Back to Basic

Cytogenetic Studies for Hematologic Malignancies in Korea

Introduction to Cytogenetic Studies External QA in Hematologic Malignancies Clinical Aspects of Molecular Cytogenetics: FISH, Array CGH

Back to Basic혈액종양 관련 세포유전학 역사

1960 Philadelphia chromosome (Ph)

1970 Banding technique1973 t(8;21) in AML-M2

t(8;14) in Burkitt lymphoma/Leukemia1977 t(15;17) in AML-M3, t(4;11) 1979 High-resolution banding technique

19801982 t(9;11) in AML-M5a, inv(3) in AML1983 inv(16) in AML-M4E 1984 t(1;19) in ALL

t(1;3) in AML with dysmegakaryopoiesis

1990 Molecular cytogenetics (FISH, CGH)

Back to Basic

1980 1983 MYC-IGH t(8;14) 1984 ABL-BCR t(9;22), IGH-BCL2 t(14;18)1988 ATRA in APL Tx

1990 1991 MLL t(4;11), E2A-PBX1 t(1;19)

PML-RARA t(15;17), AML1-ETO t(8;21) DEK-CAN t(6;9)

1993 CBFB-MYH11 inv(16), MLL-AF9 t(9;11)1997 TEL-AML1 t(12;22)

Cytogenetic & Gene Rearrangement

혈액종양 관련 세포유전학 역사

Back to BasicKimSW, et al.: Banding patterns of normal human chromosomes. The Seoul Journal of Medicine 21(2):133-137(1980).

조혈기질환에 있어서 골수세포 염색체 분석에 관한 연구 . 대한혈액학회지 23(2),1988

Karyotype No.

t(22q;9q) 7

22q- 622q-. -9 149,XXX,+9,+21 1Normal 6

21

Table 4. Karyotypic pattern in cases with CML

Back to Basic

RPMI 1640FBS (15%)Pen-StrepL-GlutaminePHAPB/BM cells

MTX(10-7M)

Thymidine (10-5M)

1) PB/BM Culture

3-5 hrs 17 hrs

2) Harvest5 hrs1) Colcemid (50 g/ml) treatment

2) Hypotonic Solution (KCl;0.075M) treatment3) Fixation (methanol:Acetic acid=3:1)

3) Slide preparation & Staining (Giemsa-Trypsin)4) Microscopy and Karyotyping 5) Printing (Photography) and Reporting

Giemsa Banding (G-Banding) TechniqueGiemsa Banding (G-Banding) Technique

72 hrs

High-resolution Banding TechniqueHigh-resolution Banding TechniqueUsing Methotrexate Cell SynchronizationUsing Methotrexate Cell Synchronization

Back to BasicComputerized Image Analyzer Cytovision®

CCD camera

Main ProgramMicroscope

Monitor PC Printer

Back to BasicConventional Cytogenetics

in Hematologic Malignancies

검사자 숙련도에 따라 슬라이드의 질적 차이가 많다분열세포 적고 , 염색체 길이 짧고 , quality 는 불량하

다 . 복잡하고 다양한 핵형을 보이는 경우가 많다

Back to Basic

Back to BasicISCN 2005

An International System for Human Cytogenetic Nomenclature (2005)

The complete citation for reference lists is:

ISCN (2005):An International System for Human Cytogenetic Nomenclature, Schaffer L.G., Tommerup N. (eds);S. Karger, Basel, 2005

ISCN (1985, 1991, 1995, 2005)

21. F. Mitelman, An International System for Human Cytogenetic Nomenclature (S. Karger, Basel, Switzerland, 1995).

Morphology (A) and karyotyping (B) of interspecies blastocysts derived from a human cord fibroblast transfer into enucleatedbovine oocyte. An interspecies blastocyst at hatching were obtained 144 hours after culture and subsequently provided forchromosome analysis using a cytovision. Karyotyping shows 46 pairs of autosome and sex chromosome of XY.

A.K. Tarkowski and J. Rossant, Haploid mouse blastocysts developed from bisected zygotes. Nature 259 (1976), pp. 663–665

Back to Basic

Philadelphia Chromosome (=Ph) der(22)t(9;22)(q34.1;q11.21)

q34ABL

q11.2BCR

Ph (formerly Ph1) may be used in text, but not in the description of the karyotype, where der(22)t(9;22)(q34;q11.2) is recommended.

Back to Basic

50,XX,+8,t(9;22)(q34;q11.2),+10,+19,+der(22)t(9;22)(q34.1;q11.21)

CML Blast Crisis - Karyotype

Back to BasicPractical Use of Cytogenetics in CML

Advantages of Cytogentics, compared to molecular DNA studies for BCR gene rearrangement

Distinguish betwn variant Ph and standard t(9;22)Dectect other abnormalities : +8, i(17q),+Ph,+19Predict or confirm blast crisisGive information regarding percentage of normal vs abn

ormal cellsValuable after BMT to follow engraftment of the donor c

ells and identify possible relapse

Back to BasicAML-M2 with t(8;21)(q22;q22)

Usually AML-M2, occasionally M1 & M4Young individuals with good remission rateBlasts containing a single thin Auer rodRT-PCR : AML1/ETO fusion transcript

Back to BasicAML-M3 with t(15;17)(q22;q21)

The most specific clinical association in AMLVariants : t(15;Var;17), t(11;17), t(5;17)RT-PCR : PML/RARA fusion transcriptResponsive to all trans-retinoic acid treatment

15 t(15;17) 17

PML RARA

PML/RARA

Back to BasicAML-M4Eo with inv(16)

Young patients, organomegaly, abnormal eosinophilsSpecifically associated with M4Eo in over 50% of casesFavorable prognosis, High incidence of CNS relapse

MYH11

CBFB CBFB-MYH11

Back to BasicAcute Myeloid Leukemias

AML with recurrent cytogenetic translocations t(8;21), AML1(CBF)/ETO t(15;17), PML/RAR inv(16), CBF/MYH11 11q23(MLL) abnormalities

AML with multilineage dysplasia AML and MDS, therapy related AML not otherwise categorized

Proposed WHO Classification

Back to BasicNormal ChromosomeNormal Chromosome

del 5qdel 5q-7, del 7q-7, del 7qdel 9qdel 9qdel 20qdel 20q+8+8complex defectscomplex defectst(1;3), t(2;11)t(1;3), t(2;11)

t(3;3), inv(3)t(3;3), inv(3)t(6;9), inv(16)t(6;9), inv(16)t(8;21)t(8;21)t(9;22)t(9;22)t(v;11)t(v;11)t(15;17)t(15;17)

MDSMDS

AMLAML

Back to Basic

Myeloid Malignancy Secondary to Radiotherapy or

Chemotherapy

Clinical featuresAML-M1,M2,M6Lower remission rate and long-term reponse

CytogeneticsUsually absence of t(8;21), t(15;17) & inv(16)

Usually complex karyotype Common abnormalities:

-5, del(5q) in chemotherapy, -7, del(7q) in radiation therapy,

Abnormalities of 3p, 11q23, 12p, and 17

Back to BasicCytogenetics in Acute Lymphoblastic Leukemia

Poorly spread, shortFuzzy chromosomesIndistinct bands

Low success rate 50% in conventional culture76% Clarkson (1985)

Back to BasicPloidy Groups in Childhood ALLPloidy Groups in Childhood ALL

Hyperdiploid >50 28%Early pre-B immunophenotypeFound at the age of 2-10 yearsLower WBC count, Favorable prognosis

Hyperdiploid 47-49 13% Diploid 46 9% Pseudodiploid 46 38% Hypodiploid <46 6%

Back to BasicLymphoid Neoplasm

B-Cell NeoplasmPrecursor B-lymphoblastic leukemia/lymphoma

t(9;22), BCR/ABL t(v;11), MLL rearranged t(1;19), PBX/E2A t(12;21), TEL/AML1

Mature B-cell neoplasm

T-Cell and NK-Cell Neoplasm Hodgkin’s Lymphoma

Proposed WHO Classification

Back to Basic

Back to BasicFrequency of AML with specific chromosome defects

Chromosome defect

Breakpoint FAB FrequencyKorea1) USA2)

del 5q 5q31 & q35 M2,M1 4        1

-7/del 7q 7q31.2 & q36  M2,M1,M4~7 5        3

t(6;9) 6p22.2 & 9q34.1 M2,M1,M4 1        2

t(8;21) 8q22.1 & 21q22.3 M2,M4,M1 22       20

+8 M2,M1,M4~6 20       18

t(9;22) 9q34.1 & 22q11.21 M1,M2 3        8

t(V;11) 11q23.3 M4,M5a,M2 2        9

t(15;17) 15q22 & 17q21 M3 19        6

inv 16  16p13.2 & q22.1 M4 2        9

Complex M2,M1,M4 23       14

Others M0 ～ M7 15       17

1) Hallym University Medical Center (1995)2) University of Minnesota Medical School

Back to Basic(External Proficiency Testing)

● 법적 근거– 생명윤리 및 안전에 관한 법률 (2005)– 유전자검사 시행기관 대상의 정확도 평가 사업을 시행

● 목적과 방법– 유전검사의 정확도를 평가하고자 하는 목적– 정도관리 물질을 각 검사기관에 발송하여 실제 검체와

같은 방법으로 검사한 결과를 상호 비교

● 대상기관– 진료목적으로 유전자검사를 시행하는 모든 검사기관– 순수 연구를 목적으로 하는 검사나 , 그 시행기관은

제외

국내 유전검사 정확도 평가사업

Back to Basic국내 세포유전검사 현황

● 외부정도관리 사업 : 검사의 정확도 ( 숙련도 ) 평가 , External Quality Assurance (Proficiency Tests)

– 대한임상검사정도관리협회 (1997 년 )– 한국유전자검사평가원 (2006 년 ) ( 참고 ) 미국 CAP, 유럽 EMQN

● 국내 세포유전학검사실– Conventional cytogenetics : 65 대학 ( 종합 ) 병원 (43), 검사전문센터 (8), 여성전문병원 (13),

기타 (1)– FISH 시행기관 : 22

BCR/ABL(19), AML1/ETO(19),

TEL/AML1(15), MLL(15), X/Y(22)

Back to Basic

● 사업목표 - 염색체 이상의 정확한 검출 정확한 명명법 준수 (ISCN 2005)

● 사업내용 (10 cases/year) - Metaphase 사진을 이용한 핵형분석 말초혈액 , 양수 , 골수 검체 등 핵형분석용 사진 : 각 증례별 분열중기세포 (metaphase) 5

개씩 전혈 또는 골수 검체을 이용한 염색체검사 실시

● 증례선정 : Cytogenetics Resource Committee

세포유전검사 외부정도관리 사업

Back to Basic

Back to Basic

Back to Basic

● 평가기준 :

- 참고기관의 80% 이상이거나 , 참여기관 다수의 의견일치 (good or acceptable) 외부정도관리위원회 검토

● 평가분석요소 - M : modal chromosome number S : sex chromosome designation A : recognition of abnormalities N : karyotype nomenclature (ISCN 2005)

● 등급 : Good / Acceptable // Unacceptable

세포유전검사 결과 분석

Back to Basic

핵형 정답률

46,Y,fra(X)(q27.3) Fragile site 71%

46,XY,del(5)(p15.1) Terminal deletion 85%

46,XX,t(5;8)(q13;p23) Balanced translocation 96%

46,XX,inv(2)(p22q24) Pericentric inversion 72%

핵형 정답률

45,X,-Y,t(8;21)(q22;q22) Balanced translocation 75% 　

48,XY,+X,t(1;9)(p10;q10), t(7;9)(p22;p22),+12[5]

Complex defect(balanced translocation)

14.3%

　

Constitutional Abnormalities

Cancer Cytogenetics

세포유전검사 신빙도조사 결과 (1998)

Back to Basic세포유전검사 신빙도조사 결과 (2007)

핵형 정답률

46,XX,t(8;13)(q24.1;q14.1) Balanced translocation 93%

46,X,del(X)(q24q26) or 46,X,del(X)(q26) Turner Syndrome, Variant 95%

46,XY,+4,der(4;21)(p10;q10) Unbalanced whole arm translocation 71%

46,XY,der(21;21)(q10;q10) Robertsonian translocation 95%

46,XX,inv(11)(p15.5q13.1) Paracentric inversion 100%

46,XY,t(2;6)(p25.3;q15) Balanced translocation 98%

Constitutional abnormalities

Back to Basic

핵형 정답률

46,XY,t(8;14)(q24.1;q32)[3]/46,XY[2] Burkitt lymphoma/leukemia

95% 　

46,XY,del(13)(q12q14)[5] Multiple myeloma

95% 　

46,XX,t(9;22)(q34;q11.2)[2]/46,sl,inv(3)(q21q26.2)[3]46,XX,t(9;22)(q34;q11.2)[2]/46,idem,inv(3)(q21q26.2)[3]

Chronic myelogenous leukemia88% 　

46,XX,t(2;8)(p12;q24.1)[3]/46,XX[2]Burkitt lymphoma/leukemia

93% 　

Cancer Cytogenetics

세포유전검사 신빙도조사 결과 (2007)

46,XX,t(9;22)(q34;q11.2)[2]/46,sl,inv(3)(q21q26.2)[3]

46,XX,t(9;22)(q34;q11.2)[2]/46,sl,inv(3)(q21q26.2)[3]46,XX,t(9;22)(q34;q11.2)[2]/46,idem,inv(3)(q21q26.1)[3]

Stemline(sl) : the most basic clone of a tumor cell population listed firstSidelines(sdl) : all additional deviating subclones, sdl1, sdl2, sdl3 Idem : used only for a stemline with a single sideline

Back to Basic

세포유전검사 정도관리사업 계획

ISCN 2005 교육 프로그램세포유전 검사방법 워크샵FISH 정도관리 시범사업 다양한 환자검체 확보 노력표준시행지침서

주관기관 : 삼성서울병원 유전검사실

Back to Basic

Clinicalfeature

No single ‘gold standard’

“Real” disease entity

Genetic featuresMolecular &cytogenetics

Morphology

WHO Classification

Immunophenotype

Back to BasicChromosomal basis of Malignancy

– Numerical abnormalities– polyploid : triploid(69,XXY), quadriploid(92,XXYY)– aneuploid : Trisomy, monosomy

– Structural abnormalities– translocations– deletions– inversions– duplications etc.

– Net loss of chromosomal material• inactivation of tumor suppressor genes

– Net gain of chromosomal material• activation of protooncogenes

– Relocation of sequences with no gain or loss of genetic material• new fusion genes : interfering regulatory control of genes

Back to BasicMolecular Cytogenetics ( 분자세포유전학 )

• Blurring the boundaries with cytogenetics and molecular biology• Bridging the gap between cytogenetic and molecular approaches • Based on fluorescent in situ hybridization (FISH)

Advances in FISH-based techniques• An important aim ⇒ Resolution↑• The two crucial elements

Target (resolution)Metaphase spreads (~5Mb)Interphase nuclei (50kb~2Mb)Chromatin strands using fiber FISH (5kb~500kb)DNA microarray (single-nucleotide level)

Probe

Back to BasicPrinciples of fluorescence in situ hybridization.

Back to Basic

FISH probes for different applications

The new cytogenetics: blurring the boundaries with molecular biology

Exciting advances in fluorescence in situ hybridization and array-based techniques are changing the nature of cytogenetics, in both basic research and molecular diagnostics.

Nature Review Genetics 6, 782-92 (Oct 2005)

Back to BasicAdvances in FISH-based techniques

• Advances in metaphase spread analysisMultiplex-FISH (M-FISH)Spectral karyotyping (SKY) (Spectral karyotype imaging, SKI)

Combined binary ratio labeling (COBRA)

• Comparative genomic hybridization (CGH) on chromosomes• Applied to target metaphase chromosomes• Interphase cytogenetics

Back to BasicConventional Copy Number Analysis Tools

Karyotyping (G-banding)

Provides a global view of metaphase chromosomal characteristics (number, type, shape etc)

Each chromosome has a characteristic banding pattern that helps to identify them

Spectral Karyotyping (SKY)

Allows simultaneous visualization all the chromosomal pairs in different colors using chromosome specific probes

More accurate than G-banding

Fluorescent in situ Hybridization (FISH)

More specific and sensitive than karyotyping Uses fluorescent probes to detect and localize the presence or absence of specific DNA sequences on chromosomes Resolution: 5 Mb – Metaphase 2 Mb – Interphase 0.5 Mb – Fibre FISH

Back to BasicComparison of cytogenetic techniques for identifying chromosomal abnormalities.

Back to Basic

Multiplex FISH

Nat Rev Genet. 2005;6(10):782-92

Combining cytogenetic approaches to understand a complex chromosomal rearrangement

Array CGH

Banding analysis

Conventional CGH

Back to BasicSummary of Cytogenetics Technologies

Karyotyping– Provides a visual examination of the entire genome– the best coverage but not the best resolution (≥ 5 Mb)– Banding resolution differs from preparation to preparation

FISH– Sensitive (high resolution)– Only provides information on tested regions, other aberrations wi

ll NOT be tested, i.e. not genome view

Array-based Copy Number / CGH AnalysisSingle step global genome scan prevents FISHing expedition – BAC (Bacterial Artificial Chromosome) Array

• Requires well characterized and high resolution clones– High Resolution Oligonucleotide Microarray

• Highest resolution.• Precise identification of gains and losses of genetic material.

Back to BasicFISH for hematologic malignancies

Classification No.Normal/No mitotic cell

Cy* (+), FISH(+)

Cy (+), FISH(-)

Cy (-)FISH(+),

Precursor B-cell Leukemia 144 20 64 11 49

Precursor T-cell Leukemia 22 8 7 1 6

Acute biphenotypic Leukemia 18 1 10 - 7

Mixed lineage (undifferentiated) 4 - 　 3 1 - 　

Others (CML, in blastic phase) 1 - 　 1 - 　 - 　

Total 188 29 84 1362

(32.8%)

*p16 signal 결손이나 중복 : 20 cases TEL/AML1 gene rearrangement : 14 cases MLL gene rearrangement : 5 cases BCR/ABL gene rearrangement : 4 cases

Unpublished, SMC data

Back to BasicFISH profiles

ALL : MLL, BCR/ABL, ETV6/AML1, IgH, MYC, p16, E2A, chromosomes 4, 10, 17

AML :ETO/AML1, PML/RARA, MLL, CBFB/MYH11, P53, Chromosomes 5, 7, 20

CLPD : IGH, IGH/BCL2, IGH/CCND1, P53, MYB, ATM, 13q14.3, chromosome 12

Back to Basic

Accuracy : detection of clonal abnormalities– Variations due to specimen quality, analysts, techniques– Supported by FISH, proficiency tests, periodic check of

positive rate etc.

Turn-around time (TAT)– Technical TAT : 3-4 days– Ideal TAT : before final report of BM study– Practical TAT : 7~21 days

FISH tests– The more, the better?– Expecting Array CGH era

Clinical considerations