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Cytogenetics

Gail H. Vance, MD

Indiana University School of Medicine

April 2013

Disclosure(s)

In accordance with ACCME guidelines, any individual in a position to influence and/or control the content of this ASCP CME activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this CME activity.

The individual below has responded that he/she has no relevant financial relationship(s) with commercial interest(s) to disclose:

Gail H. Vance, MD

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Lecture Overview

History

Chromosome Structure/Identification/Nomenclature

Chromosome Abnormalities

Numerical (Meiosis, Mitosis, Nondisjunction)

Structural (Inversions, deletions, translocations).

Syndromes

FISH

Cancer Cytogenetics (Numerical/Structural/Nomenclature)

Gene Amplification

Leukemia/Lymphoma/MM

Chromosomal Microarray analysis

4

Cytogenetic Resources

Genetics in Medicine: Thompson & Thompson-7th edition WB Saunders, 2007.

Human Genetics: Vogel & Motulsky, Springer,1997.

Chromosome Abnormalities and Genetic Counseling, Gardner & Sutherland, Oxford, 2012.

WHO-Tumours of Haematopoietic and Lymphoid Tissues, 2002/2008

The Principles of Clinical Cytogenetics. Gersen and Keagle, Humana Press, 2005.

Human Chromosomes, 4th Edition, Miller and Therman, Springer Press, 2001.

Websites http://atlasgeneticsoncology.org/

Mahaffey VJ et al. Leukemia Research 2004; 28:1351-1356

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Prehypotonic Era

Hypotonic Era (1950s) Determined actual number of

chromosomes

Staining Era (1960s) Discovery of trisomy 21 in DS Discovery of Philadelphia chromosome

Banding Era (1970s)

Molecular Era (1980 to date) Extended chromosome analysis Molecular cytogenetics

History of Cytogenetics

6

Genomic era 2000-present

Chromosomal Microarrays

CGH arrays

SNP arrays

History of Cytogenetics

Molecular cytogenetics in clinical diagnostics prognostics

cytogenetics.org.uk

emedicine.medscape.com

fluidigm.com

Cytogenetics FISH Arrays

Sequencing

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History of Cytogenetics

1923 T.S. Painter establishes human chromosome number as 48.

1953 T.C. Hsu accidentally discovers hypotonic treatment to spread chromosomes and lyse RBCs

1956 J. Tijo and A. Levan determine correct number of chromosomes as 46.

9

History of Cytogenetics

1959 J. Lejeune identifies trisomy 21 as first clinical cytogenetic syndrome.

1960 P. Nowell identifies small G-group chromosome in CML as the Philadelphia chromosome.

1968 Caspersson utilizes quinicrine mustard to develop Q-banding method.

1971 Paris, 1st nomenclature conf.

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History of Cytogenetics

1973 G-banded chromosomes, J. Rowley identifies the Philadelphia chromosome as t(9;22)(q34;q11.2).

1990 Molecular Cytogenetics era; FISH

1992 Metaphase Comparative Genomic Hybridization (CGH).

1996 24-color karyotype (Spectral Image)

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History of Cytogenetics

2001 CGH BAC arrays

2006-7 CGH Oligo arrays

2007 Molecular karyotyping with SNPs- constitutional abnormalities

2010 Molecular karyotyping in Cancer

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Human Chromosomes

Chromosome means “colored body”.

Contain most of the human DNA

Chromosomes reside in the nucleus

NIHGR website

13

Reasons for Referral for Testing

Prenatal Analysis (Amniotic fluid/CVS)

Advanced maternal age (35 yrs and greater)

Abnormal serum triple/quad screen

Abnormal cell-free fetal DNA

Abnormal ultrasound

Family hx of chromosome abnormality.

Maternal anxiety

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Reasons for Referral for Testing

Peripheral blood

Family hx chromosome abnormality

Multiple miscarriages

Birth defect(s)

Unexplained mental retardation

Confirmation of prenatal diagnosis

Ambiguous genitalia

Abnormal growth and development.

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Reasons for Referral for Testing

Cancer (Bone marrow/unstimulated PB/tumor

Anemia (MDS)

Leukemia/Lymphoma

Myeloproliferative disorder (CML,PCV)

Solid tumor

Multiple myeloma

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Chromosome Structure Key Points

A chromosome is formed from a DNA strand.

After replication (S-phase), the strand is duplicated, two sister chromatids formed.

Each chromosome undergoes a series of compression and compaction steps to form the metaphase chromosome.

The nucleosome is the first level of compaction.

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Chromosome Identification

Chromosomes are identified by

Overall size

Placement of the centromere

G-banded pattern

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Centromere Consists of highly repetitive DNA

Contains -satellite DNA on which the kinetochore forms

Interacts with microtubule proteins to move chromosomes during mitosis and meiosis –serves as a mitotic checkpoint

Telomere Maintains the integrity of chromosomes and

prevents them from sticking together

Naming parts of the chromosome

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Chromosome banding techniques were developed in the early 1970’s

Prior to banding chromosomes were identified according to size and shape named A-G groups

Banding allows one to identify homologs and to better differentiate one chromosome from another

Chromosome banding and staining

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Group A Group B

Group C

Group D Group E

Group F Group G

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Chromosome Identification

G-banding Giemsa stain is used to identify the alternating dark and

light bands of the chromosome. Stains AT-rich DNA. Less affinity for GC-rich areas.

Q-banding Produced by a fluorescent stain, similar to G-bands

C-banding Stains centromere and heterochromatin

R-banding Reverse of G-banding. Used in Europe. Good for tips of

the chromosome.

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Chromosome Banding

Q-bright and G-positive bands are AT-rich

G-positive bands-late replicating/LINES

Gene poor

Q-dull and G-negative bands are GC-rich.

G-negative bands-early replicating/SINES

Gene rich

G-banding

resolution

Adapted from ISCN 1995

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G-banded

metaphase

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G-banded Karyogram

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Q-banded karyogram

29 C-banded metaphase

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Which one of the following procedures is used as a routine technique for karyotyping using light microscopy?

A. C-banding

B. Fluorescence in situ hybridization

C. G-banding

D. Q-banding

E. NOR staining

Questions

31

Which of the following pairs of staining techniques results in opposite staining patterns of chromosomal loci?

A. C &G

B. G & Q

C. Q & C

D. Q & R

E. None of the above.

Question

32

Which of the following is NOT characteristic of G-positive bands on the chromosome?

A. Early replicating

B. Correspond to Q-bright areas

C. Includes heterochromatic regions

D. AT-rich

E. Contain LINE repetitive sequences.

Question

Nomenclature

• Method of communication between cytogeneticists.

• Adhere to nomenclature standards outlined in “International Standards of Cytogenetic Nomenclature”-ISCN, 2013.

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Karyogram: display of mitotic chromosomes of an individual, lined up from the largest to the smallest (1-22) and according to location of centromere

Karyotype: is the use of nomenclature to describe the chromosomal complement

Idiogram: diagrammatic representation of a chromosome

Terminology

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Arms of the chromosome

Short arm = p

Long arm = q

Nomenclature

46,XX – normal female karyotype

46,XY – normal male karyotype

47,XXY – compatible with Klinefelter syndrome

47,XX,+21 – compatible with a female with Down syndrome

46,XY,t(1;2)(q21;q22) – A male with a translocation between the long arms of chromosomes 1 and 2

Karyotype Nomenclature

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Chromosome Abnormalities

Numerical abnormalities Mitotic or meiotic non-disjunction

Inheritance of a marker chromosome

Anaphase lag

Irregular cell cycle/abnormal spindle proteins

Structural Abnormalities Inherited abnormality

Chromosome instability

Abnormal DNA repair/breaks abnormal reunion

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Constitutional Numerical Abnormalities

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Meiosis/Mitosis

Meiosis: One replication and two divisions results in haploid gamete (23 chromosomes.)

Meiosis I: Reduction from 46 to 23 chromosomes

Meiosis II: Equatorial division/ same as mitosis but only 23 chromosomes.

Mitosis and Meiosis

Mitosis

Meiosis (germ cells only)

2N

2N

N

N

N

N

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Female Meiosis

Begins in utero

Progresses to prophase I (diplotene)

Arrested at 9th month gestation (dictyotene).

Begins again in puberty at ovulation.

Meiosis II completed upon fertilization

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Male Meiosis

Begins at puberty in seminiferous tubules

Goes through completion in 64 days

Continues late in life

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Nondisjunction

Errors in mitosis or meiosis called nondisjunction.

Mitotic errors, after formation of zygote result in mosaicism.

Meiotic errors occur most frequently in meiosis I.

Meiosis I errors, gamete contains both paternal and maternal chromosomes.

Meiosis II errors, gamete with both copies of paternal or maternal chromosomes.

a) Nondisjunction

at meiosis I

Nondisjunction

disomic

gametes

nullisomic

gametes Adapted from Gardner & Sutherland, Chrom. Abnl and Genetic Counseling

b) Nondisjunction

at meiosis II

Adapted from Gardner & Sutherland, Chrom. Abnl and Genetic Counseling

disomic

gametes

Nondisjunction

nullisomic

gametes

normal gametes

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Nondisjunction

Nondisjunction can lead to:

Monosomy or one copy of a chromosome.

Trisomy or three copies of a chromosome.

Mosaicism or more than one cell line.

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Aneuploidy

Change in chromosome number that is not exact multiple of haploid set

Most spontaneously aborted.

Due to nondisjunction or failure of normal separation of chromosome pair (meiosis I) or chromatids (meiosis II) pre-fertilization.

Mosaicism-post fertilization.

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Polyploidy

Chromosome number is a multiple of the haploid number (23).

Euploid = 46 chromosomes

Triploid = 69 chromosomes

Tetraploid = 92 chromosomes

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Triploidy

69,XXY; 69,XXX; or 69,XYY

1-3% of all recognized pregnancies

15-20% of chromosomally abnl SAB.

<1/20,000 liveborns; survival days to few months

85% are diandric (2 paternal/1 maternal set of chromsomes)

15% are digynic (2 maternal/1 paternal set of chromosomes)

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Triploidy

Mechanisms

Haploid egg + 2 sperm = dispermy

Haploid egg + diploid sperm (MI or MII error)

Haploid sperm + diploid egg (failure to release polar body)

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Tetraploidy

92,XXXX or 92,XXYY

6-7% of spontaneous abortions

Due to replication without meiotic division

Less than 10 reported liveborns,

Survival in days

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Autosomal and Sex Chromosome Aneuploidy Syndromes

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Trisomy 21 (Down Syndrome)

Incidence: 1/700 (maternal age related)

Etiology: 90% maternal errors

75% MI 25% MII

3-5% paternal errors

Features: Mild to moderate mental retardation 30-40% risk of heart defects 1% risk for leukemia Alzheimer disease with age Male infertility

4% cases due to Robertsonian translocation

errors 2% mosaicism 94% trisomy 21

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Trisomy 18 (Edwards Syndrome)

Incidence: 1/7,500 (maternal age related)

Etiology: 90% maternal error

33% MI 66% MII

Features: Mental retardation Failure to thrive Neural tube defects Heart defects Short sternum Rocker bottom feet 2nd & 5th digits overlap 3rd & 4th Hypertonia

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Trisomy 13 (Patau syndrome)

Incidence: 1/15,000 (maternal age related)

Etiology: 70% maternal error

MI most common

Features: Mental and growth retardation Holoprosencephaly Cleft lip/Cleft palate Postaxial Polydactyly Clenched fists Rocker bottom feet Eye malformations Heart defects

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XXY (Klinefelter syndrome)

Incidence: 1/1,000 (maternal age related)

Etiology: 50% maternal errors

MI most common 50% paternal errors

Features: Tall, long, thin arms Gynecomastia Hypogonadism Infertility Germ cell tumors 15% mosaicism

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XXX

Incidence:

1/1,000 females (maternal

age related)

Etiology:

90% maternal errors

MI most common (75%)

Features:

Tall stature

Behavior problems

Risk of learning disabilities

Ovarian failure

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XYY

Incidence:

1/1,000 males

Etiology:

100% paternal error

MII most common

Features:

No abnormal features

Risk of learning disabilities

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45,X (Turner syndrome)

Incidence: 1/1,000 females 50% with 45,X 15% with structurally abnormal X 15% with mosaicism

Etiology: 80% missing paternal X

Features: Short stature Heart defect; coarctation of the aorta Cystic hygroma / Webbing of neck Gonadal dysgenesis Broad Chest Short 4th metacarpal Hand/Foot edema Renal abnormalities

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Structural Abnormalities of Chromosomes

Translocation

Insertion

Inversion

Deletion

Duplication

Derivative chromosome

Ring chromosome

Marker chromosome

Isochromosome

Paracentric Inversion

E

Adapted from Elsevier Scientific

Pericentric Inversion

Adapted from Elsevier Scientific

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Two chromosome breaks required.

Chromosome segment rotated 180 .

Usually no loss or gain of genetic material.

Occasionally break occurs in a gene or region critical for normal development or cellular regulation.

Two types: Paracentric

centromere outside of rotated segment

Pericentric centromere inside rotated segment

Inversions

72 Ring

Insertion

Deletion Isochromosome

46,XY,t(7;15)(p22;q22)

Segregation of Reciprocal Translocations

Alternate

4

20 der(4)

der(20)

Normal

4

20

Balanced

der(4) der(20)

20 4

20

4

Segregation of Reciprocal Translocations

Adjacent-1

Unbalanced

der(4) 20

4 der(20)

4

der(20) der(4)

20

4 20

20 4

Segregation of Reciprocal Translocations

Adjacent-2

Unbalanced

4

der(4) 20

der(20)

20

der(20) der(4) 4

4 20

4 20

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Segregation Patterns in Metaphase I of Meiosis I

Alternate – gametes are normal or balanced and zygotes are viable

Adjacent-1 – gametes are unbalanced and zygotes maybe viable

Adjacent-2 – gametes are unbalanced and zygotes are presumably nonviable

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CHROMOSOME DELETION SYNDROMES

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Cri-du-Chat (Cry of the cat)

Deletion: 5p-5p15.2 critical region

Incidence: 1/20,000-1/50,000 1% of patients with IQ < 20 10-15% parental translocation

Features: Cat cry Micrognathia Low-set ears Microcephaly Hypotonia/Hypertonia Hypertelorism Mental retardation Heart defects

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Wolf-Hirschhorn Syndrome

Deletion: 4p-4p16.3 critical region

Incidence: 87% de novo 10-15% parental translocation

Features: IUGR Failure to thrive Microcephaly Greek helmet facies Cleft palate Heart defects Mental retardation Coloboma Seizures

Greek Helmet

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MICRODELETION SYNDROMES

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Smith-Magenis Syndrome

Critical Region: 17p11.2

Incidence: 1/25,000

Features: Brachycephaly Prognathia Speech delay Mental retardation Growth Delay Behavior problems Hearing loss Scoliosis Heart defects Renal anomalies

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Prader-Willi Syndrome

Critical Region: 15q11.2-q13

Incidence: 1/10,000-1/15,000 70-80% paternal deletion

25% maternal uniparental disomy Imprinting mutation

Features: Hypotonia Mental retardation Short stature Food preoccupation w/ obesity Hyperphagia Hypogonadism Small hands and feet

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Angelman Syndrome

Critical Region: 15q11.2-q13

Incidence: 1/10,000 70% maternal deletion

5% paternal uniparental disomy 5% imprinting mutation 10% UBE3A mutation

Features: No speech Ataxia Mental retardation Seizures Microcephaly Inappropriate laughter Hypotonia Large mouth

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Williams Syndrome

Critical Region: 7p11.23 Elastin gene

Incidence: 1/20,000

Features: Mild mental retardation Periorbital fullness Supravalvular aortic stenosis Loquacious personality Anxiety Attention deficit disorder Hypercalcemia Stellate iris pattern

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VCF/DiGeorge Syndrome

Critical Region: 22q11.2

Incidence: 1/2,000-1/4,000 94% de novo

Features: Conotruncal heart defects Cerebral Palsy Learning disabilities Psychiatric illness Palate insufficiency Speech delay Nasal speech/tubular nose Long fingers Hypocalcemia Thymic aplasia/hypoplasia Parathyroid hypoplasia T-cell deficiency

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Miller Dieker Syndrome

Critical Region: 17p13.3

Features: Lissencephaly (smooth brain) Microcephaly Severe mental retardation Spasticity Seizures Vertical forehead furrows IUGR

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Fluorescence in situ hybridization (FISH)

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Initiated in 1990

“Southern on a glass slide” -non radioactive probes

Multiple probe types

Multiple tissue applications

FISH: Molecular Cytogenetics

91 NIHGR

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• Centromere probes – Specific for given chromosome.

• Locus specific probes – Specific chromosome regions known to be

involved in genetic syndromes or cancers.

• Subtelomeric probes – Used to identify cryptic rearrangements in the

subtelomeric regions of chromosomes.

• Whole chromosome paints – Specific for chromosomes 1 through 22, plus

X,Y.

FISH Probes

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FISH on Tissues

Amniotic fluid / CVS

Non-neoplastic Tissue

Stimulated peripheral blood

Bone marrow or unstimulated PB cultured/uncultured, smears,

PET BM clot

Tumors- biopsy or tissue section Fresh, cultured, paraffin-embedded (formalin-fixed)

tissue

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Types of Probe Strategies

Dual/Triple color; dual fusion

Dual color; single fusion

Extra signal rearrangement

Break-apart rearrangement

Multi-color FISH

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Chromosome 9 Chromosome 22

ABL

gene BCR

gene

Arrows indicate

breakpoints

within the two

involved genes

Dual Color, Single Fusion

Dual Color, Extra Signal

Dual Color, Dual Fusion

VYSIS

FISH in CML

96 VYSIS

BCR/ABL Probes

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Break-apart Strategy

Inv(16), MLL, MYC, EWS

Adapted from Vysis Website

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ETO/AML1 CBF split

D8Z2

Favorable prognosis

Intermediate prognosis

Unfavorable prognosis

t(8;21) inv(16)

MLL split

t(11;var)

(q23;var)

+8

D7S486/D7Z1

7q-

BCR/ABL

t(9;22) 5q-

EGR1/D5S23

t(15;17)

PML/RAR

Adapted from: Vance et al.

Leukemia Research, May 2007

Some FISH Probes for AML

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FISH Applications

Prenatal Lab

Detection of specific trisomies (13,18, 21) and numerical sex abnormalities (XY), follow with G-banding to confirm.

Leukocyte Lab

Detection of interstitial or terminal deletions (submicroscopic) or additional unknown material, and marker chromosomes.

Cancer Lab

Detect numerical and structural anomalies, diagnosis, remission, minimal residual disease, relapse, and transplant engraftment.

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Chromosomal rearrangements involving telomeres emerging as an important cause of human disease

~7% of unexplained MR have subtelomere rearrangements

50% of rearrangements are familial

Knight , Lancet 354:1676,1999

Subtelomere FISH

101 Adapted from Korf:

Human Genetics

Subtelomere Probes

TTAGGG

102 Adapted from Stohler, et al. AJMG, 2007

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Cancer Cytogenetics

An area of cytogenetics based on the identification or recurring abnormalities in malignant cells

104

Conventional (G-banded) cytogenetic studies are utilized to:

Identify malignant disorders with specific chromosome abnormalities.

Assess the effectiveness of treatment.

Monitor remission.

Cancer Cytogenetics

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Modal Number

Diploid – 46 chromosomes

Hypodiploid – <44 chromosomes

Near Haploid – close to 23 chromosomes

Hyperdiploid – >46 chromosomes

High Hyperdiploid- >50 chromosomes

Pseudodiploid – 46 chromosomes with structural abnormalities

Numerical Abnormalities in Cancer

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Translocation

Insertion

Inversion

Deletion

Duplication

Isochromosome

Ring Chromosome

Marker Chromosome

Derivative Chromosome

Structural Abnormalities in Cancer

108

t(X;18) – Synovial Sarcoma

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Chromosome Nomenclature

110

A clone is defined as a cell population derived from a single progenitor. A clone exists if two or more cells containing the same structural abnormality or supernumerary marker chromosome.

OR

Three cells missing the same chromosome (ISCN, 2009, p.88).

Cytogenetic Definition of a Clone

111

Frequently found as the sole karyotype abnormality and associated with a particular tumor. Ex: t(9;22)(q34;q11.2)

Primary Aberration (Stem Line)

112

Rarely found alone; develops in cells already carrying a primary abnormality. Ex: t(9;22)(q34;q11.2),+8,i(17)(q10)

Secondary Aberration (Side Line)

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Chromosomal

Manifestations of Gene

Amplification

114

Gene Amplification

• Importance:

– One mechanism for the activation of

cellular oncogenes.

– Reflects the genetic instability of the

tumor cell.

– Frequently characterizes aggressive

subsets of tumors.

115

Gene Amplification (cont’d)

• Earliest, in vitro example of selective

(pressure) amplification was demonstrated

in mouse cells by Schimke in l978 with

amplification of the dihydrofolate reductase

gene (DHFR) conferring resistance to

methotrexate (MTX).

Cancer Genet & Cytogenet

1980; 2:339-348

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Gene Amplification (cont’d)

• Three “chromosome” structures associated

with amplification include:

– Homogenously staining region

(HSR)

• Integrated region of amplification

– Double minute chromosome (DM)

• Small, paired, acentric extra-

chromosomal body

– Episomes

• Submicroscopic extra-chromosomal

body

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Examples of Amplified Oncogenes

• MYCN (2p24) in neuroblastoma – Prototype for oncogene amplification in cancer.

– MYCN amplification also seen in retinoblastoma, small cell lung cancer, glioblastomas and astrocytomas.

– Amplified copies present as DM or HSR. • Amplicons usually range from 100-200 kb. • Proportion of tumors with amplified MYCN, ~20%. • Usually associated with a poor prognosis.

• ERBB2/HER2 (17q21) in breast cancer

– First observed in breast cancer cell line MAC117.

– Observed in ~18-20% of primary breast tumors.

– Amplification correlated with decreased overall survival

and time to relapse.

– Specific therapy, trastuzumab (Herceptin),

lapatinib/capecitabine, pertuzumab.

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HER2 Testing by FISH

119

First identified in 2003 during routine screening by FISH with ETV6/RUNX1 probe set.

In karyotype, often associated with an additional marker or ring chromosome.

Observed in both adult and pediatric patients.

Incidence estimated at 1.5-2.0% of childhood ALL

Associated with poor prognosis

Amplification of RUNX1 in ALL

Adapted from Leukemia

2003;17:547-553

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121

t(12;21) [VYSIS]

Amplified RUNX1 on Marker

122

ABL1 Amplification in T-Cell ALL

• First reported in 2004 by C. Graux, et al*.

– “Fusion of NUP214 and ABL1 on amplified

episomes in T-cell acute lymphoblastic leukemia”

– Screened for possible ABL1

rearrangements in CML by FISH. • 5/90 (5.6%) had extrachromosomal amplification (>10

copies) of ABL1.

• Amplification was extrachromosomal but not

observed by G-banding (episomes).

• FISH mapping confirmed amplification of ABL1,

LAMC3, and NUP214 (CAN) localized to 500-kb region

on 9q34.

* Nature Genetics 2004, 36:1084-89.

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Leukemia

Myeloid leukemias

CML and pre-B-ALL t(9;22)(q34;q11.2) BCR/ABL1

AML-M2 t(8;21)(q22;q22) CBFA2T1/RUNX1

AML-M2 t(7;11)(p15;p15) HOXA9/NUP98

AML-M3 t(15;17)(q24;q21.1) PML/RARA

AML-M3, atypical t(5;17)(q35;q21.1) NPM1/RARA

AML-M3, atypical t(11;17)(q23;q21.1) PLZF/RARA

AML-M3, atypical t(11;17)(q13;q21.1) NuMA1/RARA

AML-M3, atypical t(17;17)(q11.2;q21) STAT5B/RARA

AML-M4eo inv(16)(p13.1;q22) MYH11/CBFB

CMML t(5;12)(q33;p13) PDGFRB/ETV6

B cell leukemias and lymphomas

precursor-B ALL t(12;21)(p13;q22) ETV6/RUNX1

precursor-B ALL t(1;19)(q23;p13.3) PBX1/TCF3

Burkitt lymphoma t(8;14)(q24;q32.3) MYC/IGH@

Burkitt lymphoma t(2;8)(p12;q24) IGK@/MYC

Burkitt lymphoma t(8;22)(q24;q11.2) MYC/IGL@

Mantle cell lymphoma t(11;14)(q13;q32.3) CCND1/IGH@

Follicular lymphoma t(14;18)(q32.3;q21) IGH@/BCL2

Diffuse large B cell lymphoma t(3;14)(q27;q32.3) BCL6/IGH@

Lymphoplasmacytic lymphoma t(9;14)(p13;q32.3) PAX5/IGH@

T cell leukemias/ lymphomas

T-ALL

7p14-15

7q35

14q11.2

TRG@

TRB@

TRA/B@

T-ALL

t(7;11)(q35;p13)

TRB@/LMO2

ALCL

t(2;5)(p23;q35)

ALK/NPM1

127

World Health Organization (WHO) Classification for Leukemias 2001 Acute myeloid leukemia with recurrent cytogenetic

abnormalities – AML with t(8;21)(q22;q22) (AML1/ETO) AML with inv(16)(p13.1q22) (CBFB/MYH11) APL with t(15;17)(q22;q21.1) (PML/RARA) AML with 11q23 (MLL) abnormalities

Subtypes of Acute Leukemia

128

New Subtypes of AML WHO Classification 2008

AML with recurrent genetic abnormalities AML with t(8;21)(q22;q22)(RUNX1/RUNX1T1)

AML with inv(16)(p13.1q22)(CBFB/MYH11)or t(16;16)(p13.1;q22)

AML with t(15;17)(q22;q21.1)(PML/RARA)

AML with t(9;11)(p22;q23)(MLLT3/MLL)

AML with t(6;9)(p23;q34)(DEK/NUP214)

AML with inv(3)(q21q26.2(RPN1/MECOM(EVI1)or t(3;3)(q21;q26.2)

AML with t(1;22)(p13;q13)(RBM15/MKL1)

AML with mutated NPM1

AML with mutated CEBPA

t(8;21)(q22;q22.3)

RUNX1T1 (ETO)/RUNX1 (AML1)

5-12% cases AML (M2)

Loss of a sex chromosome

AML

• t(15;17)(q24;q21.1)

PML/RARA

– 5-8% of AML (M3)

– Disseminated

intravascular

coagulation

– STAT FISH test

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131

Chromosome 16 Abnormalities

inv(16)(p13.1q22) and t(16;16)(p13.1;q22)

Generally classified as acute myelomonocytic leukemia

(AML-M4) with bone marrow eosinophilia (AML-M4Eo) FAB or

Acute myeloid leukemia with abnormal bone marrow eosinophils inv(16)(p13.1q22) and t(16;16)(p13.1;q22) by WHO classification

Inv(16) and t(16;16) account for approximately 10-12% of AML

Genes involved-MYH11 at 16p13.1 (Smooth muscle myosin heavy chain gene)and CBFB (Core binding factor beta) at 16q22

Creates a transcriptionally active fusion gene

132

133

Chromosome 16 Abnormalities

del(16)(q22)

Patients may present with MDS and then transform to AML

Less frequent than inv(16) and t(16;16)

Patients have loss of genes on 16q (LOH mechanism for leukemogenesis)

Mixed Lineage Leukemia • MLL - 11q23 involved with multiple translocation

partners

– Accounts for 5-6% of cases.

– Seen in infant leukemia and secondary

leukemia with topoisomerase inhibitors.

135

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1

3

5

6 7

8 9 10

11

12

13 14 15

16

17 18

19 20 21 22

Y

X

1p32

4q21 5q12

5q31 9q34

6q27

1q21

3p21

1q32

3q25

3q28

9p22

10p11.2

10p13

11q23

14q24

15q15

16p13

17q25

17q21 17q23

19p11 19p12 19p13

22q11.2

2p21

Xq13

12q13

Chromosomal partners for 11q23

Adapted from www.pathology.washington.edu

136

137

ALL Metaphase

Acute Lymphoblastic Leukemia

138

Acute Lymphoblastic Leukemia

Adult ALL – 5 year survival is approximately 40%

Childhood ALL- 5 year survival 75-80%

ALL accounts for 75-80% of childhood leukemia; less common in adults

139

Hyperdiploidy

>50 chromosomes, account for 25% of total cases.

Frequently see gains of X, 4, 6, 10, 14, 17, 18, and 21.

t(12;21)(p13;q22) ETV6/RUNX1

Accounts for 22% of cases.

Has a favorable prognosis

T cell-ALL

Accounts for ~12% of cases.

Has an intermediate prognosis.

MLL(11q23)

Accounts for 8% of cases

ALL t(9;22)

Account for 3% of cases

Worst prognostic

group-however

changing with TKIs

for 9;22

ALL Classifications in Children

140

High Hyperdiploid Karyogram

141

t(12;21) [VYSIS]

RRGF

t(12;21)(p13;q22)

der(19)t(1;19)(q23;p13.3) Pre-B ALL

PBX1/TCF3

20% ALL with pre-B phenotype

~6% of childhood ALL

Hypodiploid ALL 43 or fewer chromosomes

Rare, ~3% of patients with ALL

Subgroups

Near haploid 24-31 chromosomes

Low hypodiploid 32-43 chromosomes

Associated with a poor prognosis

Infant Leukemia Leukemias diagnosed in the first 12-months of life.

May be lymphoid or myeloid.

Accounts for 2.5-5% of ALL and 6-14% of pediatric AML.

Infant ALL associated with rearrangement of MLL gene,11q23

Most common karyotype for ALL is t(4;11)(q21;q23) (AFF1(AF4)/MLL).

145

MLL [VYSIS]

NORMAL BCR/ABL1/ASS [VYSIS]

NORMAL

ALL Panel

t(12;21) [VYSIS]

NORMAL

CEP 4(SO), CEP 10 (SG)

CEP 17(SA) [VYSIS]

NORMAL

ALL Panel

Burkitt Lymphoma

FAB L3 morphology

Classification: 1) Endemic; 2)Sporadic;

3)Immunodeficiency-associated

Chromosome translocations lead to deregulation

of MYC oncogene

Burkitt Lymphoma

148

Breakpoints on chromosome 8 are 3’ and breaks on chromosome 2 and 22 occur 5’ of the Kappa and Lambda gene constant region segments.

Light chains move to chromosome 8.

Variant Translocations

Atlas of Cancer Cytogenetics

149

FISH for Burkitt Lymphoma

Multiple Myeloma Cytogenetics

16-20% t(11;14)(q13;q32) CCND1/IGH@

15% t(4;14)(p16.3;q32) FGFR3-WHSC1/IGH@

4% t(6;14)(p21;q32) CCND3/IGH@

5-8% t(14;16)(q32;q22) IGH@/MAF

t(16;22)(q22;q11.2) MAF/IGL@

45% hyperdiploid 3,5,7,9,11,15,19,21

Blood 109, 31:32-3133,

2007

151

t(11;14) [VYSIS]

NORMAL

RB1(SG), CEP12(SO) [VYSIS]

NORMAL D13S319 [VYSIS]

NORMAL

FISH Panel for Multiple Myeloma

152

Comparative Genomic Hybridization (CGH)

Analyzes gains and losses of genetic material (unbalanced abnormalities).

May be performed on metaphases or on spotted DNA/RNA (arrays).

Total test DNA is labeled with one color, normal “competing” DNA with a second color.

The two DNAs are mixed and hybridized to normal metaphase cells or normal DNA on a spotted array.

153

Courtesy of Signature Genomics

154

CGH Metaphase

155

Composite of CGH Metaphase

156 Courtesy of Signature Genomics

157 Courtesy of Signature Genomics

158 Courtesy of Signature Genomics

159 Courtesy of Signature Genomics

160

Oligo-Based CGH Arrays

Greater density of probes

Greater coverage

No dye-reversal or mismatch of sex for control DNA

Multi-plexed

Filters set

163

Targeted Array CGH Diagnostic Applications

Microscopic chromosome abnormalities Aneuploidy (mosaicism)

Unbalanced translocations

Chromosomal origin of centric and noncentric markers.

Submicroscopic alterations Microdeletions

Microduplications

Unbalanced subtelomeric rearrangements (del, der)

Will not detect balanced alterations (rcp, inv, rob, ins)

ACMG Practice Guidelines-Genet Med 2010:12(11):742-745

SNP Arrays

• Single nucleotide polymorphisms (SNP) -DNA sequence variations in which a single nucleotide differs on paired chromosomes (or between individuals).

• Specialized software aligns SNPs in chromosomal order.

• Single test DNA is labeled and hybridized to the array.

• SNP-based arrays quantitatively determine copy number (gains/losses), but compare result to a historic reference control.

• Capable of detecting allelic copy neutral changes such as loss of heterozygosity (LOH) and Uniparental disomy - segment or entire chromosome from one parent is missing and replaced by gain of the other.

164

del(13)(q14.1q31.2) (46 Mb)

B-allele frequency

Log R ratio

←no het→

decreased intensity

BB

AB

AA

Courtesy of T. Smolarek CCHMC 165

Copy Number Variations

• DNA segment (>1 kb) with a variable copy number compared to normal reference genome.

• Occur on every chromosome; cover ~12% of genome.

• Complicates the interpretation of microarray data.

• Determine if CNV is pathogenic or benign

166

Copy Number Variants Guidelines

Pathogenic Benign

Inherited from an affected parent Inherited from a healthy parent

Similar CNV in affected relative Similar CNV in healthy relative

Gene-rich area Gene-poor area

Expanded or altered CNV from affected parent

Completely contained within genome imbalance

Identified in database as pathogenic Identified in database as probably benign

167

Copy Number Variant Resources

• Databases

– DECIPHER- http://decipher.sanger.ac.uk/

– Database of Genomic Variants- http://projects.tcag.ca/variation/

• Pub Med

• OMIM

• UCSC Browser

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Summary of Cytogenetic Resolution

• Karyotyping ~5-10 Mb of DNA

• FISH ~50-200 kb of DNA

• Multicolor FISH ~ 5- 10 Mb of DNA

• Metaphase CGH ~ 5-10 Mb of DNA

• aCGH –BAC platform ~200 kb of DNA

• aCGH –Oligonucleotide array ~50 kb of DNA

169

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© Association for Molecular Pathology, 2013 170