : 2 metaphase cells

The same chromosomal loss : 3 metaphase cells

(7)

Nomenclature

ISCN 2005

(International System for Human Cytogenetic Nomenclature)

centromere p (short arm)

q (long arm) Dark band

Light band

deletion

del(7)(q22)

(8)

Nomenclature

• Clone size [ ]

– 46,XY[20]

– 46,X,-Y,t(8;21)(q22;q22),+mar[20]

• Different clones (/)

– 46,XX,t(9;22)(q34;q11.2)[17]/46,XX[3]

• Chimerism secondary to BMT

– 46,XY[3]//46,XX[17]

– 46,XY,t(9;22)(q34;q11.2)[4]//46,XX[16]

– //46,XX[20]

– 46,XY[20]//

(9)

Nomenclature

• Stemline, sideline, clonal evolution

– Cytogenetically related clones (subclones) – In order of increasing complexity

46,XY,t(9;22)(q34;q11.2)[3]/92,slx2[5]/93,sdl1,+8[2]

48,XX,t(12;16)(q13;p11.1),t(14;19)(q23;p11),+17,-19,+20,+21[32]

/49,idem,+6[17]

idem = same (Latin)

46,XX,t(9;22)(q34;q11.2)[3]/47,sl,+8[17]/48,sdl1,+9[3]/49,sdl2,+11[12]

stemline (sl)

1^st sideline (sdl)

2^nd sideline3^rd sideline

(10)

DNA fragments for target DNA Labelling with fluorochrome

Fluorescence In Situ Hybridization

Target DNA Probes

Detection under fluorescence microscope Probes

Specimen slide

Procedure of fluorescence in situ hybridization

(FISH)

(11)

Metaphase Chromosomes

Interphase Nuclei

Centromere

Repetitive sequence probe

Telomere

Painting probe

Unique sequence / Locus specific

probe

FISH probes

(12)

Application of FISH probes

Aneuploidy detection

Origin of marker chromosome

Translocations

Complex rearrangements

Microdeletion Microduplication

Gene amplification Breakpoint mapping

Origin of additional material

Repetitive sequence

Probes

Painting Probes

Locus specific

Probes

Cryptic rearrangements

Gene rearrangements

(13)

Dual color Single fusion

Design of locus specific probes for gene rearrangements

Dual color Dual fusion

A gene B gene

Dual color Extra signal Dual color Break apart

A gene B gene A gene

B gene

A B

der(A) der(B)

A gene

? gene

A ?

(14)

9q34 region

LSI ABL

~ 650 kb

ASS gene ABL gene

BCR/ABL Dual color Dual fusion Translocation Probe

LSI BCR

~ 1500 kb BCR gene

22q11.2 region

(15)

CML, Glivec treatment for 1 month

nuc ish(ABLx3),(BCRx3),(ABL con BCLx2)[280/400]

% cells with BCR/ABL rearrangement : 70%

(BCR/ABL dual color dual fusion translocation probe, Vysis)

(16)

LSI ABL

~ 650 kb

ASS gene ABL gene

9q34 region

LSI BCR

~ 300 kb

BCR gene

22q11.2 region

m-bcr M-bcr

BCR/ABL Dual color Extra signal (ES)

Translocation Probe

(17)

ALL

46,XX,t(9;22)(q34;q11.2)[20]

minor BCR/ABL rearrangement

(BCR/ABL ES dual color translocation probe, Vysis)

(18)

FISH probes for Cancer diagnostics

IGH/MYC, MYC(8q24), IGH(14q32), IGH/CCND1, IGH/MALT1, API2/MALT1, MALT1, IGH/BCL2, BCL6(3q27), ALK(2p23) Lymphoma

BCR/ABL1, TEL/AML1, MLL, P16(9p21), IGH/MYC, MYC ALL

MYCN(2p24.3), HER2(17q21.1), EWSR1(22q12),

SYT(18q11.2), CHOP(12q13), FKHR(13q14), 1p36, PTEN, EGFR, CCND1, ZNF217

Solid tumor

IGH/FGFR3, IGH/MAF, TP53, RB1, IGH/CCND1, IGH Myeloma

TP53, ATM, Tri 12, RB1, D13S25(13q14) CLL

EGR1(5q31), D7S486(7q31), D20S108(20q12), Tri 1q(1q25), Tri 8

MDS/AA

BCR/ABL1, HER2/CEP17, TP53 CML

BCR/ABL1, MLL AMLL

BCR/ABL1, AML1/ETO, PML/RARA, RARA, MLL, CBFB, Tri 8, EGR1(5q31), D7S486(7q31), D20S108(20q12)

AML

(19)

XY Chimerism

Post-PBSCT (2005.1.11) of relasped common cell ALL nuc ish(DXZ1,DYZ3)x1[276]//(DXZ1x2)[124]

% cells with XY : 69 %

(20)

• Probe cocktail of 24 chromosomes

• 1-2 Mb resolution

• Powerful tool for

– Complex rearrangements in cancer cells

– Cryptic rearrangements – Marker chromosome

Multicolor FISH (SKY, M-FISH)

(21)

Comparative genomic hybridization (CGH) Metaphase CGH vs Array CGH

Metaphase CGH Array CGH

Copy number change !

(22)

Techniques Resolution Unbal. Balan. Marker Mosaicism Disease Rearr. Rearr. Chr. (%) Monitoring G-banding 5-8Mb + + Limited 1 +

LS-FISH 0.5kb + + + 1 +

M-FISH/SKY 2-3Mb +/- + + 10 +

Meta. CGH 3-10Mb + - + - Limited

Array CGH 1kb-1Mb + -/+ + 30 Limited

Cytogenetic analysis and array CGH

(23)

Chronic myelogenous leukemia

5’BCR 3’ABL

Chronic phase

Quantitation (FISH,PCR)

+Ph,+8,i(17q), t(3;21),+19,+21

Blast crisis

Chromosomal analysis

t(9;22) : 92%

t(9;22;v) : 6%

Cryptic : 2%

FISH

Treatment

(24)

CML, Glivec treatment over 1 year

% cells with BCR/ABL rearrangement and ABL1/BCR deletion on der(9) : 21.25%

?

(25)

CML, Glivec treatment

% cells with BCR/ABL rearrangement and 3’BCR deletion on der(9) : 36.2%

(26)

Chronic myeloproliferative disorder

del(20q), del(13q), +8, +9, -7/del(7q),

del(5q), del(11q) P.Vera

15%→40% → 100%

Idiopathic myelofibrosis

60%

Essential

thrombocythemia 5-10%

Clonal evolution AML

(27)

Myelodysplastic syndrome

+8, -5/del(5q), -7/del(7q), del(20q), -Y, complex tri 1q

Clonal evolution RA

25%

RCMD 50%

RAEB 50-70%

RARS 20%

AML

(28)

Chromosomal abnormalities according to

Morphological & immunological charact. in Acute leukemias

Pre Pre-B ALL Common ALL Pre-B ALL B-cell ALL

Lymphoid stem cell

Early thymocyte ALL Common T ALL Mature T ALL t(4;11)

t(9;22) t(11;19)

t(9;22) t(12;21) Hyperdiploidy

del(12p) del(6q)

t(1;19) t(9;22) t(12;21)

t(8;14) t(2;8) t(8;22)

9p abn. t or del 9p

t(10;14) t(11;14)

t or del 14q11 t(8;14)(q24;q11) Myeloid

stem cell AML t(8;21) : M2 -5

t(15;17) : M3 del(5q) Inv(16) : M4e -7

t(9;11) : M4/5 del(7q) t(6;9) : M1 +8 t(9;22) : M0 del(9q) t(5;12) : M0

Hematopoietic stem cell

(29)

Acute myelogenous leukemia

(30)

Cytogenetic classification systems in the 3 major collaborative cytogenetic studies of adult AML

(J Clin Oncol 2005;23:6285)

(31)

AML-M1 with normal karyotype

46,XY[25].ish ins(8;21)(q22;q22q22)(ETO+,AML1+;AML1+)[5]

nuc ish(ETOx2),(AML1x3),(ETO con ML1x1)[370/400]

(AML1/ETO dual color dual fusion translocation probe, Vysis)

(32)

AML

Karyotype : 48,XX,-5,+11,-18,+3mar[16]/46,XX[4]

FISH : Amplification of MLL gene (MLL probe, Vysis)

(33)

AML de novo vs t-AML/t-MDS

(34)

Acute lymphoblastic leukemia

(35)

Acute lymphoblastic leukemia

(J Clin Oncol 2005;23:6306)

Adult Childhood

(36)

100 80 60 40 20

0 15-20 21-30 31-40 41-50 51-60 61-70 >70

Age (years)

% BCR-ABL rearrangement

Fig. Frequency of BCR-ABL rearrangement in adult ALL according to age distribution (P <0.001)

(data from Asan Medical Center)

t(9;22)(q34;q11.2)

(37)

Childhood ALL

Karyotype : 57,XY,+X,+4,+5,+6,+8,+10,+14,+17,+18,+21,+21

(38)

Common ALL, cryptic translocation of t(12;21)

(TEL/AML1 Dual color Extra signal translocation probe, TEL-Green/AML1-Red ES, Vysis)

Normal

Typical TEL/AML1

rearrangement.

(1F 1G 2R)

TEL/AML1 rearrangement

Deletion of non-translocated TEL allele (1F 2R)

TEL/AML1 rearrangement Gain of AML1 gene

(1F 1G 3R)

(39)

B-CLL

Karyotyping

< 50%

del(13q/RB1) del(11q/ATM)

trisomy 12 del(17p/TP53)

del(6q)

FISH

> 80%

(40)

Dysregulation of Cyclin D1, D2, D3

IGH translocation

Myeloma

TP53RB1 PTENMYC

RAS

Multiple myeloma

t(11;14)(q13;q32), CCND1/BCL1 t(4;14)(p16;q32), FGFR3

t(14;16)(q32;q23), MAF t(6;14)(p21;q32), CCND3

MUM2 (1q21), MYC (8q24), PAX (9p13), BCL7A (12q24), BCL2 (18q21), IRF4 (6p25), MAFB (20q12)

Monosomy 13/del(13q)

Del(17p)

(41)

Multiple myeloma

Karyotyping 30-50%

FISH 80-90%

Chromosomal abnormalities (+) Grade of BM infiltration

Extend of lytic bone lesions Plasma cell labelling index Higher progression rate Shorter EFS

Significantly lower rate of CR (27% vs 48%)

Complex clonal aberrations advanced stages

elevated β2-microglobuline

Non-deviding myeloma cells

Immuno-FISH Acurrate

Quantitation

Retrospective analysis

(42)

Karyotype (2006-903) :

52,XX,+3,+4,+6,+add(8)(q13),+9,-13,+15,+19[2]/46,XX[18]

(43)

Karyotype (2006-1024) :

47,X,-X,ins(1;?)(p22.1;?),i(1)(q10),t(4;10)(p16;q22),t(11;14)(q13;q32),der(16)t(1;16)(q11;q24), +21,+mar[4]/46,XX[16]

(44)

FISH (2006-MM-2) :

nuc ish(FGFR3x3),(IGHx3),(FGFR3 con IGHx2)[359/400],(RB1x1)[336/400]

RB1 (13q14) probe

IGH(14q32, Green) & FGFR3 (4p16.3, Orange) probe Fusion of IGH/FGFR3

IGH

FGFR3

(45)

• Relationship of patient survival and chromosome anomalies

detected in metaphase and/or interphase cells at diagnosis of

myeloma (Dewald GW et al, Blood 2005;106:3553)

(46)

Cytogenetic analysis : mandatory Conventional cytogenetics and FISH

to guide therapeutic strategies and prognostic stratification

High risk prognostic group

del(13q), t(4;14), t(14;16), del(17p)

More investigational therapy High-dose melphalan, ASCT

“Tailor medicine”

MMSET/FGFR3 inhibitor : CHIR258 Cyclin D1 inhibitor : flavopyridol Thalidomide, lenalidomide, bortezomib

Melphalan-based, ASCT

(+) (-)

Clinical implications of

chromosomal abnormalities in MM

(47)

Chromosomal abnormalities in lymphoma

• Conventional cytogenetics : “gold standard”, but in lymphoma

– Lack in availability of fresh material

– Low mitotic index and/or percentage of neoplastic cells – Cytogenetic complexity

– Technically more demanding

• PCR

– limited when chromosomal breakpoints are spread over a large genomic region, t(11;14)

– t(14;18) and t(2;5) : found in healthy individuals, false (+)

• FISH

– Lymphoma-associated translocations – Paraffin-embedded tissue

(48)

(J Mol Diagn 2006;8:141)

Chromosomal abnormalities in lymphoma

(49)

Karyotype (2006-855) : 46,XX,t(3;14)(q27;q32)[13]/46,XX[2]

(50)

Karyotype (2006-855) : 46,XY,dup(1)(q21q32),t(8;14)(q24;q32)[20]

(51)

Burkitt lymphoma

FISH (2006-IGH/MYC/CEP8-1) :

nuc ish(CEP8x2),(MYCx3),(IGHx3),(MYC con IGHx2)[342/400]

IGH/MYC gene rearrangement

IGH(14q32, Green), MYC (8q24, Orange), CEP8 (Aqua) probe Fusion of IGH/MYC

IGH MYC

CEP8 CEP8

(52)

Low-grade MALT lymphoma

nuc ish (API2x3)(MALT1x3)(API2 con MALT1x2) API2/MALT1 gene rearrangement

(API2/MALT1, dual color dual fusion translocation probe, Vysis)

(53)

Chromosomal analysis in Cancer

1.

혈액종양의 진단 , 분류, 예후 추정

– 만성골수성백혈병, 급성백혈병, 골수형성이상증후군, 골수증식질환 – 기타 혈액종양 : 다발성골수종, 만성림프세포증식질환

– 기타 혈액질환 : 재생불량성빈혈

2.

악성종양세포의 골수 침윤이 의심되는 경우

– 림프종, 고형종양

3.

혈액종양의 치료 경과 추적

– 항암치료 후 관해상태 판정 – 골수이식(조혈모세포이식) 후

– 글리벡 등 target therapy 후 반응 평가

4.

혈액종양의 진행 (clonal evolution), 재발

5.

림프종 , 고형종양 조직

(54)

FISH in Cancer

1. 염색체의 수적, 구조적 이상 확인

• 유전자 재배열

• 유전자/염색체 결실

2. Marker 염색체의 유래를 확인 3. 유전자 증폭을 확인

4. 치료경과 추적 및 최소잔존질병 검색 5. 골수이식 후 키메리즘 판정

Any specimen types : BM, blood, body fluid, biopsy, swab, Smear slide, cytospin, paraffin-embedded

→ Retrospective analysis

Low mitotic index and/or percentage of neoplastic cells Inadequate speciem for chromosomal analysis or failure