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The first signs of alert

Gene action sheds light on the origin and indicates the evolution of tumors

At first glance the glass slides curiously guarded in a room at the Ludwig Institute for Cancer Research in São Paulo appear to be the same as any other microscope slides, the same as those used by college students in their search for microbes contained in a drop of water. But, when observed with the help of a laser or ultraviolet light, these rectangular glass plates, no bigger than a domino piece, reveal precious information that is improving the understanding and the treatment of cancer. It is these small glass chips known as DNA microarrays that allow for the simultaneous identification of which of the almost 30,000 human genes find themselves active in a determined group of cells or body tissue. This is an essential property in the investigation into complex illnesses such as cancer, because it allows for the comparison of genes that work within healthy cells with those brought into action in cancerous cells, thus revealing a type of digital fingerprint of a tumor.

Using DNA microarrays developed at the laboratories of the Ludwig Institute and the University of São Paulo (USP), São Paulo researchers have analyzed the gene activity in four types of cancer – breast, prostrate, stomach and the esophagus – and have made discoveries capable of altering the treatment of some tumors. In one of their pieces of research, they identified a combination of three genes that allows them to know ahead if the medicine doxorubicin, the chemotherapy most widely used in the public health network against breast cancer, has or has not made the desired affect. In relation to prostrate cancer, the second illness that kills most men in the country, they have verified that there is a relationship between the working of certain groups of genes and the seriousness of the tumor.

This same technique also allows for a better understanding of how the adenocarcinoma of the esophagus comes about. This is the type of cancer that has increased in the West over the last three decades and today affects around 1% of the developed countries populations. This tumor seems to initially come about in the stomach and only afterwards invades the esophagus, and not the contrary, which had previously been believed. Based on a comparison of the genetic activity of the stomach cells, it was also possible to distinguish those that originated a cancer from those that should remain healthy or those that are characteristic of people who suffer from gastritis. These are promising results because they can help in the precocious detection of these tumors, generally discovered in an advanced stage.

Benefits to women
Of these four discoveries, that which helps to redirect the treatment of breast cancer has had the greatest impact on people’s health. The main cause of death among women, the breast tumor should attack 467,000 Brazilian women – and kill almost 50,000 – only during this year, according to estimates from the National Cancer Institute (Inca). In an attempt to minimize the physical and psychological scars brought about by the illness, doctors indicate a treatment based on medicines to reduce the size of the tumor – and, consequently, the volume of the breast to be removed. The problem is that not always does the pre-operational treatment most adopted by the Public Health System (SUS) work. In approximately 20% of women, the intravenous administration of the medicines doxorubicin and cyclophosphamide do not produce the desired effect of reducing the size of the tumor, something that is only discovered afterwards when the patients have already gone through the treatment, which in general brings about the falling of hair, as well as intense nausea and a general feeling of being unwell.

Faced with this situation, Maria Mitzi Brentani and Maria Aparecida Koike Folgueira, associate professors of Oncology at USP?s College of Medicine, decided to search for a test that would reveal the women for whom these medicines would work, before they would receive the medication. Thus, time is gained, public money is saved and there is a reduction of the discomfort for these people. “If doxorubicin doesn’t work for a woman, the doctor can attempt other medications or anticipate the surgery”, explains Maria Mitzi.
In collaboration with teams from the Brazilian Institute for Cancer Control, the Hospital das Clinicas and the Cancer Hospital, both in São Paulo, and the Amaral Carvalho Hospital in the town of Jau, in the interior of the state, Maria Mitzi and Maria Aparecida collected breast tumor samples from 51 women aged between 31 and 67 years. They then extracted the cellular genetic material and analyzed the genetic expression using microarrays loaned by the Ludwig Institute. Also known as DNA chips, these microarrays – glass laminas chemically treated upon which a robot deposits a gene in each point – were made up with 4,608 genes identified through the Human Cancer Genome Project, a project financed by FAPESP and the Ludwig Institute that had sequenced the genes of twenty types of cancer.

From all of the genes evaluated, some 228 behaved in a different manner – some showed that they were more expressed than others – in women in which the treatment had been efficient and had reduced the size of the tumor by at least 30%. In partnership with Dirce Carraro, from the Genetic Expression Analysis Laboratory of the Ludwig Institute, Helena Brentani, from the Bioinformatics Laboratory of the Cancer Hospital, and Paulo José da Silva e Silva, from USP’s Mathematics and Statistics Institute (IME), the researchers identified a trio of genes – PRSS11, CLPTM1 and MTSS1 – capable of pointing towards the women for whom the treatment would be efficient.

In order to know if this combination of genes functioned in practice as a predictive test, they made use of it in the evaluation of cancer samples from a further 14 women. The gene trio has permitted the separation, with 85% certainty, women for whom doxorubicin and cyclophosphamide work from those who would not benefit from the therapy, in accordance with the study’s results that are shortly to be published in the magazine Clinical Cancer Research. Currently, the team from USP’s School of Medicine are testing a larger number of breast tumor samples in an attempt to prove the efficiency of their method. “To approximate scientific research results to daily clinical activities is what we’ve been searching for over the last twenty years”, says Ricardo Renzo Brentani, a chaired professor of oncology at USP’s School of Medicine and the director of the Ludwig Institute. Considered to be one of the top national authorities in the study of cancer, Brentani is also the director president of FAPESP and the coordinator of the Antônio Prudente Center for Research and Cancer Treatment, funded by FAPESP.

This was not the first time that the DNA microarrays developed at the Ludwig Institute have shown their use. Since 2001, the team headed by the biochemist Luiz Fernando Lima Reis, also from the Ludwig Institute, has been adopting this tool in a study on the origin and evolution of the esophagus and stomach tumors. In an article that merited the cover story of the August edition of the magazine Cancer Research, Drs. Lima Reis and Luciana Gomes showed the most probable origin of adenocarcinoma of the esophagus, one of two types of cancer typical to this muscular that carries food from the mouth to the stomach. In general this tumor develops in the region of the esophagus that is connected to the stomach, the cardia, and affects both organs. Up until the publication of this article, it was believed that the cancerous cells came about in the esophagus – as a consequence of the reflux of acidic secretions of the stomach, which would cause chronic irritation of the cells – and thereafter would spread out in the direction of the entrance to the stomach.

The research results place this idea in check. Lima Reis has discovered that the standard of gene expression in the adenocarcinoma of the esophagus is very similar to that of the adenocarcinoma of the stomach, the most common form of gastric cancer. “This is a signal that, in reality, this cancer must originate in the stomach and, in the more advanced phases, moves into the esophagus”, the researcher explains. It may seem obvious, once it is understood that the classification of the tumor is the same – adenocarcinoma, a malignant tumor in the covering tissue of a gland or one whose form approximates to that of a gland – and which brings about similar alterations in the format of the cells. However, the indication that the origin may well be in another organ makes all the difference when the objective is to precociously detect the tumor in the esophagus, in general identified in the most advanced stages, when the only solution is the complete extraction of the organ. “We spent a lot of time looking for the adenocarcinoma of the esophagus in the initial stages in the wrong place”, says Lima Reis. “Perhaps we should be looking for the precocious signs of this tumor also in the stomach, close to the esophagus orifice, the cardia.”

In February of last year, another study from professor Lima Reis’ team had also conquered the cover of the same scientific magazine, one of the most important publications on cancer. The researchers investigated the expression of 376 genes from 99 samples of stomach mucus – in which there were healthy tissue; that with gastritis, pre-tumor tissue and that of adenocarcinoma – and they found one hundred trios of genes that, with greater or lesser precision, foretold to which type of tissue the sample pertained. If these results were to be confirmed in longer duration studies, in which the patients were accompanied right from the first signs of alteration of their gastric mucus to the oncoming of a tumor, these gene combinations could well be used in tests to foretell the evolution of gastric tumors that, it is supposed, come about starting from the chronic inflammation brought about by the bacteria Helicobacter pylori.

Sergio Verjovski-Almeida, from USP’s Chemical Institute, has opted for a different strategy. To the common DNA microarrays – that only contain genes or strips of genes, the so called exons, segments of the DNA molecule that stores information for the production of proteins -, the researcher incorporated new elements: adding in the so called introns, strips of DNA that do not guide the manufacture of proteins, but generate another type of genetic material, a type of RNA capable of controlling the activity of other genes. Using a DNA chip composed of 2,000 introns and 2,000 exons, Verjovski-Almeida and his team have evaluated the genetic expression of 27 prostrate cancer samples and have verified that the introns were capable of foretelling the degree of malignancy of the tumor better than the exons, in accordance with a study published last year in the magazine Oncogene.

Added to those of other countries, these results are beginning to approximate the DNA microarrays to the clinical treatment of patients. As yet still in a timid manner, pharmaceutical companies are initiating the production of the first laboratory tests using the technology of DNA chips. In the United States there are six DNA microarray tests destined towards the identification of breast, ovary and leukemia cancers – or to foretell how these tumors might evolve – that are still awaiting approval from the FDA, the agency that regulates foods and medicines. Before going on the market, however, these tests will have to show themselves to be truly trustworthy.