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

혈액종양 관련 세포유전학 역사 Philadelphia chromosome (Ph) 1970 Banding technique 1973 t(8;21) in AML-M2 t(8;14) in Burkitt lymphoma/Leukemia 1977 t(15;17) in AML-M3, t(4;11) 1979 High-resolution banding technique 1980 1982 t(9;11) in AML-M5a, inv(3) in AML 1983 inv(16) in AML-M4E 1984 t(1;19) in ALL t(1;3) in AML with dysmegakaryopoiesis 1990 Molecular cytogenetics (FISH, CGH)

Cytogenetic & Gene Rearrangement 혈액종양 관련 세포유전학 역사 Cytogenetic & Gene Rearrangement 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)

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):133-137(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

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

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

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

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)

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 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

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.

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)

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

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

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

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

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

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

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

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

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

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

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

(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

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)

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

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

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%　

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%

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%　

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

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

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

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

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

Principles of fluorescence in situ hybridization.

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) 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

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

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: 5 Mb – Metaphase 2 Mb – Interphase 0.5 Mb – Fibre FISH 44

Comparison of cytogenetic techniques for identifying chromosomal abnormalities.

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

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

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

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

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