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

2. 혈액종양 관련 세포유전학 역사 Philadelphia chromosome (Ph) 1970 Banding technique
t(8;21) in AML-M2
t(8;14) in Burkitt lymphoma/Leukemia
t(15;17) in AML-M3, t(4;11)
High-resolution banding technique
1980
t(9;11) in AML-M5a, inv(3) in AML
inv(16) in AML-M4E
t(1;19) in ALL
t(1;3) in AML with dysmegakaryopoiesis
Molecular cytogenetics (FISH, CGH)

3. Cytogenetic & Gene Rearrangement
혈액종양 관련 세포유전학 역사
Cytogenetic & Gene Rearrangement
1980
MYC-IGH t(8;14)
ABL-BCR t(9;22), IGH-BCL2 t(14;18)
ATRA in APL Tx
1990
MLL t(4;11), E2A-PBX1 t(1;19)
PML-RARA t(15;17), AML1-ETO t(8;21)
DEK-CAN t(6;9)
CBFB-MYH11 inv(16), MLL-AF9 t(9;11)
TEL-AML1 t(12;22)

4. KimSW, et al. : Banding patterns of normal human chromosomes
KimSW, et al.: Banding patterns of normal human chromosomes. The Seoul Journal of Medicine 21(2): (1980).
Table 4. Karyotypic pattern in cases with CML
Karyotype
No.
t(22q;9q) 7
22q- 6
22q-. -9 1
49,XXX,+9,+21
Normal 21
조혈기질환에 있어서 골수세포
염색체 분석에 관한 연구.
대한혈액학회지 23(2),1988

5. Giemsa Banding (G-Banding) Technique
High-resolution Banding Technique Using Methotrexate Cell Synchronization
1) PB/BM Culture
RPMI 1640
FBS (15%)
Pen-Strep
L-Glutamine
PHA
PB/BM cells
3-5 hrs
MTX(10-7M)
17 hrs
72 hrs
Thymidine
(10-5M)
2) Harvest
1) Colcemid (50 g/ml) treatment
2) Hypotonic Solution (KCl;0.075M) treatment
3) Fixation (methanol:Acetic acid=3:1)
5 hrs
3) Slide preparation & Staining (Giemsa-Trypsin)
4) Microscopy and Karyotyping
5) Printing (Photography) and Reporting

7. Computerized Image Analyzer Cytovision®
CCD camera
Main Program
Microscope
Monitor PC
Printer

8. Conventional Cytogenetics
in Hematologic Malignancies
검사자 숙련도에 따라 슬라이드의 질적 차이가 많다
분열세포 적고, 염색체 길이 짧고, quality는 불량하다.
복잡하고 다양한 핵형을 보이는 경우가 많다

10. ISCN 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)

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

12. Morphology (A) and karyotyping (B) of interspecies blastocysts derived from a human cord fibroblast transfer into enucleated
bovine oocyte. An interspecies blastocyst at hatching were obtained 144 hours after culture and subsequently provided for
chromosome 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

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

14. 50,XX,+8,t(9;22)(q34;q11.2),+10,+19, +der(22)t(9;22)(q34.1;q11.21)
CML Blast Crisis - Karyotype
50,XX,+8,t(9;22)(q34;q11.2),+10,+19, +der(22)t(9;22)(q34.1;q11.21)

15. Practical 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,+19
Predict or confirm blast crisis
Give information regarding percentage of normal vs abnormal cells
Valuable after BMT to follow engraftment of the donor cells and identify possible relapse

16. AML-M2 with t(8;21)(q22;q22) Usually AML-M2, occasionally M1 & M4
Young individuals with good remission rate
Blasts containing a single thin Auer rod
RT-PCR : AML1/ETO fusion transcript

17. AML-M3 with t(15;17)(q22;q21) 15
t(15;17) 17
RARA
PML
PML/RARA
The most specific clinical association in AML Variants : t(15;Var;17), t(11;17), t(5;17) RT-PCR : PML/RARA fusion transcript Responsive to all trans-retinoic acid treatment

18. AML-M4Eo with inv(16)
Young patients, organomegaly, abnormal eosinophils
Specifically associated with M4Eo in over 50% of cases
Favorable prognosis, High incidence of CNS relapse
MYH11
CBFB
CBFB-MYH11

19. Acute Myeloid Leukemias
Proposed WHO Classification
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

20. MDS AML Normal Chromosome del 5q -7, del 7q del 9q del 20q +8
complex defects t(1;3), t(2;11)
MDS
t(3;3), inv(3)
t(6;9), inv(16)
t(8;21)
t(9;22)
t(v;11)
t(15;17)
AML

21. Myeloid Malignancy Secondary to Radiotherapy or Chemotherapy
Clinical features AML-M1,M2,M6 Lower remission rate and long-term reponse Cytogenetics Usually 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

22. Cytogenetics in Acute Lymphoblastic Leukemia
Poorly spread, short Fuzzy chromosomes Indistinct bands Low success rate 50% in conventional culture 76% Clarkson (1985)

23. Ploidy Groups in Childhood ALL
Hyperdiploid >50 28%
Early pre-B immunophenotype
Found at the age of 2-10 years
Lower WBC count, Favorable prognosis
Hyperdiploid %
Diploid %
Pseudodiploid %
Hypodiploid < %

24. Lymphoid Neoplasm B-Cell Neoplasm T-Cell and NK-Cell Neoplasm
Proposed WHO Classification
B-Cell Neoplasm
Precursor 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

26. Frequency of AML with specific chromosome defects
Breakpoint
FAB
Frequency
Korea1) 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
23       14
Others
M0～M7
15       17
1) Hallym University Medical Center (1995)
2) University of Minnesota Medical School

27. (External Proficiency Testing)
국내 유전검사 정확도 평가사업
(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

28. 국내 세포유전검사 현황 ● 외부정도관리 사업 : 검사의 정확도(숙련도) 평가,
External Quality Assurance (Proficiency Tests)
대한임상검사정도관리협회(1997년)
한국유전자검사평가원(2006년)
(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)

29. 세포유전검사 외부정도관리 사업 ● 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) 5개씩
전혈 또는 골수 검체을 이용한 염색체검사 실시
● 증례선정 : Cytogenetics Resource Committee

32. 세포유전검사 결과 분석 참여기관 다수의 의견일치(good or acceptable) 외부정도관리위원회 검토
● 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

33. 세포유전검사 신빙도조사 결과 (1998) Constitutional Abnormalities 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%
Cancer Cytogenetics 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%　

34. 세포유전검사 신빙도조사 결과 (2007) Constitutional abnormalities 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
46,XX,inv(11)(p15.5q13.1)
Paracentric inversion
100%
46,XY,t(2;6)(p25.3;q15)
98%

35. 세포유전검사 신빙도조사 결과 (2007) Cancer Cytogenetics 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
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 leukemia
88%　
46,XX,t(2;8)(p12;q24.1)[3]/46,XX[2]
93%　

36. 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 first
Sidelines(sdl) : all additional deviating subclones, sdl1, sdl2, sdl3
Idem : used only for a stemline with a single sideline

37. 세포유전검사 정도관리사업 계획 ISCN 2005 교육 프로그램 세포유전 검사방법 워크샵 FISH 정도관리 시범사업
다양한 환자검체 확보 노력
표준시행지침서
주관기관: 삼성서울병원 유전검사실

38. No single ‘gold standard’
WHO Classification
“Real” disease entity
No single ‘gold standard’
Morphology
Immunophenotype
Genetic features
Molecular &
cytogenetics
Clinical
feature

39. Chromosomal 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

40. Molecular 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

41. Principles of fluorescence in situ hybridization.

42. 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, (Oct 2005)
Figure 2 | Fluorescence in situ hybridization probes for different applications.
a | Painting probes stain entire chromosomes.In a normal diploid interphase nucleus, two chromosome territories are typically visible.
b | Regional painting probes can be generated by chromosome microdissection. Differentially labelled probes that are specific for each of the arms of a particular chromosome are shown.
c | Centromeric-repeat probes are available for almost all human chromosomes. Because of their ease of use and high signal intensities, these probes are popular for counting chromosome copy numbers in interphase nuclei.
d | Large-insert clones are available for most genomic regions. Subtelomeric probes, which are often used to screen for cryptic translocations that are not usually visible in conventional chromosome-banding analyses, are shown in this example.
e | Special probe sets can be designed to facilitate diagnosis of known structural rearrangements. In this example, the probe set includes a breakpoint-spanning probe (red) and two breakpoint-flanking probes (green and blue). Probe sets of this type allow structural rearrangements to be detected even in interphase nuclei.
f | Genomic DNA is used as the probe in comparative genomic hybridization (CGH) to establish copy number. An analysis of chromosome 8 is shown as an example. Simultaneous visualization of both test DNA (green region) and normal reference DNA (red region) fluorochromes shows balanced regions in orange (equal amounts of green and red fluorochromes). In regions of gain in copy number, green fluorescence dominates, whereas regions of loss appear red. Quantification is carried out using software that calculates a ratio profile for the two colours. The standard representation of the ratio profile consists of a centre line (black), which reflects a balanced ratio of green and red fluorescence. The green line represents the upper threshold for significant gain, the red line the lower threshold for significant loss.
g | For high-resolution analysis, DNA fibres can be used as the target for probe hybridization. The simultaneous hybridization of two different probes is shown, labelled green and red. The yellow colour indicates an overlap between these two probes.
h | Microarrays can be used as targets for hybridization to provide resolutions down to the single-nucleotide level. A BAC array is shown, to which test DNA and reference DNA are hybridized. Individual clones show different colours after hybridization depending on whether the corresponding DNA in the test sample is lost (red on the array), gained (green on the array) or neither (yellow on the array), in a similar way to conventional CGH.
FISH probes for different applications

43. Advances in FISH-based techniques
Advances in metaphase spread analysis
Multiplex-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

44. Conventional 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: Mb – Metaphase Mb – Interphase Mb – Fibre FISH 44

45. Comparison of cytogenetic techniques for identifying chromosomal abnormalities.

46. Combining cytogenetic approaches to understand a complex chromosomal rearrangement
Banding analysis
Multiplex FISH
Conventional CGH
Array CGH
Nat Rev Genet. 2005;6(10):782-92

47. Summary 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 will NOT be tested, i.e. not genome view
Array-based Copy Number / CGH Analysis
Single 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. 47

48. FISH 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 10 -
Mixed lineage (undifferentiated) 4 -　 3
Others (CML, in blastic phase)
Total
188 29 84 13
62 (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

49. FISH 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

50. Clinical considerations
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