One in eight women will be diagnosed with breast cancer during her lifetime. The earlier cancer is detected, the better the chance of successful treatment and long-term survival. However, early cancer diagnosis is still challenging as testing by mammography remains cumbersome, costly, and in many cases, cancer can only be detected at an advanced stage. A team based in the Dept. of Biomedical Engineering at McGill University’s Faculty of Medicine has developed a new microfluidics-based microarray that could one day radically change how and when cancer is diagnosed.
For years, scientists have worked to develop blood tests for cancer based on the presence of the Carcinoembryonic Antigen (CEA), a protein biomarker for cancer identified over 40 years ago by McGill’s Dr. Phil Gold. This biomarker, however, is also found in healthy people and its concentration varies from person to person depending on genetic background and lifestyle. As such, it has not been possible to establish a precise cut-off between healthy individuals and those with cancer.
‘Attempts have been made to overcome this problem of person-to-person variability by seeking to establish a molecular ‘portrait’ of a person by measuring both the concentration of multiple proteins in the blood and identifying the signature molecules that, taken together, constitute a characteristic ‘fingerprint’ of cancer,’ explains Dr. David Juncker, the team’s principal investigator. ‘However, no reliable set of biomarkers has been found, and no such test is available today. Our goal is to find a way around this.’
Dr. Mateu Pla-Roca, the study’s first author, along with members of Juncker’s team, began by analysing the most commonly used existing technologies that measure multiple proteins in the blood and developing a model describing their vulnerabilities and limitations. Specifically, they discovered why the number of protein targets that can be measured simultaneously has been limited and why the accuracy and reproducibility of these tests have been so challenging to improve. Armed with a better understanding of these limitations, the team then developed a novel microfluidics-based microarray technology that circumvents these restrictions. Using this new approach, it then became possible to measure as many protein biomarkers as desired while minimising the possibility of obtaining false results.
Juncker’s biomedical engineering group, together with oncology and bioinformatics teams from McGill’s Goodman Cancer Research Centre, then measured the profile of 32 proteins in the blood of 11 healthy controls and 17 individuals who had a particular subtype of breast cancer (oestrogen receptor-positive). The researchers found that a subset of six of these 32 proteins could be used to establish a fingerprint for this cancer and classify each of the patients and healthy controls as having or not having breast cancer.
‘While this study needs to be repeated with additional markers and a greater diversity of patients and cancer subsets before such a test can be applied to clinical diagnosis, these results nonetheless underscore the exciting potential of this new technology,’ said Juncker.
Looking ahead, Juncker and his collaborators have set as their goal the development of a simple test that can be carried out in a physician’s office using a droplet of blood, thereby reducing dependence on mammography and minimizing attendant exposure to X-rays, discomfort and cost. His lab is currently developing a hand-held version of the test and is working on improving its sensitivity so as to be able to accurately detect breast cancer and ultimately, many other diseases, at the earliest possible stage. McGill University

Virginia Commonwealth University School of Medicine researchers have discovered that changes in the gene expression of a key enzyme may contribute to high blood pressure and increase susceptibility to forming blood clots in pregnant women with preeclampsia.
These findings could provide clues to the best treatment approaches for high blood pressure and the formation of blood clots that can block blood flow to a pregnant woman’s internal organs and lead to organ failure.
Researchers have been working to determine the root cause of preeclampsia on the molecular level and have now identified that epigenetic mechanisms may be at play. Epigenetics refers to changes in gene expression that are mediated through mechanisms other than changes in the DNA sequence.
In a study published, the VCU team reported that thromboxane synthase – an important inflammatory enzyme – is increased in the blood vessels of expectant mothers with preeclampsia. The thromboxane synthase gene codes for this enzyme, which is involved in several processes including cardiovascular disease and stroke. This enzyme results in the synthesis of thromboxane, which increases blood pressure and causes blood clots.
‘The present work is unique because it opens up a new concept as to the cause and subsequent consequences of preeclampsia relating to epigenetics,’ said corresponding author Scott W. Walsh, Ph.D., professor in the VCU Department of Obstetrics and Gynecology. ‘It is the first study to show that epigenetic alterations in the blood vessels of the mother are related to preeclampsia.’
According to Walsh, one of the main epigenetic mechanisms is methylation of the DNA, which controls the expression of genes. The increase of this enzyme in the blood vessels is related to reduced DNA methylation and the infiltration of neutrophils into the blood vessels. Neutrophils are white blood cells that normally help fight infection.
In the future, Walsh said some potential treatments for preeclampsia may include inhibition of thromboxane synthase, blockade of thromboxane receptors or dietary supplementation with folate. He said that folate supplementation could increase methylation donors to protect against adverse changes in DNA methylation that affect expression of the thromboxane synthase enzyme. Virginia Commonwealth University

Scientists have identified which strains of the Toxoplasma gondii parasite, the cause of toxoplasmosis, are most strongly associated with premature births and severe birth defects in the United States. The researchers used a new blood test developed by scientists at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, to pinpoint T. gondii strains that children acquire from their acutely infected mothers while in the womb.
Pregnant women can become infected with T. gondii through contact with cat faeces that contain infectious forms of the parasite or by eating undercooked meat. Women who become infected while pregnant may miscarry, give birth prematurely, or have babies with eye or brain damage.
‘If undetected or untreated, congenital toxoplasmosis can have serious consequences for a child’s quality of life,’ noted NIAID Director Anthony S. Fauci, M.D. ‘The findings from this study support the value of screening for toxoplasmosis to identify patients who could benefit from treatment.’
Currently available blood tests can determine whether a person has ever been infected with any strain of Toxoplasma parasite. The experimental test developed at NIAID improves upon the older tests because it can detect the presence of strain-specific antibodies that distinguish infecting strains from one another. The test was developed by Michael Grigg, Ph.D., of NIAID’s Laboratory of Parasitic Diseases, and his colleagues. It was applied to blood samples collected between 1981 and 2009 as part of the National Collaborative Chicago-Based Congenital Toxoplasmosis Study. The study of congenitally infected children was initiated by NIAID grantee Rima McLeod, M.D., of the University of Chicago, who is the first author of the new study.
At least 15 distinct T. gondii strain types have been found throughout the world. In France, where research has been done to establish which strains are most common, a strain called type II predominates. Type II parasites can be distinguished from all other strains, which are collectively termed not exclusively type II strains (or NE-II).
Using the new test, the researchers found evidence of either type II or NE-II infections in 183 of the mother-child pairs in the national congenital toxoplasmosis study. Statistical analysis revealed that NE-II parasites were more likely to be associated with premature birth, and infants infected with these strains were more likely to have severe manifestations of disease than infants infected by type II parasites. For example, severe eye damage was seen in 67 percent of NE-II cases (59 out of 88), while such eye damage was present in only 39 percent of type II cases (18 out of 46). The researchers noted, however, that the association is not absolute, and that mild, moderate or severe disease can result regardless of the infecting strain.
‘We knew that, in mice, certain parasite strains are clearly associated with severe disease,’ said Dr. Grigg. ‘But we didn’t know if the same association between strain type and disease severity would hold true for people. Until now, we had not systematically determined whether infected people in the United States had European-type strains or other types, and we also hadn’t determined whether strains found here would have more severe disease symptoms associated with them.’
When she helped start the congenital toxoplasmosis study in 1981, optimal drug treatment regimens were unknown, said Dr. McLeod. Now, thanks in part to controlled clinical trials run under the auspices of the study, the condition can be successfully treated and many babies who are diagnosed before or shortly after birth and who are treated suffer few or no ill effects. When the researchers looked at the clinical histories of those children in the long-term study who had been diagnosed with congenital toxoplasmosis during gestation and whose mothers had received drug treatment prior to giving birth, the association between NE-II and severe disease at birth vanished. ‘Our study demonstrates that outcomes are equally good following postnatal treatment for type II and NE-II parasites, although not all outcomes are favorable for all children,’ she said.
In France, all pregnant women are screened for Toxoplasma infection. Prompt treatment is offered to any woman who becomes infected while pregnant, thus lessening the chance that the parasite will damage the fetus, Dr. McLeod noted. ‘In the United States, obstetrical screening for Toxoplasma infection is rarely practiced. This new study underscores the value of identifying all patients who will benefit from treatment and suggests that widespread screening and treatment of pregnant women who are infected could prevent infants from suffering eye and brain damage due to congenital toxoplasmosis,’ she said. EurekAlert

Researchers at the Hospital de Mar Research Institute (IMIM) have discovered that the protein LOXL2 has a function within the cell nucleus thus far unknown. They have also described a new chemical reaction of this protein on histone H3 that would be involved in gene silencing, one of which would be involved in the progression of breast, larynx, lung and skin tumours.
Led by Dr Sandra Peiró, the study is a significant advance in describing the evolution of tumours and opens the door to researching new treatments that block their activity. ‘LOXL2’s action on the intra-cellular level and its interaction with histone H3 stimulates tumour growth. The fact that the protein LOXL2 is an enzyme and is overly expressed in many types of cancer makes it a very good therapeutic target. Now that we know how it acts, we need to keep working to develop chemical inhibitors that counteract its activity’, the researcher explained.
Previous studies had identified the extra-cellular function of the protein LOXL2, and it was being studied as a possible therapeutic target for avoiding metastasis in certain kinds of tumours. However, this study has described the presence of this protein at the level of the cell nucleus for the first time.
The process of gene expression in cells consists of transforming the information of the DNA into the proteins necessary to carry out different functions. The DNA molecule has been found to form a certain structure due to its interaction with some proteins called histones. When these histones are modified, the structure of the DNA is also modified and the final result is the expression or non-expression of a certain group of genes.
In the case of tumour cells, the protein LOXL2 acts upon one of these histones (histone H3) and modifies it, eliminating the lysine 4 amino group, a change never described before. As a result of its action, the genes modulated by histone H3, modified by LOXL2, stop expression, preventing the cells from behaving normally and favouring tumour development.
The work of Sandra Peiró’s team is the conclusion of three years of effort focused on the biochemical characterisation of the protein LOXL2 and the study of its role in the modification of histone H3. Since this modification had never been described before, the data obtained open many lines of research. The location on the genomic level of the protein LOXL2 and histone H3, modified by LOXL2, and the possible existence of some enzyme that might neutralise its function, are two of the questions that the group aims to tackle in the years to come. IMIM (Hospital del Mar Research Institute)

Researchers at National Jewish Health have discovered a novel genetic mechanism of immune deficiency. Magdalena M. Gorska, MD, PhD, and Rafeul Alam, MD, PhD, identified a mutation in Unc119 that causes immunodeficiency known as idiopathic CD4 lymphopenia. Unc119 is a signalling protein that activates and induces T cell proliferation. The mutation impairs Unc119 ability to activate T cells.
‘A better understanding of the molecular mechanisms associated with this mutation will improve diagnosis and pave the way for development of new therapies,’ said Dr. Gorska.
Nearly a decade ago Drs. Alam and Gorska identified Unc119 as a novel activator of SRC-type tyrosine kinases, important regulators of cellular function. Since then, they have published numerous papers where they characterised the function of this protein in various aspects of the immune system.
Idiopathic CD4 lymphopenia is a rare and heterogeneous syndrome defined by low levels of CD4 T cells in the absence of HIV infection, which predisposes patients to infections and malignancies. Recent research by others had linked the syndrome to reduced activation of the SRC-type kinase known as Lck. The latter kinase is involved in T cell development, activation and proliferation.
So, Drs. Alam and Gorska thought Unc119, an activator of Lck, might be involved. They kept an eye out for patients with CD4 lymphopenia coming to National Jewish Health, which specialises in immune-related disorders as well as respiratory and cardiac diseases. They identified three patients with CD4 lymphopenia, then sequenced their Unc119 gene as well as the Unc119 gene in several patients who suffered low CD4 T cell counts as a result of other conditions.
One of the three patients, a 32-year-old woman with a history of recurrent infections, had a missense mutation in her Unc119 gene. The same mutation was not present in other lymphopenia patients nor in any genetic database.
The researchers then performed several studies with the woman’s blood cells, to understand the mutation’s effect. They introduced the mutated gene into normal T cells and examined the outcome.
The mutation prevents Lck activation and its downstream signalling. It also reduces the amount of Lck found near the plasma membrane, where it plays a major role in propagating signals from the T-cell receptor. Proliferation of T cells, which normally occurs on stimulation of T-cell receptors, was profoundly reduced in cells from the patient.
‘Since we originally published our findings earlier this year, we have received inquiries from many physicians with lymphopenia subjects,’ said Dr. Alam. ‘Working with them, we expect to find several more patients with this novel mutation, which should help us better understand its effect, improve diagnosis and possibly find therapies.’
At this point there is no treatment for CD4 lymphopenia caused by this mutation other than close monitoring of the patient and treatment of resulting infections and malignancies. National Jewish Health