What is the study of cytogenetic analysis?

Cytogenetic analysis (karyotype or chromosomal or map) is the study of the chromosomes of cells. The chromosomes contain genes which are made up of DNA, the molecule that contains all the necessary information for the construction of a certain individual and the functioning of the organism.

In human cell there are 46 chromosomes, 23 chromosomes come from the father (the sperm) and 23 from the mother (egg cell). Sperm and egg cells are germ cells and are the only ones to contain only 23 chromosomes. If the sperm carries the X chromosome, a female will be born, if it’s bearing the Y chromosome, a male will be born. The karyotype of a normal female will then be 46, XX while that of a male 46, XY. To study the chromosomes we have to use culture techniques during cell division to view them.


What is the use of cytogenetic analysis?

Cytogenetic analysis is for testing whether there are no changes in the number and/or structure of the chromosomes that may be responsible for diseases characterized by Down syndrome, infertility/sterility (e.g. Turner and Klinefelter syndromes), psychomotor and language syndrome, growth and development. Even the exposure to early abortion may be a result of a chromosomal error in one of the parents (3-5% of cases).


When you should undergo the cytogenetic analysis?

  • Prenatal cytogenetics is performed in pregnancies  in which there is an increased risk of chromosomal abnormalities in the fetus, maternal age equal to or greater than 35 years (completed before the child’s birth), sum of the incorrect number of chromosomes, parents carrying structural rearrangements that do not show clinical signs, parents with error numbers of sex chromosomes (e.g. 47, XXX; 47, XXY), birth defects highlighted in ultrasound examinations, indications arising from biochemical tests (e.g. bi-test), repeated miscarriages. By withdrawing trans-abdominal chorionic villus sampling can be performed during the first trimmest (9-12 weeks) or an amniocentesis during the second trimester (15-18 weeks). For CVS cells are taken from the placenta (chorionic villus sampling) which have the same origin (and therefore the same genetic) of the fetal, while amniocentesis is for studying fetal cells found in the amniotic fluid (amniocentesis).
  • Cytogenetic postnatal. The karyotyping is performed in patients with suspected chromosomal syndrome, parents and family members of those with chromosomal abnormalities, parents of malformed or subjects with suspected chromosomal syndrome who have died without diagnosis, where there is mental retardation and / or birth defects, retardation, in infants born or dead, couples with repeated miscarriages, male infertility, women with primary or secondary amenorrhea (absence or interruption of the menstrual cycle).
  • Cytogenetics of abortion material. Approximately 15-20% of all recognized pregnancies results in a miscarriage and more than 50% have an altered number and / or structure of chromosomes that is due to the interruption of pregnancy. The cytogenetic study of tissues abortifacients is therefore of fundamental importance to understanding the cause of the interruption of pregnancy, and to support the pair (as in most cases the error chromosomal is purely coincidental and does not entail an increased risk that the event repeats).
  • Cytogenetics of tumors. Cytogenetic analysis can also be performed to study the tumors, both hematological (e.g. leukemia) and solid (e.g. lung, breast, liver, bladder). Certain chromosomal rearrangements are tumor-specific and therefore allow a correct diagnosis compared with a clinical suspicion or doubt. For example, the finding of the Philadelphia chromosome in bone marrow aspirate from a patient with suspected leukemia, is used to diagnose chronic myelogenous leukemia, or the presence of the t (X; 18) in a cell culture prepared from solid tumor biopsy, allows to diagnose a Synovial Sarcoma.


New technologies: Fluorescent in situ Hybridization (FISH)

The development of sophisticated techniques defined in Molecular Cytogenetics, such as the Fluorescent In Situ Hybridization (FISH), allows us to perform more in-depth cytogenetic studies because it allows the location of a specific DNA sequence on fixed preparations of chromosomes, nuclei interphase and tissue sections, obtained from any type of biological material (blood, biopsies, amniotic fluid, gametes), whether it be fresh, cryopreserved or paraffin. The FISH technique is based on the properties of DNA denaturation in a reversible manner (opening of the double helix) and provides the link between a specific DNA fragment to the region of interest – labeled with fluorescent compounds (probe) – and the complementary DNA sequence preparation that is mounted on a microscope slide: the chromosomal region of interest is so easily identified with a fluorescence microscope.

FISH is an indispensable complement to the traditional cytogenetics as it is characterized by a greater power of resolution, which makes it possible to characterize chromosomal abnormalities in number and structure, which cannot be defined through the techniques of classical cytogenetics and identify cryptic rearrangements, which are not visible even after banding high resolution. FISH is not applied to the A routine karyotype analysis, but only in selected cases based on specific suspicions diagnostic or to deepen certain cytogenetic abnormalities.

One of the latest applications is in the field of oncology: in many cases, in fact, especially for the crops of solid tumors, that do not get cell growth and division and thus cannot highlight the chromosomes and analyze them. In addition, the level of resolution of the study conducted with the traditional cytogenetics, does not allow us to identify abnormalities that may relate to only one gene.

Starting in 2000 have been developed DNA probes able to recognize specific abnormalities, for example bladder cancer for which four probes are used that recognize the chromosomes 3, 7, 17:09 labeled with different fluorochromes (Multicolor FISH) .

The Cytogenetics Laboratory of the Clinical Institute Humanitas is a reference center for the study of bladder cancer. After a study conducted to test the effectiveness of this test, UROVYSION® analysis in clinical practice was introduced, in collaboration with the Unit of Urology. The test is non-invasive and allows detection in 48 hours, via tumor cells obtained from a simple urine sample. The test has a high predictive power, which means that the FISH identifies chromosomal abnormalities typical of cancer before there is evidence of the disease or positive cystoscopic investigation of other diagnostic markers such as CTM (malignant tumor cells). In 2001, the test has been approved by the Food and Drug Administration (FDA) for the monitoring of disease recurrence in patients who have already been diagnosed with cancer and had undergone surgery to remove and / or BCG therapy, and in 2004 for the diagnosis in patients with hematuria.

The FISH technique may also provide information on the most appropriate therapy for a certain type of cancer in an individual patient (Targeted Therapy). It is known for example, that patients with breast cancer who have a positive FISH for amplification of a gene called HER-2 / neu, whose protein is exposed on the cell membrane of the tumor, respond to therapy with a particular drug, trastuzumab, an antibody that binds to the receptor neutralizing (immunotherapy). The test is called PATHVYSION® and is approved by the FDA. FISH can also be used to study the amplification of another gene called EGFR in lung cancer and colon cancer. Also in this case you can use different drugs depending on whether the patient's tumor is found or not, depending on the amplification of the gene. In these cases it is not therapy that uses antibodies but small molecules that inhibit cell division (biological therapy).

New frontiers are opened with the application of FISH for other cancers such as melanoma, where the differential diagnosis of dysplastic nevus is particularly difficult if based solely on morphological criteria.

Because of the high sensitivity, specificity and anticipatory power, the FISH technique is particularly effective in the study of both hematological and solid tumors. In particular, not only is it diagnostic / prognostic but is crucial in the choice of therapy based on the genomic profile of the tumor.