UNDERSTANDING YOUR CLINICAL CYTOGENETICS REPORT

INTRODUCTION

Cytogenetics is the branch of genetics that studies chromosome structure and function. Chromosomes (Greek: chromos = colored; soma = body) are present in all nucleated cells and contain DNA with associated acidic and basic proteins. Chromosomes undergo a cycle of condensation and decondensation throughout the cell cycle. Chromosomes are maximally decompacted during interphase and achieve maximum compaction during the metaphase stage of mitosis, just prior to the separation of sister chromatids [Figures 1, 2]. The establishment of the correct diploid chromosome complement (karyotype) of 46 in man (Tjio and Levan, 1956) led to the discovery of major human constitutional chromosomal syndromes and later the observation of acquired chromosome abnormalities in hematologic malignancies and solid tumors. The 46 chromosomes in the normal human karyotype are arranged in decreasing order of size as 23 matching or homologous pairs in a karyogram [Figure 3], with 22 pairs of autosomes and the sex chromosomes (XX female, XY male). One of each pair of autosomes and the X are of maternal origin, while the Y and the remaining autosomes are of paternal origin. Each chromosome is composed of two chromatids joined by a centromere, which is the site of attachment of the spindle fibers. The spindle fibers draw the chromatids to opposite poles during cell division. The position of the centromere is constant for a given chromosome, with three subgroups [Figure 4]:

Metacentric – centromere in the middle of the chromosome (chromosomes 1, 3, 16, 19, 20) Submetacentric – intermediate position of the centromere (chromosomes 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 17, 18, X) Acrocentric – centromere close to one end of the chromosome (chromosomes 13, 14, 15, 21, 22, Y)

The centromere divides the chromosome into short (p) and long (q) arms. The telomeres “cap” the p and q arms and are important for structural integrity of the chromosome. Individual chromosomes are identified by their relative size, the position of the centromere and the banding pattern (see banding technique).

ELEMENTS OF THE STANDARD CYTOGENETIC REPORT Specimen Type

It is important that representative tissue be sent to the laboratory for analysis. Only those cells involved in the neoplastic process will harbor the abnormalities being sought. Specimens should be collected under sterile conditions. Bone marrow aspirate, bone core and peripheral blood (if circulating blast percentage is higher than 10%) samples are routinely analyzed for the diagnosis of acute myeloid leukemia. Heparinized (sodium heparin) samples should be transported (at ambient temperature) to the laboratory in a timely manner (ideally within 24 hours) for tissue culture. Bone marrow smears and touch preparations of the bone marrow core biopsy can also be used for directed fluorescence in situ hybridization (FISH) analyses (see below). Tissue samples may also be analyzed in the setting of extramedullary leukemia (myeloid sarcoma, chloroma). Sample Set-up and Processing

Conventional cytogenetic analysis requires cells to be actively in cell division. Direct and short-term (24-48 hours) unstimulated cell culture and mitotic arrest with a spindle inhibitor such as Colcemid is followed by treatment with hypotonic saline to facilitate cell membrane disruption and fixation with methanol/acetic acid to create permanent preparations that can be “dropped” onto slides for staining and microscopic analysis. Banding Technique

Slide preparations are treated chemically (enzymatic digestion) and stained with a DNA-binding dye (Giemsa) to reveal chromosome specific patterns of light and dark bands that are microscopically visible (G-banding). G-band dark bands appear to contain relatively few active genes, are AT-rich and replicate late in S-phase. Light bands contain about 80% of active genes, are relatively GC-rich and replicate early in S-phase. Band Level and Identification

An International System for Human Cytogenetic Nomenclature (ISCN 2016) describes a standardized numbering pattern for chromosome identification based on the banding pattern. This uniform system of nomenclature is the result of multiple consensus conferences that began in 1960 and continue today. A band is defined as a chromosomal area that is distinguishable from adjacent segments by appearing lighter or darker. A standardized numbering system for bands seen with G-banding is shown diagrammatically in the human idiogram [Figure 5]. This system permits accurate description of breakpoints in chromosomal rearrangements. Each chromosome in the ideogram is divided into a number of chromosome regions using the ends, centromere, and most prominent G-bands as landmarks. Regions or bands are numbered consecutively from the centromere outward along the chromosome arm. In designating a band, four items are required: the chromosome number, the arm symbol, the region number, and the band within that region. These items are given in consecutive order without spacing or punctuation. For example, 5q33 means chromosome 5, long arm, region 3, band 3.

In a cytogenetics report, the primary quality indicator is the number of observable bands. The banding level of resolution in hematologic malignancies is in the 300 to 400 band range. Conventional cytogenetic analysis provides a low DNA resolution scan of the entire genome: 100 genes/band, 9 x 106 base pairs/band and 9Mb band width are observed at the 350 band level.

Number of Cells Counted/Analyzed

To determine if abnormalities are present, a minimum of 20 cells (metaphase preparations) are analyzed. In each cell, the number of chromosomes is enumerated and each chromosome homolog is identified band for band. Computerized systems for image capture and analysis assist in karyogram preparation and allow for electronic storage of images, however, trained cytogenetic technologists are responsible for this individual cell analysis. A minimum of two karyograms are produced per case, with two additional karyograms per abnormal cell line. ISCN Results

The ISCN provides specific guidelines for reporting cytogenetic results. In the karyotypic description, the first item recorded is the total number of chromosomes followed by a comma (,). The sex chromosome constitution is given next. Thus, a normal female karyotype is written as 46,XX. The normal male karyotype is 46,XY. Punctuation is very important in cytogenetic nomenclature. All chromosome numbers are followed by a comma and no spaces are used to separate chromosome number from sex chromosome content. To specify structurally altered chromosomes, single and three letter abbreviations are used. The number of the chromosome or chromosome involved in the rearrangement is specified within parentheses immediately following the symbol indicating the type of rearrangement. If two or more chromosomes are altered, a semicolon (;) is used to separate their designations. If one of the rearranged chromosomes is a sex chromosome, this is listed first; otherwise, the rule is that the lowest chromosome number is cited first. The breakpoints, given within the parentheses, are specified in the same order as the chromosomes involved, and a semicolon is again used to separate the breakpoints. Punctuation is never used in intrachromosomal rearrangements, that is, to separate breakpoints in the same chromosome. When reporting results, the number of cells analyzed is given in square brackets at the end of the karyotypic description. If multiple clonal populations are present, the karyotypic description of each clone is separated by a slant line (/). The internationally accepted operational definition states that a clone exists if two or more cells are found with the same structural abnormality or chromosomal gain. If the abnormality is a missing chromosome, the same change must be present in at least three cells analyzed. The stemline indicates the most basic clone in a cell population. All additional deviating clonal findings are termed sidelines.

The modal number is the most common chromosome number in the population analyzed. The modal number is hypodiploid when the mode is less than 46 chromosomes, and hyperdiploid when the mode is greater than 46 chromosomes. Hypodiploidy and hyperdiploidy are both

examples of aneuploidy, where the incorrect number of chromosomes are present. Loss of a chromosome is described as monosomy. Three copies of a chromosome is trisomy, four copies is tetrasomy. Karyotypes with a normal chromosome number, but contain numerical and/or structural abnormalities, are described as pseudodiploid.

A Glossary of Common ISCN Abbreviations/Punctuation Used to Describe Karyotypes

add Indicates additional material of unknown origin attached to a chromosome region or a

band. Latin, additio.

46,XX,add(12)(p13)

~ Approximate sign, indicates uncertainty in chromosome or band designation. Arrow, meaning from to [ ] Square brackets. Surrounds number of cells that constitute a clone. c A constitutional anomaly is indicated by the letter c after the abnormality designation.

48,XX,+8,+21c Tumor cells with a constitutional trisomy 21 and an acquired trisomy 8. : Colon. Used to indicate a chromosome break. :: Double colon. Used to indicate a chromosome break and reunion. , Comma. Separates chromosome numbers, sex chromosomes and chromosome

abnormalities in the karyotype. cp Composite karyotype. Contains all clonally recurring abnormalities in setting of

karyotypic heterogeneity.

45~48,XX,del(3)(p12)[2],-5[4],+8[2],+11[3][cp7] The total number of cells in which the clonal changes were observed is given in square brackets after the karyotype, preceded by the symbol cp. The number of cells with a particular abnormality is shown in square bracket after each abnormality. The range of chromosome numbers in metaphase cells is written first.

del Deletion. A deletion is a loss of a chromosome segment [Figure 6].

46,XX,del(5)(q13) 46,XX,del(5)(pterq13:)

Terminal deletion with a break (:) in band 5q13. The remaining chromosome consists of the entire short arm of chromosome 5 and part of the long arm lying between the centromere and band 5q13.

46,XX,del(5)q13q33) 46,XX,del(5)(pterq13::q33qter) Interstitial deletion with breakage and reunion (::) of bands 5q13 and 5q33. The segment lying between these bands has been deleted.

der Derivative chromosome. A structurally rearranged chromosome generated

either by a rearrangement involving two or more chromosomes or by multiple rearrangements within a chromosome with a net result of deletion and duplication of genetic material.

45,XY,der(1;7)(q10;p10)

A derivative chromosome consisting of the long arm of chromosome 1 and the short arm of chromosome 7. The missing chromosomes 1 and 7 are not indicated because they are replaced by the derivatives. There is one normal chromosome 1, one normal chromosome 7, and the der(1;7) present, with the net result being loss of 1p and 7q.

46,XY,+1,der(1;7)(q10;p10)

Two normal chromosome 1s, one normal chromosome 7 and the der(1;7) with the net result of duplication of 1q and loss of 7q.

47,XY,t(9;22)(q34;q11.2),+der(22)t(9;22)(q34;q11.2)

A derivative chromosome 22 resulting from the t(9;22). dic Dicentric, indicates the presence of two centromeres. dmin Double minute. dup Duplication. A gain of a chromosome segment observed at the original

chromosome location [Figure 7].

46,XX,dup(1)(q21q31) ins Insertion. A chromosomal segment is inserted at a different point in the same

chromosome or in a different chromosome. i Isochromosome. A mirror image duplication of a chromosome arm is formed. The

breakpoints in isochromosomes are assigned to the centromeric bands p10 and q10 according to the morphology of the isochromosome [Figure 8].

46,XY,i(17)(q10)

An isochromosome for an entire long arm of chromosome 17. There is loss of the short arm of chromosome 17 and one normal copy of chromosome 17 present.

idem Denotes the stemline karyotype in a subclone. Latin = same. 47,XX,del(5)(q13q33),+8 46,idem,-7

Idem refers to the karyotype listed first, additional changes to the stemline (basic clone), in this example, monosomy 7, are then listed.

ider Isoderivative chromosome. Designates an isochromosome formation for one of the arms

of a derivative chromosome.

46,XX,ider(17)(q10)t(15;17)(q24;q21.1) An isochromosome for the long arm of the der(17) generated by a t(15;17). idic Isodicentric chromosome. An isochromosome with two centromeres present. inc The symbol inc placed at the end of the karyotype string denotes that the karyotype

presented is incomplete, usually due to the poor chromosome quality. It is possible that the karyotype may contain unidentified structural or numerical changes in addition to the abnormalities listed.

48,XY,+8,inc

ins Insertion. A chromosomal segment is inserted at a different point in the same

chromosome or in a different chromosome. inv Inversion. A portion of a chromosome is rotated 180° [Figure 9].

46,XX,inv(16)(p13q22) A pericentric inversion, the inverted segment includes the centromere.

46,XX,inv(3)(q21.3q26.2) A paracentric inversion, the inverted segment does not include the centromere. mar Marker chromosome. A structurally abnormal chromosome that cannot be

unambiguously identified or characterized by conventional chromosome analysis. min Minute acentric fragment. - Minus sign, loss. Placed before a chromosome or abnormality. x Multiplication sign, multiple copies. or Alternative interpretation. p Short arm of chromosome. ( ) Parentheses. Surrounds chromosome numbers and abnormalities.

+ Plus sign, gain. Placed before a chromosome or abnormality. pter Terminal end of short arm of chromosome. qter Terminal end of long arm of chromosome. ? Question mark. Notes uncertainty in chromosome or band designation. r Ring chromosome. ; Semicolon. Separates altered chromosomes and breakpoints in structural rearrangements

involving more than one chromosome. / Slant line. Separates karyotype designations of different clones. // Slant line, double. In chimerism secondary to bone marrow transplant, the recipient cell

clones are listed first, followed by the donor cell line. The recipient and donor cell lines are separated by a double slant line.

46,XY[3]//46,XX[17]

Three cells from the male recipient were identified along with 17 cells from the female donor.

t Translocation. Interchange or relocation of chromosome segments between two or more

chromosomes. No visible loss of genetic material in reciprocal, balanced translocation [Figure 10].

46,XX,t(8;21)(q22;q22)

Interpretation

The interpretation of results should be clear to a non-geneticist physician and should include a narrative description of the abnormalities observed including modal chromosome number in each clone, and numerical and structural abnormalities. The report should comment on the clinical significance of the abnormalities observed, including clinically relevant genes involved, possible disease association and prognostic significance. The term complex aberrant is designated to describe a karyotype with multiple unrelated cytogenetic abnormalities. In AML, karyotypes with 3 or more aberrations are classified as complex, adverse risk according to the recommendation of the European Leukemia Net (ELN). A monosomal karyotype is defined as the presence of at least 2 autosomal monosomies or a single autosomal monosomy associated with at least one structural abnormality.

FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ANALYSES

Fluorescence in situ hybridization (FISH) detects and localizes specific DNA sequences using fluorescently labeled complementary DNA probes. The principle is based on the property of double-stranded DNA to denature on heating to form single-stranded DNA. On cooling, the single-stranded DNA reanneals with its complementary sequence to reform double-stranded DNA. If an appropriately labelled segment of DNA (probe) is added to denatured chromosomes on a microscope slide during the process of reannealing, some of the labelled DNA will hybridize to its complementary sequence in the chromosome. Detection of the labelled DNA under the microscope identifies the chromosomal site of the complementary sequence. FISH studies may be performed on previously fixed metaphase and interphase cell preparations, touch preps, smears and formalin-fixed, paraffin-embedded tissue. Interphase FISH is exceptionally useful as an adjunct to conventional cytogenetic analysis. Because mitotic cells are not required, interphase analysis makes it practical to examine large numbers (50-500) of cells and cells from samples that have low (or no) mitotic index. FISH is not a replacement for conventional cytogenetic analyses as FISH detects only its intended targets and may give no information about additional abnormalities that may signal disease progression or secondary disease. The test is rapid, with a turn-around of a few hours to overnight. Building upon sequence data available from the Human Genome Project, probes can be produced for the study of almost any chromosomal site. The majority of probes used for clinical purposes are commercially manufactured and sold as analyte-specific reagents (ASRs) that must be validated by each laboratory. Most FISH probes fall into one of three categories: repetitive sequence, whole chromosome, or unique sequence. The most widely used repetitive sequence probes are for the alpha satellite sequences at the centromeres of human chromosomes. Each chromosome’s alpha-satellite sequence is sufficiently divergent to allow for the development of centromere specific probes (exceptions are chromosomes 5, 13, 14, 21, 22). Whole chromosome probes or “painting” probes are composed of unique and moderately repetitive sequences from an entire chromosome or chromosomal region. Unique sequence DNA probes can be used to examine a particular area for copy number or location. Analytic strategies include counting centromere specific probes to confirm chromosome number, break-apart probes that detect involvement of genes in structural rearrangement and dual-color dual fusion probes to detect translocation events. [Figure 11].

ISCN FISH nomenclature

FISH reporting has also been standardized by the ISCN committees. Several brief interphase FISH examples will be provided to illustrate the results of the different analytic strategies described above. To indicate the number of signals, the symbols nuc ish are followed immediately in parentheses by the location designation, a multiplication sign (x), and the number of signals seen. nuc ish(DXZ1 x 2)[200]

Two copies of the locus DXZ1 alpha satellite probe on the X chromosome, observed in 200 interphase nuclei.

nuc ish(D8Z1 x 3)[175/200]

Three copies of the D8Z2 alpha satellite probe on chromosome 8, observed in 175/200 interphase nuclei.

nuc ish(D17Z1 x 2,TP53 x 1)[50/200]

Two copies of the D17Z1 alpha satellite probe on chromosome 17 and one copy of TP53 (mapped to the short arm of chromosome 17) in 50/200 interphase nuclei.

46,XY[20].nuc ish(D7Z1,D7S486 x 1)[25/200]

Conventional chromosome analysis and FISH performed, each reported with the string, separated by a period (.). Normal karyotypic findings in 20 cells. FISH analysis detected monosomy 7 in 25/200 interphase nuclei. Probes on chromosome 7 include the alpha satellite centromeric probe and locus-specific probe in commonly deleted region of 7q.

Interphase FISH may be used to determine donor versus recipient in the transplant setting. See the glossary for discussion of double slant line use. nuc ish(DXZ1 x 2)[500]// 500 cells all representing the XX recipient. //nuc ish(DXZ1,DYZ3) x 1[500] 500 cells representing the XY donor. nuc ish(DXZ1 x 20[125]//(DXZ1,DYZ3 x 1)[375] 125 recipient XX cells and 375 donor XY cells found using the X and Y centromere probes. The normal signal pattern in dual color, breakapart rearrangement probes appears to be two “fusion” signals, reflecting the two color make-up of the probe. nuc ish(MLL x 2)[200]

Two MLL probe fusion signals in interphase cells, indicating no disruption of the KMT2A gene.

nuc ish(MLL x 2)(5’ MLL sep 3’ MLL x 1)[200] Two MLL probe signals, but one has separated into the 5’ probe and the 3’ probe, presumably because of a translocation involving the KMT2A gene.

If loci on two separate chromosomes are tested, they are expected under normal circumstances to be spatially separated and results are expressed as follows:

nuc ish(ABL1,BCR) x 2[200] However, if they have become juxtaposed on two chromosomes using dual-fusion probes, the results are expressed with the first set of parentheses indicating the number of signals and the second set of parentheses describing the relative position of the signals to one another: nuc ish(ABL1,BCR) x 3(ABL1 con BCR x 2)

REFERENCES

Atlas of Genetics and Cytogenetics in Oncology and Haematology. http://atlasgeneticsoncology.org. Frohling S, Dohner H. Chromosomal abnormalities in cancer. N Engl J Med. 2008; 359(7):722-734. Gersen S, Keagle M, eds. Principles of Clinical Cytogenetics. 2nd ed. Totowa, New Jersey: Humana Press; 2005. Heim S, Mitelman F, eds. Cancer Cytogenetics. 4th ed. Oxford: Wiley-Blackwell; 2015. McGowan-Jordan J, Simmons A, Schmid M, eds. ISCN 2016: An International System for

Human Cytogenomic Nomenclature. Basel: S Karger; 2016. Korf BR, Irons MB, eds. Human Genetics and Genomics. 4th ed. Oxford: Wiley-Blackwell; 2013. Mascarello JT, Hirsch B, Kearney HM et al. Section E9 of the American College of Medical Genetics Technical standards and guidelines: fluorescence in situ hybridization. Genet Med.

2011;13(7):667-675. Mikhail FM, Heerema N, Rao KW et al. Section E6.1-6.4 of the ACMG technical standards and guidelines: chromosome studies of neoplastic blood and bone marrow-acquired chromosomal abnormalities. Genet Med. 2016;18(6):635-642. Tobias ES, Connor M, Ferguson-Smith M, eds. Essential Medical Genetics. 6th ed. Oxford: Wiley-Blackwell; 2011. Zneimer SM, ed. Cytogenetic Abnormalities: Chromosomal, FISH and Microarray-Based

Clinical Reporting. Oxford: Wiley-Blackwell; 2014.

LEGENDS

Figure 1 Mitosis. Schematic representation of two pairs of chromosomes undergoing cell division: (a) interphase, (b) prophase, (c) metaphase, (d) anaphase, (e) telophase, (f) cytokinesis, and (g) interphase of the next cell cycle. Figure 2 G-banded metaphase preparation. Figure 3 Normal male karyogram:. 46,XY. Figure 4 The functional and structural components of metaphase chromosomes. Figure 5 Schematic presentation (ideogram) of the G-banded human male chromosome complement. Figure 6 Deletion Figure 7 Duplication Figure 8 Isochromosome Figure 9 Inversion Figure 10 Translocation Figure 11 Common FISH signal patterns ** Figures 1 and 4 were sourced from Gersen and Keagle text. Figures 5-10 were sourced from Heim and Mitelman text. Figure 11 is vendor-supplied.

Figure 1 Figure 2

Figure 3 Figure 4

Figure 5 Figure 6

Figure 7 Figure 8

Figure 9 Figure 10

Figure 11