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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)
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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
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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
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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
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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.
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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
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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
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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
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● 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)
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● 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
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● 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
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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
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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
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세포유전검사 정도관리사업 계획
ISCN 2005 교육 프로그램세포유전 검사방법 워크샵FISH 정도관리 시범사업 다양한 환자검체 확보 노력표준시행지침서
주관기관 : 삼성서울병원 유전검사실
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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.
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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.
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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
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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
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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