Back to Basic혈액종양의 분자세포유전학Molecular Cytogenetics in hematologic Malignancy

Nucleus

Cell

Chromosome

DNA

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1960 Philadelphia chromosome (Ph)

1970 Banding technique1970 t(9;22) in CML

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

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)

Historical Backgroud

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1980 1983 MYC-IGH t(8;14) 1984 BCR-ABL1 t(9;22) IGH-BCL2 t(14;18) 1990 1991 MLL-AFF1 t(4;11) E2A-PBX1 t(1;19)

PML-RARA t(15;17) RUNX1-RUNX1T1 t(8;21)

DEK-NUP214 t(6;9)1993 CBFB-MYH11 inv(16) MLL-MLLT3 t(9;11)1997 TEL-AML1 t(12;22)

Cytogenetic & Gene Rearrangement

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KimSW, 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

Cytogenetic Studies for Hematologic Malignancies in Korea

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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

abnormal cellsValuable after BMT to follow engraftment of the donor

cells 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 : RUNX1-RUNX1T1 fusion transcript

Back to BasicAPL with t(15;17)(q22;q21), PML/RARA

G-banding : t(15;17)(q22;q21) & Variants of t(15;Var;17), t(11;17), t(5;17)RT-PCR, FISH : PML/RARA fusion transcript

PML RARAPML/RARA

From highly fatal to highly curable

ATRA (all-trans retinoic acid) & ATO (arsenic trioxide)

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 Leukemia and Related Neoplasms

AML with recurrent genetic abnormalities t(8;21), RUNX1-RUNX1T1 t(15;17), PML/RAR inv(16), CBF/MYH11 t(9;11), MLLT3/MLL t(6;9), DEK/NUP214  inv(3) or t(3;3), RPN1-EVI1  t(1;22), RBM15-MKL1 

Acute myeloid leukemia with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia, not otherwise specified Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm

Proposed WHO Classification

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Normal ChromosomeNormal Chromosomedel 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 BasicMyeloid 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

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Cytogenetics in Acute Lymphoblastic Leukemia

Poorly spread, shortFuzzy chromosomesIndistinct bands

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

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Ploidy Groups in Childhood Ploidy Groups in Childhood ALLALL 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 BasicPrecursor Lymphoid Neoplasm

B lymphoblastic leukemia/lymphoma, NOS B lymphoblastic leukemia/lymphoma with recurrent genetic

abnormalities – t(9;22)(q34;q11.2); BCR-ABL1 – t(v;11q23); MLL rearranged – t(12;21)(p13;q22) TEL-AML1 (ETV6-RUNX1) – hyperdiploidy – hypodiploidy – t(5;14)(q31;q32) IL3-IGH – t(1;19)(q23;p13.3);TCF3-PBX1

T lymphoblastic leukemia/lymphoma

Proposed WHO Classification

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Clinicalfeature

No single ‘gold standard’

“Real” disease entity

Genetic featuresMolecular &cytogenetics

Morphology

WHO Classification

Immunophenotype

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Back to BasicAML 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 BasicCytogenetics 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– M-FISH(Multi-FISH, Spectral karyotyping)

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 BasicClinical Application of FISHClinical Application of FISH

Microdeletion syndromes• Prader-Willi/Angelman syndrome• DiGeorge/CATCH 22 syndrome • Williams syndrome etc.

Marker chromosome

Hematologic malignancies• t(9;22): abl/bcr• t(15;17): PML/RARa • 11q23: MLL• t(12;21): TEL/AML1

BMT follow-up using X/Y

46,XY,t(9;22)(q34;q11.2)

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1. Preparation of tumor sample 1. Preparation of tumor sample 2. Tumor cell culture2. Tumor cell culture

Culture MediaCulture MediaFBS (11-20%)FBS (11-20%)AntibioticsAntibiotics AdditivesAdditivesTumor cellsTumor cells

2. Harvest 2. Harvest 1) Colcemid (0.01-0.1mcg/mL) treatment 1) Colcemid (0.01-0.1mcg/mL) treatment 2) Hypotonic (0.050-0.075M KCl) treatment2) Hypotonic (0.050-0.075M KCl) treatment 3) Fixation3) Fixation 4) Slide preparation4) Slide preparation3. Staining and analysis 3. Staining and analysis

Solid Tumor CytogeneticsSolid Tumor Cytogenetics

InsulinInsulinTransferrinTransferrinHydrocortisoneHydrocortisoneEDGFEDGF

Back to BasicSolid Tumor Abnormalities

der(1) breast carcinomasdel(1p) colorectal adenomadel(3)(p12p14) breast carcinomasdel(3)(p14p23) small cell carcinoma of lungs

del(3p)renal cell carcinoma, non-small cell lung cancer

del(7)(q21q31) uterine leiomyomas del(9)(p13) malignant melanomadel (11)(p13) Wilm's tumordel(22q) or -22 meningiomas i(6)(p10) retinoblastomai(8)(q10) uveal melanomai(12)(p10) testicular germ cell tumorsi(17)(q10) primitive neuroectodermal tumorinv(10)(q11q21) papillary carcimoma of the thyroidt(1;17)(p36;q12) neuroblastoma

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inv(10)(q11q21) papillary carcimoma of the thyroidt(1;17)(p36;q12) neuroblastomat(2;13)(q35;q14) alveolar rhabdomyosarcomat(3;8)(p21;q12) pleomorphic ademoma of salivary glandst(3;12)(q27-28;q13-15) sporadic typical lipomast(9;22)(q22;q11-12) extraskeletal myxoidchondrosarcoma

t(11;19)(q21;p11)mucoepidermoid carcinoma, adenolymphoma of salivary gland

t(11;22)(p13;q12)intraabdominal desmoplastic small round cell tumor

t(11;22)(q24;q12)Askins tumor, Ewings sarcoma, esthesioneuroblastoma

t(12;14)(q14-15;q23-24) uterine leiomyomast(12;16)(q13;p11.2) myxoid liposarcomat(X;1)(p11;q21) malignant papillary renal cell carcinomat(X;18)(p11.2;q11.2) synovial sarcoma

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1983 19851988 1991 19940

5000

10000

15000

20000

25000

1983 19851988 1991 1994

Solid TumorsMalig LymphomaHemat Malignancy

“Catalog of Chromosome Aberrations in Cancer ”

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CONTENTS

1) A New Approach to an Old Problem 2) Cytogenetic Methods 3) Cytogenetic Nomenclature 4) Nonrandom Chromosome Abnormalities in Cancer - An Overview

5-10) Hematologic Malignancies : AML, MDS, CML, CMPN, ALL, Mature B- and T-Cell Neoplasms and Hodgkin Lymphoma

11-23) Solid Tumors : Upper Aerodigestive tract, Lung, Digestive Tract, Urinary Tract, Breast, Genital Organs, Endocrine Glands, Nervous System, Eye, Skin, Bone, Soft Tissue Tumors