In both techniques, DNA from a reference or control sample and DNA from a test or patient sample are differentially labelled with two different fluorophores and used as probes that are cohybridized competitively onto nucleic acid targets. In conventional CGH, the target is a reference metaphase spread. Figure 2. DNA from the sample to be tested is labeled with a red fluorophore Cyanine 5 and a reference DNA sample is labeled with green fluorophore Cyanine 3. Equal quantities of the two DNA samples are mixed and cohybridized to a DNA microarray of several thousand evenly spaced cloned DNA fragments or oligonucleotides, which have been spotted in triplicate on the array.
After hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores. If the intensities of the flurochromes are equal on one probe, this region of the patient's genome is interpreted as having equal quantity of DNA in the test and reference samples; if there is an altered Cy3:Cy5 ratio this indicates a loss or a gain of the patient DNA at that specific genomic region.
Array CGH has been implemented using a wide variety of techniques.
- Hitlers Last Day: Minute by Minute.
- Log in to Wiley Online Library.
- Medieval Islamic Civilization: An Encyclopedia.
- London: The Biography.
- Canine Cytogenetics - From band to basepair - Europe PMC Article - Europe PMC?
- The Future of the Mind: The Scientific Quest to Understand, Enhance, and Empower the Mind;
Therefore, some of the advantages and limitations of array CGH are dependent on the technique chosen. The use of BACs provides sufficient intense signals to detect single-copy changes and to locate aberration boundaries accurately. These techniques include ligation -mediated polymerase chain reaction PCR , degenerate primer PCR using one or several sets of primers, and rolling circle amplification. These arrays currently yield a high spatial resolution, but the number of cDNAs is limited by the genes that are encoded on the chromosomes, and their sensitivity is low due to cross-hybridization.
The amount of oligos is almost infinite, and the processing is rapid, cost-effective, and easy. Although oligonucleotides do not have the sensitivity to detect single copy changes, averaging of ratios from oligos that map next to each other on the chromosome can compensate for the reduced sensitivity. There are two approaches to the design of microarrays for CGH applications: whole genome and targeted. Whole genome arrays are designed to cover the entire human genome.
They often include clones that provide an extensive coverage across the genome; and arrays that have contiguous coverage, within the limits of the genome.
Whole-genome arrays have been constructed mostly for research applications and have proven their outstanding worth in gene discovery. They are also very valuable in screening the genome for DNA gains and losses at an unprecedented resolution. Targeted arrays are designed for a specific region s of the genome for the purpose of evaluating that targeted segment. It may be designed to study a specific chromosome or chromosomal segment or to identify and evaluate specific DNA dosage abnormalities in individuals with suspected microdeletion syndromes or subtelomeric rearrangements.
The crucial goal of a targeted microarray in medical practice is to provide clinically useful results for diagnosis, genetic counseling, prognosis, and clinical management of unbalanced cytogenetic abnormalities. Conventional CGH has been used mainly for the identification of chromosomal regions that are recurrently lost or gained in tumors, as well as for the diagnosis and prognosis of cancer. Furthermore, conventional CGH can be used in detecting chromosomal abnormalities and have been shown to be efficient in diagnosing complex abnormalities associated with human genetic disorders.
CGH data from several studies of the same tumor type show consistent patterns of non-random genetic aberrations. For example, gains of chromosomal regions lq, 3q and 8q, as well as losses of 8p, 13q, 16q and 17p, are common to a number of tumor types, such as breast, ovarian, prostate, renal and bladder cancer Figure. Other alterations, such as 12p and Xp gains in testicular cancer, 13q gain 9q loss in bladder cancer, 14q loss in renal cancer and Xp loss in ovarian cancer are more specific, and might reflect the unique selection forces operating during cancer development in different organs.
Cri du Chat CdC is a syndrome caused by a partial deletion of the short arm of chromosome 5. For example, Levy et al. CGH analysis revealed a loss of chromosomal material from 5p These results demonstrate that conventional CGH is a reliable technique in detecting structural aberrations and, in specific cases, may be more efficient in diagnosing complex abnormalities. Array CGH applications are mainly directed at detecting genomic abnormalities in cancer. However, array CGH is also suitable for the analysis of DNA copy number aberrations that cause human genetic disorders.
Earlier diagnosis is of benefit to the patient as they may undergo appropriate treatments and counseling to improve their prognosis. Genetic alterations and rearrangements occur frequently in cancer and contribute to its pathogenesis. Detecting these aberrations by array CGH provides information on the locations of important cancer genes and can have clinical use in diagnosis, cancer classification and prognostification. In a recent study, array CGH has been implemented to identify regions of chromosomal aberration copy-number variation in several mouse models of breast cancer, leading to identification of cooperating genes during myc-induced oncogenesis.
Array CGH may also be applied not only to the discovery of chromosomal abnormalities in cancer, but also to the monitoring of the progression of tumors. Differentiation between metastatic and mild lesions is also possible using FISH once the abnormalities have been identified by array CGH. Prader—Willi syndrome PWS is a paternal structural abnormality involving 15q, while a maternal aberration in the same region causes Angelman syndrome AS.
- Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors!
- How to Survive the Recession and the Recovery.
- Route 15.
- Cycles in US Foreign Policy since the Cold War.
- Login using!
As a proof of principle Vissers et al. In all three cases, the abnormalities, ranging from 1. When using overlapping microarrays, it is also possible to uncover breakpoints involved in chromosomal aberrations. Though not yet a widely employed technique, the use of array CGH as a tool for preimplantation genetic screening is becoming an increasingly popular concept. It has the potential to detect CNVs and aneuploidy in eggs, sperm or embryos which may contribute to failure of the embryo to successfully implant, miscarriage or conditions such as Down syndrome trisomy This makes array CGH a promising tool to reduce the incidence of life altering conditions and improve success rates of IVF attempts.
The technique involves whole genome amplification from a single cell which is then used in the array CGH method. It may also be used in couples carrying chromosomal translocations such as balanced reciprocal translocations or Robertsonian translocations, which have the potential to cause chromosomal imbalances in their offspring.
A main disadvantage of conventional CGH is its inability to detect structural chromosomal aberrations without copy number changes , such as mosaicism , balanced chromosomal translocations , and inversions. CGH can also only detect gains and losses relative to the ploidy level. Although CGH has proven to be a useful and reliable technique in the research and diagnostics of both cancer and human genetic disorders, the applications involve only gross abnormalities. Because of the limited resolution of metaphase chromosomes, aberrations smaller than 5—10 Mb cannot be detected using conventional CGH.
Array CGH overcomes many of these limitations. The standard resolution varies between 1 and 5 Mb, but can be increased up to approximately 40 kb by supplementing the array with extra clones. However, as in conventional CGH, the main disadvantage of array CGH is its inability to detect aberrations that do not result in copy number changes and is limited in its ability to detect mosaicism.
From Wikipedia, the free encyclopedia. Molecular Pathology — Annu Rev Genom Hum Genet — The move towards array comparative genomic hybridization CGH. European Journal of Pediatrics — Human Genetics. Genes, Chromosomes and Cancer — Nature genetics — NZ Med J — Cancer Science. BioMed Research International Aneuploidy may induce genome instability and lead to acquisition of genetic cell heterogeneity triggering selective pressures in clones selection. Therefore, the survival of cells with CIN to the cancer treatment could be due to higher adaptive potential of these cells in which specific chromosomal aberrations confer cellular fitness advantages Despite aneuploidy commonly occurs in many cancers where it is an indicator of tumor growth, it often leads to a reduction in the cell proliferation rate 32 , This apparent contradiction is known as the: aneuploidy paradox.
The rate of CIN determines the effect of aneuploidy on tumors; whereas low rates of CIN missegregation of a small number of chromosomes per division are weakly tumor promoting, higher rates of CIN missegregation of more than five chromosomes cause cell death and tumor suppression Thus, cell death induced by CIN sufficiently high arises from an increase in the number of chromosomes missegregated per cell division.
Coherently with this, Komarova described a mathematical model showing that a low rate of CIN optimizes the tumor heterogeneity and survival and that increase of CIN rate is associated with decreases tumor fitness These observations suggest that increasing the rate of CIN over a critical threshold could be efficient to stop the tumor cell proliferation.
This paradoxical situation is attracting considerable attention for therapeutic purpose, although the therapeutic targeting of CIN in cancer is still at preclinical stages.
Chromosome Abnormalities and Cancer Cytogenetics
Several oncogenes and tumor suppressor genes are known to localize within centrosomes, which altered function triggers centrosome abnormalities Tubulin is another important target, as this protein acts during cellular growth, division, and migration. Taxanes paclitaxel and docetaxel and vinca alkaloids vinblastine, vincristine, and vinorelbine , well-known FDA-approved compounds clinically used for targeting tubulin, have been demonstrated to be successful to induce mitotic arrest Additional cancer therapy strategy that has attracting attention in recent years is synthetic lethality 39 , defined as a condition in which perturbation in two different genes together results in cell death but mutation of either alone is compatible with cell life.
Although the evaluation of CIN rate in tumor samples is not routinely performed in the clinical setting, direct and indirect methods to measure CIN have been adopted; these methods are based on both the determination of cell-to-cell variability in chromosome number and structure within the tumor cell population, as well as on the assessment of the rate at which these chromosomal changes occur Therefore, the methods are able to capture the dynamic nature of CIN. The CIN rate is directly related to the estimation of the mitotic error frequency in fixed cells or in formalin-fixed and paraffin-embedded FFPE tumor tissues.
Some of main defects are summarized in Fig. However, these technical approaches are difficult to apply in tumors with a low proliferation index and in tumors in which the anaphases are not clearly observed. CIN is not only a consequences of compromised mitotic apparatus but it may also arise after DNA damage or as a consequence of impaired replication fork progression Defective DNA damage response and repair results in chromosomal aberration such as deletions, amplifications, inversions and translocations It allows to quantify the variations in chromosome copy number across the cell population by using fluorescently labeled DNA probes that bind to centromeres of specific chromosomes.
Thus, FISH evaluates the chromosomal state of hundreds of cells, inferring the rate of change in chromosome number from the cell-to-cell variability Another important method is the single-cell comparative genomic hybridization CGH This assay allows the selection of individual cancer cells based on their deviation from normal cells. However, single-cell CGH is not amenable to high-throughput analysis and it is characterized by a considerable economic burden.
Then, CIN status can be measured by the stemline scatter index SSI , which is a measure of the clonal heterogeneity of the constituent tumor cells Finally, a more detailed picture of the genomic landscape can be obtained through Next Generation Sequencing NGS systems, which assume massively parallel sequencing techniques Recently, different models have been used to measure CIN in vivo like mouse models that were engineered to mimic genetic alteration driving CIN, mouse embryonic fibroblast MEFs from mouse models of genetically induced CIN 48 and organoid cultures, that allow to monitoring chromosome segregations using three-dimensional live-cell imaging Measuring CIN in vivo would more accurately show the effect of CIN for instance during development, the possible role of the immune system and inter-tissue interactions.
However, drawbacks of in vivo CIN measurement are the limited time available for imaging, the high cost and the relatively low rate of cell division in vivo Despite the studies performed so far, none of the methods used to study CIN is entirely satisfactory, thus novel approaches for an accurate detection and assessment of CIN will be critical both in clinical setting and to therapeutic targeting of CIN in the future.
NB is an embryonic tumor that can be present in fetus. Indeed, some newborn patients exhibit tumors after only a few days of life; usually, these patients have a very good OS.
- Cytogenetics and Chromosomal Structural Diversity | SpringerLink?
- Forgotten Statistics: A Refresher Course with Applications to Economics and Business;
- Making Vintage Accessories.
- A Postgenomic Perspective on Molecular Cytogenetics.
- Materials and Methods.
This, of course, is in contrast with the presence of structural CNVs in tumor of HR patients older than one year of age, suggesting an evolution of tumor aggressiveness associated with CIN. The lapse time between fetus life and infant at one age of year suggest a time-dependent increase of chromosome damages As mentioned above, cell replication errors and abnormal chromosome segregation during mitosis could originate the abnormal chromosome pattern triggering the cell to increase their aggressiveness. Masecchia et al 52 used a learning machine algorithm to show that numerical chromosome aberrations occurs early than structural ones.
It is to note that NB is originating from neural crest cells, a group of cells located on the neural tube and undergoing to epithelial to mesenchymal transition EMT during the embryonic life. These cells are migrating in the early phases of embryonic development and some of them take part to the formation of gastric ganglia and adrenal gland, two sites in which NB growths and develops. Mouse and zebrafish models have been developed demonstrating that MYCN oncogene is one of the major actors in the NB development This study needs a more accurate animal model.
Considering the strong relationship between CIN and aneuploidy, about a decade ago, a computational method was developed to represent aneuploidy in relation to the expression of genes localized in aberrant chromosomal region functional aneuploidy profile. Thus, the functional aneuploidy as a measure of the total status of chromosomal imbalance, was inferred using gene expression data of a given tumor Carter et al 55 showed 70 genes whose expression was correlated with total functional aneuploidy in several cancer types: the CIN70 gene signature able to measure the state of karyotype and to predict clinical outcome in several human cancers.
The CIN70 signature was obtained including most of genes involved in cellular processes critical for genome integrity maintenance such as DNA replication, chromosomal condensation, segregation, de-condensation and structure and genes of cell cycle, spindle apparatus and mitosis. Carter's study provided a means to assess the potential role of CIN in tumors initiation. This dataset provided expression data of 45 genes out of 70 genes included in the CIN70 signature. Above the heatmap are the indicated patient clinical features and the patients' stage.
On the left of the panel is a key of the staging colors applied. On the right of the panel are the 45 genes out of CIN Moreover, Carter et al 55 produced another CIN selecting the top 25 genes best predicting clinical outcome. So that, our preliminary data show that CIN25 gene signature is strongly associated with NB with poor outcome. Food and drug administration approved drugs targeting chromosome instability-associated genes in neuroblastoma.
All drugs are small molecule drug inhibitor of the respective genes except daunorubicin of which the type of interaction is unknown. The disease report links associated with the pathology lists were treated with the illustrated drug according to the indications derived from www. CIN is an old genomic aspect that is recently emerged as causative of cancer.
Today with the advent of NGS, there is an additional possibility to study in deep this phenomenon.
Cytogenetic and Genome Research
So that, it should be useful to initiate a CIN screening on several cancers which can be exploited for targeted cancer therapy. While numerous studies demonstrated that CIN may promote tumorigenesis, primarily through the functional loss of key players governing chromosome stability, it has also been shown that CIN beyond tolerable levels actually leads to cell death and tumor suppression These observations collectively suggest elevating CIN as a potential chemotherapeutic strategy in a genetically sensitized background, but additional studies would be needed to validate the efficacy and effectiveness of this approach to cancer treatment.
The authors would like to thank Dr. GPT conceived the present review. PF and MRE selected the articles and identified useful information within these articles. PF submitted the manuscript. All authors read and approved the final version of the manuscript. Crit Rev Oncol Hematol.
J Clin Oncol. Br J Cancer. Tonini GP: Growth, progression and chromosome instability of Neuroblastoma: A new scenario of tumorigenesis? BMC Cancer. Cancer Res. Nat Rev Cancer. Trends Genet. Nat Med. Nature Genet. Trends Cell Biol. Cancer Metastasis Rev.