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)

DNA in any chromosome (50 to 250 million bases), highly coiled and condensed DNA from a single cell if stretched - 2m DNA sequence for a single protein is a gene. Each chromosome contains a few thousand genes, Few 100 to ~2 million bases.

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 banding molecular cytogenetics Loss of heterozygosity Microarrays These analyses identify a broad range of chromosomal abnormalities in solid tumours including: altered ploidy gain or loss of individual chromosomes, deletions, amplications structural rearrangements Molecular techniques are considered high through put RLGS, RDA detect allelic balances that occur by somatic recombination or copy number change FISH/CGH - are sensitive to physical changes in genome structure or copy number

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

Cytogenetics Karyotyping All chromosomes screened Clonal Evolution Locates areas that might contain critical genes involved in tumourigenesis FISH Specific part of a gene or chromosome. Does not screen all the chromosomes for abnormalities

Conventional Cytogenetics 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

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

Terminology Parts 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 Polyploidy 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,-7 Clonal ??? How many metaphases have -7? 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[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 der - derivative 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 Inversions 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]

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

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

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 1F2R2G Inverted DAPI Metaphase FISH using dual colour dual fusion RUNX1/RUNX1T1 probe Inverted DAPI

Inversions Reversal of segment of chromosome Pericentric Paracentric 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 Pericentric Paracentric 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 Interstitial All result in unbalanced karyotype 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 9q loss of 9p that houses the PAX5 gene and CDKN

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

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

Questionable Identification 46,XY,?t(11;19)(q23;p13) Metaphase FISH using MLL break apart

Clones and subclones A 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 subclones A patient of CML ?blast crisis 46,XY,t(9;22)(q34;q11.2)[6]/47,idem,+8[14]

Clones and subclones A 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 CD19 46,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

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