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Studies in red

Genetic defects begin to explain the origin of sanguinity diseases of the elderly

Twenty years ago some uncommon cases of anemia began to call the attention of the doctors at the Blood Bank of the Campinas State University (Unicamp). Instead of young adults, as was the habit, it was the elderly who showed an expressive reduction in the level of hemoglobin, the molecule found in the interior of the red blood cells responsible for the transportation of oxygen and for the red color of live blood. More intriguing: the anemia of the elderly did not recede in the face of conventional treatment, based on vitamins and iron supplements. The hematology doctors Irene Lorand Metze and Sara Saad, who dealt with these cases, verified: the cause of this anemia was not the shortage of nutrients essential for the production of red blood cells or erythrocytes such as iron and the vitamins of complex B. The origin of the problem was much more complex: it lay in the stem cells of the bone marrow, from which the three families of sanguinity cells originate – the red blood cells, the white blood cells and the platelets. Indeed, they were not dealing with anemia resistant to treatment, but of one of the forms of a group of rare illnesses called myelodysplasia or myelodysplastic syndromes, whose treatment up until today challenges medical science, although its causes are much better understood.

With characteristics similar to acute myelocytic leukemia – the most frequent form of acute leukemia in adults –, the myelodysplasias alter the blood composition by way of two opposing mechanisms. Both occur in the bone marrow, the material that fills the large bones of the body, in which the blood cells are formed and develop before being launched into the veins and arteries. The first mechanism brings about the death in mass of the cells running through the blood. The second leads these cells to multiply in an uncontrolled manner – and the cells of the next generation reach the blood stream immature and incapable of functioning as they should. The effect is the same: the blood contains mature red blood cells in insufficient number to feed the tissues with oxygen; as well there is not an adequate quantity of white cells, which combat invading microorganisms; nor even the pieces of cell known as platelets, which block hemorrhages. It is for this reason that the person who develops myelodysplasia feels himself tired and presents frequent infections or bleeding that are difficult to contain.

The analysis of more than 200 cases already attended to at Unicamp’s Blood Bank is helping to understand how these problems come about and evolve. In laboratory tests with bone marrow from patients suffering from myelodysplasia, Irene and Sara discovered alterations in the expressions of three genes that control the programmed death – or apoptosis – of the blood cells. For this reason, at the beginning of the illness the level of apoptosis is generally high and impedes the production of red and white blood cells and platelets at an adequate level for the normal functioning of the organism. In the most advanced stages, nonetheless, the opposite occurs: the apoptosis diminishes and it is the cells running through the blood called blasts that reach the circulation – there are even cases in which the blasts are produced in an adequate quantity, but do not generate mature blood cells. “It is not known if this imbalance in the cellular mortality is the consequence only of the genetic disturbances in the unwell cells or if, at least in part, it is coming from the action of the organ’s defense system, directed towards the elimination of these cells”, comments Sara, who detected another abnormal behavior of the cells in the myelodysplasia: cultivated in the laboratory, the white cells were capable of multiplying even in the absence of the chemical signals that induce cell division, different from healthy cells.

Resistance to death
While this doubt remains, the certainty is that there is a reduction in the chemical signals that set off the programmed death of the blasts and an increase in the commands that impede it, as was verified by Irene, Elisangela Ribeiro, Carmen Lima and Konradin Metze, in an article published in 2004 in the magazine Leukemia & Lymphoma. “Through the manner in which the illness progresses, these cells contain alterations that make them less susceptible to apoptosis”, explains Irene, one of the main Brazilian researchers who studied the myelodysplastic syndromes. In an article that should come out shortly in the magazine Leukemia Research – developed in conjunction with Spanish researchers from the University of Salamanca and the Hospital Miguel Servet, in Zaragoza –, Irene and her team detected indications that that the alterations in the cellular characteristic development of the myelodyplasia could well occur even in a stage prior to that of the blasts, within the very all powerful stem cells themselves. This is a finding that helps to explain why both the level of red blood cells and platelets as well as that of the white blood cells can be found below normal levels in patients with these syndromes.

As yet, all of the genetic defects of myelodysplasia are not known, but it is estimated that these alterations – such as the loss of part of chromosomes 5, 7 and 20 or the presence of an extra copy of chromosome 8 – have contributed to almost half of the cases of these illnesses. In general, the lesions in the genetic material of cells do not come about from one moment to the next. “These genetic defects, detected in 40% to 50% of the myelodysplasia patients, are the fruit of a series of lesions that accumulate along the person’s life and shows themselves around 60 years of age”, explains the hematologist Maria de Lourdes Chauffaille, from the Federal University of São Paulo and the Fleury Institute, who investigated the genetic characteristics of these illnesses.

Today it is known that some medicines used in the treatment of cancer can damage the genetic material (DNA) of cells and lead to the development of myelodysplasia. As well, it is suspected that prolonged exposure to cigarette smoke, pesticides, solvents, such as benzene and radiation, damage the DNA of cells coming from blood and, in 10% of the cases, originate in these syndromes.

Beyond 60 years of age
These cumulative effects explain why myelodysplasias are more common after 60 years of age. It is estimated that, before this age, five adults in every group of 100,000 develop one of the forms of myelodysplasia. Starting from 60 years of age, these syndromes become four to ten times more frequent: striking from 20 to 50 individuals in each group of 100,000. According to specialists in the United States, 15,000 new cases of myelodysplasia come about per year, an indication that these syndromes are as frequent as the most common form of leukemia in Western countries, chronic lymphocitic leukemia. Even in an indirect manner, the ethnicity, socio-economic and environmental conditions can influence the age of the start of the illness. In Europe and the United States the myelodysplasia appears around 70 years of age, while in Brazil they appear earlier, before sixty. “The tendency is that the number of cases increases as our population age grows older”, says Sara, from Unicamp. Estimates from the IBGE point out that over the next 15 years the Brazilian population over sixty years of age should grow by 74% and move from the current 16.3 millions to 28.3 millions people.

But it is not always that the problem lies with the cells running through the blood. The group coordinated by Maria Mitzi Brentani, from the University of São Paulo, Radovan Borojevic, from the Federal University of Rio de Janeiro, and Luiz Fernando Lopes, from the AC Camargo Cancer Hospital, in São Paulo, investigated another group of cells found in the interior of bones: the cells of the stroma, the nutritional material in which the cells running through the blood are bathed. Like the earth that sustains and nurtures a tree, the stroma fixes these cells and regulates their development. The conclusion is that the health of the stroma can make all the difference, according to a study published in August of 2004 in Leukemia Research. On a glass sheeting with the stroma of myelodysplasia patients, cells from healthy blood began to behave like myelodysplastic syndrome cells: they proliferated without control and did not mature – possibly by the production of chemical signals that induce apoptosis, such as the necrosis tumor alpha factor and the gamma interferon. As well the opposite was verified: bone marrow cells from people with myelodysplasia showed healthy development when cultivated in stroma from people without the disease.

Analyzed in conjunction, the studies over the last ten years have helped to understand why in some cases of myelodysplasia the exams, carried out with blood from a vein in the arm, show a low count of mature cells, while in others a high number of blasts appear. This reproductive disarray that can come about in a few cells, multiplies itself during the manufacture of blood. Formed by around twenty types of distinct cells, diluted in a soup of water and proteins, the blood is in constant renovation. When everything is going well in the organism, 1% of blood cells are substituted daily. Every twenty-four hours the bone marrow manufactures around 200 billion red cells, 10 billion white cells and 400 billion platelets plaques, to substitute those that have grown old and were destroyed by the spleen.

In this natural replacement process, the stem cells of the bone marrow divide themselves up successive times, initially producing identical copies of themselves. But at a determined point of this reproductive process these cells stop copying themselves and go on to generate specialized cells for a determined function, but with lesser capacity to regenerate themselves and to originate other types of cells. The all-powerful aspect, the privilege of the most primordial stem cells, is the capacity to generate any other type of blood cell.

The search for ways out
In the face of the recent discoveries concerning the origin and evolution of the myelodysplasia, the treatment alternatives continue to be restrictive, a reason for discomfort amongst doctors. The only manner of curing myelodysplasia is a bone marrow transplant, a procedure in which the stem cells are extracted from the hip bone of a healthy donor and are injected into the sternum of the sick patient. After the elimination of the abnormal cells by chemotherapy, the healthy cells repopulate the blood. But the use of highly toxic medicines and radiation in order to eliminate the abnormal cells of the medulla limit the application of transplant, in general carried out on patients below sixty years of age – the results are better with patients below forty. The reason is that after sixty people are not as resistant to the undesirable effects of the treatment carried out before they receive the bone marrow. Even when a transplant is possible, the success level is low: in only 40% of the cases, does the person remain free of the disease for five years.

Not even amongst children are the results exciting. “In these cases the major difficulty is to find donors with a comparable bone marrow in a racially mixed population such as ours”, explains the oncologist and pediatrician Luiz Fernando Lopes, from the Cancer Hospital, who at the end of the decade of the 1980s identified the first cases of infantile myelodysplasia in the country and coordinates the group that has already attended to almost 250 children with this problem. Very rare until the age of eighteen – it affects four children and teenagers in every million –, the myelodysplastic syndromes are more aggressive in this age group: eight out of every ten cases evolve within months to acute myelocytic leukemia, in which a torrent of immature white cells arrive in the blood and make the organism vulnerable to infections. “Today we know the causes reasonable well and the evolution of the myelodysplasia”, says Lopes. “But we still don’t know how to treat it efficiently.”

In cases in which a transplant is not viable, the way out is to combat the serious manifestations of the infirmity, which vary in accordance with the type of myelodysplasia – there are seven types of myelodysplasia, according to the most recent classification. When the main effect of this abnormal cell reproduction is the increase in the quantity of immature cell in the blood, the doctors administer medicines capable of eliminating them, such as cytarabine and daunoblastine, used in the treatment of leukemia. With the diminishing of the number of red blood cells, one of the options is to treat the myelodysplasia patient with a cellular growth hormone called erythropoietin, produced by the bacterium Escherichia coli that receive a copy of the gene of this protein. Another hormone is a stimulating factor of the granulocyte colonies, used to stimulate the production of white blood cells. Depending upon the degree of the anemia, monthly blood transfusions – or even weekly – can become necessary.

Currently, dozens of medicines that combat the sick cells or stimulate the proliferation of healthy blood cells are under evaluation in clinical trials carried out mainly in the United States and Europe. But as yet a medicine that brings together the three fundamental qualities has not arrived, namely: to be efficient, of low toxicity and cheap. “Over the last twenty years various meteoric therapies have crossed the dark skies of the treatment of the myelodysplastic syndromes, but only to disappear shortly afterwards”, wrote the Italian hematologists Mario Cazzola and Luca Malcovati, in a comment published in February of this year in the New England Journal of Medicine concerning the most recent promise of a medicine capable of increasing the red blood cells, named lenalidomide, a derivative of thalidomide, which is less toxic and more efficient. The results are promising, but it is still too early to commemorate. “We are hoping that other clinical studies confirm the promising effects of lenalidomide”, they conclude.

The Project
Physiology study of the immune system in neoplasias,  self-immunity and in immune-deficiencies through the cytometry of flux (nº 04/08882-3); Modality Regular Line of Research Project Assistance; Coordinator Irene Lorand Metze – Unicamp; Investment R$ 1,345,226.42 (FAPESP)