PMC

Numerical chromosome changes in 21 experimental EC tumors as identified by SKY analysis. As shown, chromosomal gains are less common, but more profound compared to chromosomal losses.

a Representative images of mitotic chromosome spreads depicting a normal karyotype and numerical changes including small-scale losses, small-scale gains and large-scale gains. b Dot plots depicting chromosome numbers in control cells and following SKP1 or CUL1 silencing in FT194 and FT246 cells. The horizontal dotted line identifies the modal number of chromosomes for control cells (n = 46). n = 2; 100 spreads analysed/condition. c Bar charts depicting the frequencies of small-scale (<10) or large-scale (≥10) chromosome gains and losses relative to the modal chromosome number in FT194 and FT246 cells. n = 2; 100 spreads analysed/condition.

Probe map of chromosome 14 and chromosome 1 with visualization of target genes, their sizes, and fluorescent dye used on visualization. (A) Map of break-apart probe of IGH gene capable of detected aberration: deletion, amplification, and breakage in gene; (B) Map of numerical probe on chromosome 1 able to detect only numerical changes: deletion and additions of material [].

Numerical chromosomal changes and uniparental disomy in pediatric ALL. (A) Frequency of pentasomy/tetrasomy/trisomy affecting each chromosome. For X chromosome, trisomy (105 cases) contains trisosomy X in male pateints (67 cases) and disomy X in female patients (38 cases). All tetrasomy X were female patients. (B) Frequency of uniparental disomy (UPD). Whole: cases with whole chromosome UPD; p arm: cases with UPD of short arm; and q arm: cases with UPD of long arm. Chr: chromosome. UPD involving X chromosome was detected only in female cases. (C) Distribution of whole and partial chromosome UPD in HD and non–HD-ALL. Whole chromosome UPD is frequently detected in HD-ALL. Thirty-four cases with whole chromosome UPD were HD-ALL. Partial UPD is frequently detected in non–HD-ALL. Fifteen of 63 cases with partial UPD were HD-ALL. (D) Frequency of deletion of p16INK4A gene in whole chromosome 9 UPD and 9p UPD. Twenty-three cases showed deletion of p16INK4A, out of a total of 30 cases with 9p UPD. One case had p16INK4A deletion from a total 18 ALL samples with whole chromosome 9 UPD.

This figure shows the chromosome specific effects in all metaphases examined. A) Percent of metaphases with structural alterations to chromosomes. B) Percent of metaphases with missing chromosomes. C) Percent of metaphases with additional chromosomes.

Gene Silencing Induces Numerical, Structural and Cohesion Chromosome Defects. (A) Representative mitotic chromosome spread from hTERT cells exhibiting the expected number (46) of chromosomes. (B) Aberrant chromosome spread displaying a small-scale numerical defect involving <15 chromosomes. (C) Large-scale numerical defect involving ≥15 chromosomes. (D) Endoreduplication resulting in paired homologous chromosomes (magnified inset) and a large-scale numerical defect. (E–G) Chromosome breakages (i.e., structural defects) increasing in severity from DNA double-strand breaks (DSBs) on individual chromatids (E; arrowheads), to extensive breaks along the length of the chromosomes (F and G; magnified insets). (H) Chromosome decompaction classified as a structural defect. Arrowhead highlights a region of chromosome decompaction. (I) Sister chromatid cohesion defects, where a spatial separation between sister chromatids is visually apparent at the primary constriction (centromere). (J) Bar graph displaying increases in the various chromosome defects indicated following gene silencing. The numbers above the bars identify the fold increase in total aberrant events for each gene relative to control (siControl). (K) Dot plot presenting the number of chromosomes enumerated following gene silencing. Kolmogorov–Smirnov (KS) tests reveal significant changes in the cumulative distribution frequencies as indicated (ns = not significant, * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, and **** = p-value < 0.0001).

Breakpoint positions (circles to the right) and numerical changes (lines; losses to the left and gains to the right) detected in the chromosome aberrations of 32 cases of bilateral ovarian cancer.

Detection of TP53 mutation in OVPA8 cell line. Sanger sequencing showed homozygous mutation c.733G>A (p.Gly245Ser in OVPA8 cells (A). NGS analysis confirmed this result (B), FISH analysis showed no numerical changes in chromosome 17 and TP53 signals. Green dots represent the chromosome 17 centromere, while orange dots represent TP53 (C).

Copy number alterations involve chromosome breaks. As aneuploidy strictly refers to numerical changes of whole chromosomes, segmental gains and losses require that part of a chromosome has obtained a different copy number from the remainder of the same chromosome. The example shows chromosome 11 from . Segmental gains and losses create a growth advantage by uncoupling the copy number of oncogenes (e.g. cycD1) and tumor suppressor genes (e.g. chk1) from general gene dosage. Frequently, the gains and losses over a single chromosome are complementary, as segments without gains are not copy number neutral but normally show losses. Intrachromosomal segment borders that delineate copy number alterations correspond to unprotected (reactive) ends that are functionally equivalent to breaks.

Examples of numerical chromosome instability (CIN) and structural CIN. A schematic depicting examples of the types of karyotypic changes associated with either numerical CIN (N-CIN) or structural CIN (S-CIN). Note that to accurately define CIN within a given population requires multiple distinct karyotypes to be identified as a single aberrant karyotype only defines a state, and not a rate. For illustrative purposes, the starting diploid cell (center) only contains three pairs of chromosomes (i.e., a partial karyotype). N-CIN involves whole chromosome gains or losses, including both small-scale changes that result in aneuploidy, as well as large-scale polyploidization events. S-CIN includes partial chromosome deletions, amplifications, inversions, or translocations (ranging in size from single genes to entire chromosome arms). These different classes of N-CIN or S-CIN are often combined to produce complex karyotypes that evolve over time. However, techniques for evaluating CIN typically only detect a subset of these karyotypic changes.

The karyotype of the CZ2 cell line. An example of classical cytogenetic analysis. (Chromosomal structural and numerical changes are indicated by arrows). The question mark means that the chromosome can not be fully identified.

Cytogenetic analyses and single-cell cloning. (A) TTAGGG-negative ends (red arrows) and a dicentric chromosome (green arrow) in SW480. (B and C) Anaphase bridges in DLD1 (B) and SW480 (C). (D) Loss of chromosomes X (arrow; centromere in green) and 18 (centromere in violet) through bridge formation in SW480. (E and F) Fragmented DNA (green) flanked by alphoid repeats (arrows; red) in SW480. (G and H) A dicentric chromosome 7 derivative (red chromosome paint in G; red arrow in H) and two normal chromosomes 12 (green chromosome paint in G) in HT29. (I) A ring chromosome positive with the 7q31 probe (green) but not with the chromosome 7 alphoid probe (red) in the HCT116 subclone M7. (J and K) A tetrapolar anaphase cell in hematoxylin-eosin staining (J) and a tripolar metaphase cell with three centrosomes (K)(γ-tubulin in orange) in SW480. (L–N) FISH analyses with centromeric probes in SW480 showing a tetrapolar metaphase cell with four copies of the X chromosome (green) and six copies of chromosome 18 (violet) (L), a tripolar anaphase cell with 2 + 2 + 0 segregation of chromosome X and 3 + 2 + 1 segregation of chromosome 18 (M), and a tripolar anaphase cell with 3 + 1 + 0 segregation of chromosome 18 (N). (O) Degenerating daughter cells of an isolated tetrapolar mitotic cell from the same cell line. (Magnifications: ×1,000, A, E, and I; ×100, B–D, F, and J–N; ×2,000, G and H; and ×40, O.)

Examples of copy number status of MYCN gene in NB tumors detected by interphase FISH examination (probes: red-MYCN locus-2p24.3 and green-control locus-2q11.2): (A) normal, (B) chromosome 2 numerical changes, (C) 2p gain, (D) amplification.

Abnormal Mitosis − Crystal Violet stain (100X).
a. Structural Alteration In Chromosomes: Stickiness Of Chromosomes.
b. Numerical Changes In Chromosome Complement: Abnormal Distribution Of Chromosomes.
c. Complete Or Partial Suppression Of Spindles: Unipolar And Tripolar Or Polypoid Spindles.
d. Combination Of Sticky Chromosomes And Abnormal Distribution Of Chromosomes And Complete Or Partial Suppression Of Spindles.

Karyotypic changes and their origins. Eukaryotic cells with euploid karyotype contain usually a diploid chromosome set. Chromosome segregation errors during mitosis result usually in whole chromosome alterations. Chromosome missegregation can occur through various defects in centromere, kinetochore, and spindle proteins. Unattached and lagging chromosomes can be detected by microscopy. DNA damage or replication stress can result in unrepaired or underreplicated DNA, which manifests itself as defect during anaphase as chromatin and/or ultrafine bridges that are visualized by immunostaining of associated proteins (such as PICH). Lack of repair and erroneous resolution of these bridges leads to chromosome breakage and subsequent losses or gains, insertions, deletions, inversions, or translocations. It should be noted that missegregation can also result in DNA damage and vice versa. DNA repair and replication defects may increase the occurrence of segregation errors. Karyotypes in cancer are frequently diverse, displaying combinations of structural and numerical changes. DNA staining (DAPI; cyan), immunostaining of a centromeric protein CENP-B (magenta), and the helicase PICH (red).

a. Idiogram of chromosome 2 on the left, accompanied by CGH+SNP results on the right. Array-CGH analysis reveals a duplication (arrow) and triplication (double arrow) of the short arm of chromosome 2. Whole chromosome LOH is demonstrated by homozygous allele tracks (AA and BB; AAA and BBB; AAAA and BBBB). b. Proposed mechanism of clonal evolution. The initial aberration is monosomy 2, followed by mitotic nondisjunction and intrachromosomal recombination, producing extra fragments of 2p. The presence of LOH for the entire chromosome 2 indicates that numerical aberration is a primary event, while structural abnormalities are secondary changes in clonal evolution. c. Idiogram of chromosome 13 on the left, accompanied by CGH+SNP results on the right. Array-CGH analysis reveals trisomy 13 by an increased log₂ ratio, and SNP array detected two homozygous allele tracks (AAA and BBB), indicating all three copies of chromosome 13 are identical. d. Proposed mechanism of clonal evolution. The initial aberration is monosomy 13, followed by mitotic nondisjunction, resulting in whole chromosome LOH and gain of chromosome 13. Karyotypes with gains of multiple chromosomes are commonly interpreted as hyperdiploid; however, the presence of LOH regions for multiple chromosomes strongly indicates a hypodiploid nature of the rearrangement, thus differentiating true hyperdiploidy from doubled low-diploidy or near-haploid clones and affecting prognostic significance.

The route to aneuploidy and its link to centrosome dysfunction. (A) Aneuploidy is caused by errors in chromosome partitioning during mitosis. Changes include whole chromosomes (numerical aneuploidy) often caused by altered expression or mutations in centrosome (i.e., CEP55) and cytokinesis (i.e., PRC1) regulators []. (B) Centrosome loss or structural changes in its components are early drivers of genomic instability causing both localized chromosome rearrangements and transient tetraploidy []. These alterations will generate intra-tumor heterogeneity and tumors containing a mix of cells with extra-numerical centrosomes or loss of its components. In general, little is known about the mechanisms of centrosome loss. However, centrosomes are normally inactivated or lost during specific developmental stages in different animals. Abbreviations: Chromosome, Chr; CIN; Chromosome instability.

Karyotypic analysis of MB cell lines. a G-banded karyotype of the hyperdiploid MB cell line UW402, showing both complex structural and numerical changes. b–c Representative karyotypes of the MB cell line UW473 showing b hyperdiploid and c near-tetraploid karyotypic populations. Arrows point to main chromosomal alterations. M marker chromosome

Diagram depicting human cells undergoing normal versus aberrant chromosome segregation that leads to CIN. Normal anaphase and cell division allows high fidelity separation of chromosomes between daughter cells and maintenance of genome stability and normal chromosomal content. Lagging DNA during anaphase can result in chromosome missegregation during cell division. Chromosomal instability (CIN) is the persistent rate of chromosome segregation errors every cell division in a cell population, CIN can be numerical which leads to unbalanced chromosome numbers or Aneuploidy, or structural in which results in damaged, broken, or rearranged chromosomes. CIN fuels the accumulation of further genomic changes promoting cancer progression, metastasis, and therapy resistance

Karyotypic analysis of MDA-MB-231 breast cancer cells. (A). A representative G-banded karyotype of MDA-MB-231 cell line. (B). Composite G-banded karyotype of the near-triploid cell line MDA-MB-231, showing structural and numerical changes. Arrows point to main chromosomal alterations. Mar—Marker chromosome. (C,D,F,G). The representative chromosome aberrations in β-actin (C,D) and γ-actin (F,G) -depleted MDA-MB-231 cells: dicentric chromosomes (C,F) and acentric fragment (C), ring chromosomes (D,G). Arrows point to aberrations. E, H. Endoreduplication in β-actin (E) and γ-actin (H) -depleted MDA-MB-231 cell.