Outline. Chromosomal analysis FISH. Chromosomal abnormalities in cancer. Clinical application of cytogenetics. Procedure Nomenclature

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2 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 - Cytogenetics and FISH in Cancer Diagnostics -

3 Procedure of chromosomal analysis Specimen Culture Hour culture High-resolution (MTX) Mitogen-add culture Direct Mitotic inhibitor Harvest Analysis of metaphases Hypotonic solution Stain (G-banding) Fixative solution Slide making

4 Karyotyping

5 Metaphase cells of bone marrow Malignant cells + Normal BM cells Poor quality of metaphase

6 Clone 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 - Cytogenetics and FISH in Cancer Diagnostics -

7 Nomenclature ISCN 2005 (International System for Human Cytogenetic Nomenclature) Dark band Light band p (short arm) centromere q (long arm) deletion del(7)(q22) - Cytogenetics and FISH in Cancer Diagnostics -

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]// - Cytogenetics and FISH in Cancer Diagnostics -

9 Nomenclature Stemline, sideline, clonal evolution Cytogenetically related clones (subclones) In order of increasing complexity 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 sideline 3rd sideline 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) - Cytogenetics and FISH in Cancer Diagnostics -

10 Procedure of fluorescence in situ hybridization (FISH) Probes Labelling with fluorochrome DNA fragments for target DNA Fluorescence In Situ Hybridization Probes Specimen slide Target DNA Detection under fluorescence microscope

11 FISH probes Metaphase Chromosomes Repetitive sequence probe Centromere Telomere Painting probe Unique sequence / Locus specific probe Interphase Nuclei

12 Application of FISH probes Repetitive sequence Probes Aneuploidy detection Origin of marker chromosome Origin of additional material Translocations Painting Probes Complex rearrangements Cryptic rearrangements Microdeletion Locus specific Probes Microduplication Gene rearrangements Gene amplification Breakpoint mapping

13 Design of locus specific probes for gene rearrangements A gene Dual color Single fusion A gene Dual color Dual fusion B gene B gene A B der(a) der(b) A gene Dual color Extra signal A gene Dual color Break apart B gene? gene A?

14 BCR/ABL Dual color Dual fusion Translocation Probe 9q34 region ASS gene ABL gene ~ 650 kb LSI ABL 22q11.2 region BCR gene ~ 1500 kb LSI BCR

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 BCR/ABL Dual color Extra signal (ES) Translocation Probe 9q34 region ASS gene ABL gene ~ 650 kb LSI ABL m-bcr 22q11.2 region BCR gene M-bcr ~ 300 kb LSI BCR

17 ALL 46,XX,t(9;22)(q34;q11.2)[20] nuc ish(ablx3),(bcrx3),(abl con BCLx2)[385/400] minor BCR/ABL rearrangement (BCR/ABL ES dual color translocation probe, Vysis)

18 FISH probes for Cancer diagnostics AML ALL AMLL CML MDS/AA CLL Myeloma Lymphoma Solid tumor BCR/ABL1, AML1/ETO, PML/RARA, RARA, MLL, CBFB, Tri 8, EGR1(5q31), D7S486(7q31), D20S108(20q12) BCR/ABL1, TEL/AML1, MLL, P16(9p21), IGH/MYC, MYC BCR/ABL1, MLL BCR/ABL1, HER2/CEP17, TP53 EGR1(5q31), D7S486(7q31), D20S108(20q12), Tri 1q(1q25), Tri 8 TP53, ATM, Tri 12, RB1, D13S25(13q14) IGH/FGFR3, IGH/MAF, TP53, RB1, IGH/CCND1, IGH IGH/MYC, MYC(8q24), IGH(14q32), IGH/CCND1, IGH/MALT1, API2/MALT1, MALT1, IGH/BCL2, BCL6(3q27), ALK(2p23) MYCN(2p24.3), HER2(17q21.1), EWSR1(22q12), SYT(18q11.2), CHOP(12q13), FKHR(13q14), 1p36, PTEN, EGFR, CCND1, ZNF217

19 XY Chimerism Post-PBSCT ( ) of relasped common cell ALL nuc ish(dxz1,dyz3)x1[276]//(dxz1x2)[124] % cells with XY : 69 %

20 Multicolor FISH (SKY, M-FISH) Probe cocktail of 24 chromosomes 1-2 Mb resolution Powerful tool for Complex rearrangements in cancer cells Cryptic rearrangements Marker chromosome

21 Comparative genomic hybridization (CGH) Metaphase CGH vs Array CGH Metaphase CGH Array CGH Copy number change!

22 Cytogenetic analysis and array CGH Techniques Resolution Unbal. Balan. Marker Mosaicism Disease Rearr. Rearr. Chr. (%) Monitoring G-banding 5-8Mb + + Limited 1 + LS-FISH 0.5kb M-FISH/SKY 2-3Mb +/ Meta. CGH 3-10Mb Limited Array CGH 1kb-1Mb + -/ Limited

23 Chronic myelogenous leukemia Chronic phase 5 BCR 3 ABL +Ph,+8,i(17q), t(3;21),+19,+21 Blast crisis t(9;22) : 92% t(9;22;v) : 6% Cryptic : 2% FISH Chromosomal analysis Treatment Quantitation (FISH,PCR) - Cytogenetics and FISH in Cancer Diagnostics -

24 CML, Glivec treatment over 1 year nuc ish(ablx2),(bcrx2),(abl con BCLx1)[85/400] % cells with BCR/ABL rearrangement and ABL1/BCR deletion on der(9) : 21.25% (BCR/ABL dual color dual fusion translocation probe, Vysis)?

25 CML, Glivec treatment nuc ish(ablx3),(bcrx2),(abl con BCLx1)[145/400] % cells with BCR/ABL rearrangement and 3 BCR deletion on der(9) : 36.2% (BCR/ABL dual color dual fusion translocation probe, Vysis)

26 Chronic myeloproliferative disorder Idiopathic myelofibrosis 60% P.Vera 15% 40% 100% del(20q), del(13q), +8, +9, -7/del(7q), del(5q), del(11q) Essential thrombocythemia 5-10% Clonal evolution AML - Cytogenetics and FISH in Cancer Diagnostics -

27 Myelodysplastic syndrome RARS 20% RA 25% RCMD 50% RAEB 50-70% +8, -5/del(5q), -7/del(7q), del(20q), -Y, complex tri 1q Clonal evolution AML - Cytogenetics and FISH in Cancer Diagnostics -

28 Chromosomal abnormalities according to Morphological & immunological charact. in Acute leukemias 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) Hematopoietic t(5;12) : M0 stem cell Pre Pre-B ALL Common ALL Pre-B ALL B-cell ALL Lymphoid stem cell 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) Early thymocyte ALL Common T ALL Mature T ALL 9p abn. t or del 9p t(10;14) t(11;14) t or del 14q11 t(8;14)(q24;q11)

29 Acute myelogenous leukemia - Cytogenetics and FISH in Cancer Diagnostics -

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 - Cytogenetics and FISH in Cancer Diagnostics -

34 Acute lymphoblastic leukemia - Cytogenetics and FISH in Cancer Diagnostics -

35 Acute lymphoblastic leukemia Adult Childhood (J Clin Oncol 2005;23:6306) - Cytogenetics and FISH in Cancer Diagnostics -

36 t(9;22)(q34;q11.2) % BCR-ABL rearrangement >70 Age (years) Fig. Frequency of BCR-ABL rearrangement in adult ALL according to age distribution (P <0.001) (data from Asan Medical Center)

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

38 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) Common ALL, cryptic translocation of t(12;21) (TEL/AML1 Dual color Extra signal translocation probe, TEL-Green/AML1-Red ES, Vysis)

39 B-CLL Karyotyping < 50% FISH > 80% del(13q/rb1) del(11q/atm) trisomy 12 del(17p/tp53) del(6q) - Cytogenetics and FISH in Cancer Diagnostics -

40 Multiple myeloma IGH translocation 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) Dysregulation of Cyclin D1, D2, D3 Myeloma Monosomy 13/del(13q) Del(17p) RB1 TP53 MYC PTEN RAS

41 Multiple myeloma Karyotyping 30-50% 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 FISH 80-90% Non-deviding myeloma cells Immuno-FISH Acurrate Quantitation Retrospective analysis - Cytogenetics and FISH in Cancer Diagnostics -

42 Karyotype ( ) : 52,XX,+3,+4,+6,+add(8)(q13),+9,-13,+15,+19[2]/46,XX[18] - Cytogenetics and FISH in Cancer Diagnostics -

43 Karyotype ( ) : 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] - Cytogenetics and FISH in Cancer Diagnostics -

44 FGFR3 RB1 (13q14) probe IGH Fusion of IGH/FGFR3 IGH(14q32, Green) & FGFR3 (4p16.3, Orange) probe FISH (2006-MM-2) : nuc ish(fgfr3x3),(ighx3),(fgfr3 con IGHx2)[359/400],(RB1x1)[336/400]

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 Clinical implications of chromosomal abnormalities in MM 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

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 - Cytogenetics and FISH in Cancer Diagnostics -

48 Chromosomal abnormalities in lymphoma (J Mol Diagn 2006;8:141)

49 Karyotype ( ) : 46,XX,t(3;14)(q27;q32)[13]/46,XX[2] - Cytogenetics and FISH in Cancer Diagnostics -

50 Karyotype ( ) : 46,XY,dup(1)(q21q32),t(8;14)(q24;q32)[20] - Cytogenetics and FISH in Cancer Diagnostics -

51 MYC CEP8 Fusion of IGH/MYC IGH CEP8 IGH(14q32, Green), MYC (8q24, Orange), CEP8 (Aqua) probe Burkitt lymphoma FISH (2006-IGH/MYC/CEP8-1) : nuc ish(cep8x2),(mycx3),(ighx3),(myc con IGHx2)[342/400] IGH/MYC gene rearrangement

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. 혈액종양의진단, 분류, 예후추정 만성골수성백혈병, 급성백혈병, 골수형성이상증후군, 골수증식질환 기타혈액종양 : 다발성골수종, 만성림프세포증식질환 기타혈액질환 : 재생불량성빈혈 2. 악성종양세포의골수침윤이의심되는경우 림프종, 고형종양 3. 혈액종양의치료경과추적 항암치료후관해상태판정 골수이식 ( 조혈모세포이식 ) 후 글리벡등 target therapy 후반응평가 4. 혈액종양의진행 (clonal evolution), 재발 5. 림프종, 고형종양조직 - Cytogenetics and FISH in Cancer Diagnostics -

54 FISH in Cancer 1. 염색체의수적, 구조적이상확인 유전자재배열 유전자 / 염색체결실 2. Marker 염색체의유래를확인 3. 유전자증폭을확인 4. 치료경과추적및최소잔존질병검색 5. 골수이식후키메리즘판정 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 Accurate, sensitive, quantitation - Cytogenetics and FISH in Cancer Diagnostics -

55 More Colorful, Brighter Whole-genome approach Target-specific approach Accurate diagnosis Risk stratification Targeted therapy Monitoring