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Genes against leukemia

Hemo-center in São Paulo develops techniques for improving diagnosis

A blood disease characterized by the presence of cancerous white cells in the medulla, leukemia affects one in 100,000 people a year. It is the commonest type of cancer in children and it usually affects them acutely and swiftly: they can become seriously ill in just a few months. In adults, on the other hand, the commonest type is chronic and it takes years to develop.

Diagnosing the disease reliably and discovering how it will take to develop, in order to establish the treatment, are ongoing objectives of the Fundação Pró-Sangue, the Hemo-center of the School of Medicine of the University of São Paulo (FMUSP), where a team is conducting the project, coordinated by the doctor Dalton Chamone, Study of Biological Markers with Possible Implications for Prognosis in Lympho-Myelo-proliferatives Diseases: the Use of Cytogenetic, Molecular Biological and Immuno-phenotypical Techniques. Financed by FAPESP, the project began in December 1996 and ended last March.

“We improved recognized techniques and brought new techniques from abroad”, says the doctor Israel Bendit, one of those responsible for the project, which involved translation research procedures: “This concept means that whatever was detected or determined on the laboratory bench will be used in the care of patients and vice-versa: if the patient has some new feature, we will try to determine how and why.”

In use
32 researchers took part (doctors, biomedical specialists and pharmacists), 12 specialists in leukemia and initiation students. The improved techniques are already being used on the 1,200 patients that the Hemocenter, the Hematology unit of the FMUSP and the Hematology Service of the Hospital das Clínicas treat each month, all under the Single Healthcare System (The Brazilian public health system – SUS in the Portuguese acronym). “This was one of our objectives”, says the doctor Pedro Llacer, also responsible for the research.

“With the spread of these more efficient diagnostic and prognostic methods, we hope that the national healthcare system will include the procedures in their procedures”, continues Llacer. The new methodology is already being used, for example, in the examinations to quantify the amount of HIV virus in Aids patients.

The researchers are looking for biological markers that, when present in the body, confirm the initial diagnosis and, often, show how the disease will develop. One example was the discovery of correlation between the percentage of medulla cells with leukemia that express CD71 (a molecule that appears on the surface of leukemia cells) and survival of the patient. The more CD71 the patient has, the longer the life expectation.

They also detected biological markers unrelated to the disease. “Apparently negative results like these are also very important”, says Llacer. “We have to publish and spread this around, so that other laboratories do not waste time looking of markers that are useless in diagnosing and making a prognosis.”

Fish Test
This field of study covers various types of liquid and solid lymphoma (tumors of the ganglia). Among the liquids are acute and chronic leukemia, myelodisplasia  (anomalies in the development of one or more lines of blood cells) and multiple myelomas (leading to lesions of the bones and kidneys because of excessive production of some antibody). At the beginning, they may be present only in one ganglion, but then they spread.

With the project, the Hemo-center, one of the main research centers for the treatment of leukemia in the country, became one of the first to develop the use on a large-scale of the Fish test, or in situ hybridization with fluorescence.

In this test, the piece of chromosome to be studied, or target sequence is put  into a   recipient with probes – complementary chromosomes, synthesized in laboratories, colored in red or green by a fluorescent substances

The probes are DNA base sequences that complement and fit perfectly into the DNA sequence being studied. When the recipient is heated, both the double string of DNA of the piece of human chromosome and the double probing string open and the probe forms a ring with the target sequence. The piece of chromosome becomes colored and fluorescent, facilitating the research.

Hybrid gene
This method is very useful in diagnosing chronic myeloid leukemia, in which the chromosome called Philadelphia appears. This is the result of the merger between pieces of chromosomes 9 and 22, also associated with some cases of acute leukemia.

According to Pedro Llacer, the Philadelphia chromosome translates movement by which the BCR and ABL genes, initially in different chromosomes, end up joining themselves, forming the hybrid gene BCR/ABL. In the bone medulla, this gene produces a protein that disrupts the multiplication of the cell where it is. Using a probe with a base sequence complementary to the hybrid gene, the research swiftly determines whether the cell has the BCR/ABL – and, therefore, whether the disease is present– and, in particular, whether the cariotype shows the presence of the Philadelphia chromosome.

By applying methods such as these, and having the correct diagnosis to hand together with a prognosis of the development of the disease, the doctor can choose between more or less aggressive treatments. If the prognosis is bad, the more aggressive treatment begins at once, without waiting for the worst consequences to appear. If the prognosis is more favorable, the patient does not need to undergo unnecessary treatment.

Bendit adds “One of the project’s objectives, that of training people, was fully achieved”.

Study of Biological Markers with Possible Implications for Prognosis in Lympho-Myelo-proliferatives Diseases: the Use of Cytogenetic, Molecular Biological and Immuno-phenotypical Techniques
Thematic project
Dalton de Alencar Fischer Chamone – USP’s Faculty of Medicine
R$ 230,165.47