Interpretation of conventional karyotyping & ISCN Nomenclature in cytogenetics

Dr Mayur Parihar

Consultant Hematopathology and Cytogenetics

Tata Medical Center Kolkata

MBBS,MD (Path),FCCG(CMC Vellore)

Cytogenetics• Cytogenetic analysis is an in vitro clinical

laboratory procedure that evaluates the chromosomes of a cell.

• Certain clinical characteristics occur consistently in association with a particular chromosome abnormality.

• This phenotype-karyotype correlation is useful.

Methods for Identifying Chromosome Aberrations

• Detected using cytogenetic and molecular methods

chromosome bandingmolecular cytogenetics– Loss of heterozygosity – Microarrays

Molecular Cytogenetics

• Powerful adjunct to conventional cytogenetic analysis

• Utilizes metaphases and non-mitotic interphase nuclei

• Can be applied to fixed archived tumour material

• Accurate, specific

Molecular CytogeneticTechniques

• FISH

• Multi-Colour FISH (M-FISH)

• Spectral Karyotyping (SKY)

• Comparative Genomic Hybridization (CGH)

• Microarrays

CytogeneticsKaryotyping

All chromosomes screened

Clonal Evolution

Locates areas that might contain critical genes involved in tumourigenesis

FISHSpecific part of a gene

or chromosome.

Does not screen all the chromosomes for abnormalities

• In vitro clinical laboratory procedure that evaluates the chromosomes of a cell.

• Chromosomes are individually distinguishable under light microscopy only during cell division

• Spontaneously proliferating cells : bone marrow, lymph nodes, solid tumors and chorionic villi.

• Cultured : PB lymphocytes, tissue biopsies

Conventional Cytogenetics

Specimen collection and handling Bone Marrow Aspirates

• Preservative free sodium heparin

• Transported at room temperature

• First few millilitres of the bone marrow tap contain the highest proportion of cells

• Processed without delay upon receipt to avoid cell death.

GTG-banded karyotype: work flow

Ann Lab Med 2014;34:413-425

Protocols have to be followed!!!

What do you mean by bands/banding

A chromosome band is a part of a chromosome that can be distinguished from adjacent segments by appearing darker or lighter by one or more techniques.

Each chromosome has a unique pattern of light and dark bands

Slide Making • Cell pellet dropped• Chromosome spreading depends upon

temperature, relative humidity, drying time• Has to be standardized each time

Capturing

• Bone marrow : correlate with BM diagnosis, IPT

• Slides are screened, looking at all metaphases

• Microscopy chromosome count done of metaphases

• Look out for specific abnormalities

• 20 metaphases are captured and analysed

Hyperdiploid Normal

NORMAL HUMAN KARYOTYPE

TerminologyParts of the chromosome

ISCN

• International System for Human Cytogenetic Nomenclature

• Each area of chromosome given number

• Lowest number closest (proximal) to centromere

• Highest number at tips (distal) to centromere

• In designating a particular band, 4 items are required 1.The chromosome number 2.The arm symbol 3.The region number and 4.The band number within that number.

• Ex: 1p31 indicates chromosome 1, short arm region3,band 1.

• According to ISCN the banding levels are varied by 400,550 and 880 levels with help of banding techniques

Idiograms

ISCN

• Normal male– 46,XY[20]

• Normal female– 46,XX[10]

Numerical

• Aneuploidy– Autosomal trisomy, 47– Sex chromosomes, 45, 47, 48, 49

• Polyploidy– Whole chromosome set– Normal Human genome is diploid that is 46

chromosomes (23x2)– Triploidy, 69 (23x3)– Tetraploidy, 92 (23x4)

Types of chromosome abnormalities

• Numerical– Aneuploidy (monosomy, trisomy, tetrasomy)– Polyploidy (triploidy, tetraploidy)

• Structural– Translocations– Inversions– Insertions– Deletions– Rings– Isochromosomes– ESAC

A 2 year old girl diagnosed as Acute Leukemia 1 month back was referred to TMC for further management.

• Fever • No history of any treatment• Hb :10.4,TLC:10,400,Platelets:2.5 lakhs• No immature cells/blasts in the peripheral

smear• Bone marrow examination : no evidence of

leukemia

55,XX,+X,+6,+8,+10,+14,+17,+18,+22,+22 [1]

CLONES• Clone is defined as a cell

population derived from a progenitor.

• The number of cells that constitute a clone is given in square brackets [ ] after the karyotype.

• Ex: 46,XX,t(8;21)(q22;q22)[20].

Clone

• Two cells/metaphases for trisomy

• Three cells/metaphases for monosomy

• Two cells/metaphases for a structural abnormality

• The number of cells that constitute a clone is given in square brackets [ ] after the karyotype.

FISH using ETV6/RUNX1 ES Probe

ETV6, Ch 12: RUNX1,Ch21:

Precursor B cell ALL

Steroids the blasts dissapear

45,XX,-7Specimen type : Heparinized Bone Marrow

Banding Resolution : 450 bphs

Cytogenetic Profile Metaphases Counted : 20

Metaphases Analyzed : 20

Metaphases Karyotyped : 8

Total Chromosome Number : 45/46

Autosomes : 44/45 Sex Chromosomes : 2(XY)

Clonal ???How many metaphases have -7?

45,XX,-7[7]/46,XX[13]Specimen type : Heparinized Bone Marrow

Banding Resolution : 450 bphs

Cytogenetic Profile Metaphases Counted : 20

Metaphases Analyzed : 20

Metaphases Karyotyped : 8

Total Chromosome Number : 45/46

Autosomes : 44/45 Sex Chromosomes : 2(XY)

45,XX,-7[1]/46,XX[19]

• Clonality not established .• The lab should record its observation saying

that monosomy 7 seen in a single metaphase/However, the clonality cannot be established

• Next step to do FISH for monosomy 7 to screen more number of cells.

ISCN

• del - deletion

• dic - dicentric

• fra - fragile site• i -

isochromosome• inv - inversion

• p - short arm• r - ring

• der - derivative• dup - duplication• h -

heterochromatin• ins - insertion• mat - maternal origin• q - long arm• t - translocation

Structural

• Breakage in at least 1 chromosome• Translocations

– 2 different chromosomes break and rejoin incorrectly• Inversions

– 2 breaks in same chromosome• Insertions

– Piece of chromosome inserted• Deletions

– Piece of chromosome missing

Translocations• Exchange of chromosome

material between two chromosomes.

• Balanced : No gain or loss of genomic material

• Unbalanced : loss or gain of genomic material.

• Denoted by t(8;21), the smaller chromosome always written first.

• In case of unbalanced translocation the word derivative is used.

46,XX,t(8;21)(q22;q22)[18]/46,XX[2]

46,XY,t(1;19)(q23;p13),-9,i(9)(q10),+mar[15]/46,XY[5]

Balanced chromosome 1 and chromosome 19 translocation

46,XY,t(9;22)(q34;q11.2),der(19)t(1;19)(q23;p13)[9]

Unbalanced translocation chromosome 1 and chromosome 19

46,XY,+1,der(1;7)(q10;p10)[14]/46,XY[6]

Loss of 7q and partial trisomy for 1q

A patient of CML on Imatinib not responding

46,XY,t(9;22)(q34;q11.2),+19,+der(22)t(9;22)[12]

46,XY,t(8;17;21)(q22;q25;q22),del(9)(q13q33)[4]

FISH showing 1F2R2GMetaphase FISH using dual colour dual fusion RUNX1/RUNX1T1 probe

Inverted DAPI

Inversions

• Reversal of segment of chromosome– If too small cannot detect by karyotype – Very rare in humans– Selected against as would get reduced fertility

• Pericentric– reversed segment includes centromere

• Paracentric– within one chromosome arm

• Paracentric inversion – main difference in karyotypes of great apes and

humans so important in evolution

Inversion

Reversal of segment of chromosome

• If too small cannot detect by karyotype

• Pericentric– reversed segment includes

centromere• Paracentric

– within one chromosome arm

– main difference in karyotypes of great apes and humans so important in evolution

47,XX,inv(16)(p13q22)

46,XY,inv(3)(q21q26)[17]

Insertions

• Segment of 1 chromosome inserted into another

A derA der B

Deletions• Terminal

– loss of end of chromosome– 46,XY,del(20)(q26) missing

long arm of 10• Interstitial

– loss of segment from within chromosome

– 46,XY,del(10)(q24q26) missing segment of 10

• All result in unbalanced karyotype

• Partial monosomy• Serious clinical effect

A patient of MDS

46,XY,del(20)(q11.2)[18]

A patient of MDS

46,XY,del(5)(q13q33)[16]

46,XY,t(4;7)(q22;p22),del(7)(q21q33),dic(9;12)(p12;p11)[14]

Isochromosome

• Two copies of the same arm

• Mirror image around centromere

• Centromeres part in wrong plane

– Monosomy for 1 chromosome arm

– Trisomy for the other arm

A patient of ALL.Isochromosome 9qloss of 9p that houses the PAX5 gene and CDKN

A patient of CML in blast crisis.Isochromosome 17qloss of 17p that houses the Tp53 gene

46,XY,dup(1)(q21q32),t(-;-)

Questionable Identification46,XY,?t(11;19)(q23;p13)

Metaphase FISH using MLL break apart

Clones and subclonesA patient of CML ?blast crisis

46,XX,t(9;22)(q34;q11.2)[9]/46,idem,inv(16)(p13q22)[8]/46,XX[3]

Clones and subclonesA patient of CML ?blast crisis

46,XY,t(9;22)(q34;q11.2)[6]/47,idem,+8[14]

Clones and subclonesA patient of AML

46,XX,t(8;21)(q22;q22)[8]/45,X,-X,t(8;21)(q22;q22)t(17;17)(q11.2q25)[12]

A patient of CML on follow up not responding

46,XY,t(9;22)(q34;q11.2)[14]/47,XY,+8[2]/46,XY[4]

Complex Karyotype• Complex karyotype is defined as presence of

three or more cytogenetic abnormalities in bone marrow not including inv(16), t(16;16), t(8;21), and t(15;17) by most of the groups

Slovak ML: German Acute Myeloid Leukemia Intergroup,Byrd JC: CALGB, Schlenk RF: SWOG;

• The MRC multicenter trial defined complex karyotype as presence of 5 or more chromosomal aberrations.

Grimwade D: MRC

The monosomal karyotype was defined by the presence of two autosomal monosmies or one single monosomy (excluding isolated loss of X or Y) in association with a structural chromosome abnormality.

Patient with AML

45,XY,inv(3)(q21q26),-7[18]/46,XY[2]

Patient with AML

45,XX,-7,t(11;17)(q23;q12)[14]/46,XX[6]

55~ 57,XX,+X,+6,+8,+10,+14,+17,+18,+22,+22 [CP10]/46,XX[10]

Composite Karyotype

• Multiple clones and subclones may be present

• Karyotypic heterogeneity

• Contains all clonally occurring abnormalities

• Gives range of modal chromosomal number

56,XXYC,+X,+4,+5,+6,+8,t(9;22)(q34;q11.2),+12,+14,+18,+21,+der(21)t(9;22)[CP14] /46,XXY[6]

A patient of AML with aberrant CD1946,XY[20] nuc.ish (RUNX1x3)(RUNX1T1x3)(RUNX1conRUNX1T1x2)[190/200]

Karyotyping results Post Transplant

46,XY,der(11)dup(11)(q14q22)t(11;22)(q24;q11.2)[6]//46,XX[14]

Conclude

• Look for clonality of the abnormalities• In case clonality not established used other

complimentary tests like FISH.• Correlate the karyotype findings with clinical

picture,morphology, IPT findings, other test reports.

• Communication with other members of the team essential across specialities.

The Cytogenetics team at the Tata Medical Center

THANK YOU