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Tracking Brain Tumors

Tracking Brain Tumors

Noninvasive molecular biology tool detected a common tumor-associated genetic mutation in cerebrospinal fluid

By SUE McGREEVEY
Wed, 2013-07-24

Researchers have tracked brain tumors by searching for evidence of mutations in cerebrospinal fluid. Image: iStock

Borrowing a tool from molecular biology, HMS researchers at Massachusetts General Hospital have detected a tumor-associated genetic mutation in the cerebrospinal fluid (CSF) of a small number of patients with brain tumors.  In a paper published in the open-access journal Molecular Therapy – Nucleic Acids, the investigators described using digital versions of the gene-amplification technology polymerase chain reaction (PCR) to analyze bits of RNA carried in membrane-covered sacs. They found a common tumor-associated mutation in a gene called IDH1, a biomarker whose presence could potentially influence patient care.

“Reliable detection of tumor-associated mutations in cerebrospinal fluid with digital PCR would provide a biomarker for monitoring and tracking tumors without invasive neurosurgery,” said Xandra Breakefield, HMS professor of neurology at Mass General and corresponding author of the paper. “Knowing the IDH1 mutation status of these tumors could help guide treatment decisions, since a number of companies are developing drugs that specifically target that mutant enzyme.”

Both normal and tumor cells regularly release membrane-covered sacs called extracellular vesicles. Found in blood, CSF and other body fluids, they contain segments of RNA, DNA or proteins. A 2008 study from the Mass General team identified a relatively large tumor-associated mutation in extracellular vesicles from the blood of brain tumor patients, but most current diagnostic technologies that analyze CSF do not capture molecular or genetic information from central nervous system tumors.  

In addition, “Tumor-specific extracellular vesicles make up only a small percentage of the total number of extracellular vesicles found in either blood or cerebrospinal fluid, so finding rare, single-nucleotide mutations in a sample of blood or CSF is very challenging,” explained Leonora Balaj, an HMS research fellow in neurology and co-lead author of the paper. “These digital PCR techniques allow the amplification of such hard-to-find molecules, dramatically improving the ability to identify tumor-specific changes without the need for biopsy.”

The current study used two forms of digital PCR—BEAMing and Droplet Digital PCR—to analyze extracellular vesicles in the blood and in the CSF of brain tumor patients and healthy controls. The scientists were searching for the presence of a single-nucleotide IDH1 mutation known to be associated with several types of cancer. Both forms of PCR detected the presence and abundance of mutant IDH1 in the CSF of 5 of the 8 patients known to have IDH1-mutant tumors. 

Two of the three mutation-positive tumors that had false negative results were low grade and the third tumor was quite small, suggesting a need for future studies of more samples to determine how the grade and size of the tumors affect the ability to detect mutations. The failure to detect tumor-associated mutations in blood samples with this technology may indicate that CSF is a better source for extracellular vesicles from brain tumors. 

The ability to noninvasively determine the genetic makeup of brain tumors could have a significant impact on patient care. “The current approach for patients who may have a brain tumor is first to have a brain scan and then a biopsy to determine whether a growth is malignant,” said Fred Hochberg, HMS associate professor of neurology and a study co-author. “Patients may have a second operation to remove the tumor prior to beginning radiation therapy and chemotherapy, but none of these treatments are targeted to the specific molecular nature of the tumor.”

Having this kind of molecular diagnostic assay—whether in spinal fluid or blood—would allow clinicians to immediately initiate treatment that is personalized for a particular patient without the need for surgical biopsy, Hochberg said. 

“For some patients, the treatment could shrink a tumor before surgical removal. For others, it may control tumor growth to the point that surgery is not necessary, which in addition to keeping patients from undergoing an unnecessary procedure, could save costs,” he said. “We still have a long way to go to improve survival of these malignancies, so every improvement we can make is valuable.”

Mass General has applied for a patent on the use of BEAMing PCR to analyze RNA from extracellular vesicles. Support for the study includes National Institutes of Health grants CA069246, CA141226, CA156009 and CA141150 and grants from the Brain Tumor Funders’ Collaborative  and the American Brain Tumor Association.

Adapted from a Mass General news release.

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Gene variant linked to weight-loss surgery success

Gene variant linked to weight-loss surgery success

Genetic factors appear to determine a patient’s response to gastric bypass

By SUE McGREEVEY
Thu, 2013-05-02

Image: CDC/Debora Cartagena

Massachusetts General Hospital researchers have identified a gene variant that helps predict how much weight an individual will lose after gastric bypass surgery, a finding with the potential both to guide treatment planning and to facilitate the development of new therapeutic approaches to treating obesity and related conditions like diabetes. The report, published online in The American Journal of Human Genetics, is the first to identify genetic predictors of weight loss after bariatric surgery.

“We know now that bypass surgery works not by physically restricting food intake but primarily through physiological effects—altering the regulation of appetite to decrease hunger and enhance satiety and increasing daily energy expenditure,” said Lee Kaplan, HMS associate professor of medicine at Mass General and director of the hospital’s Obesity, Metabolism and Nutrition Institute. He is a senior author of the report. “Genetic factors appear to determine a patient’s response to gastric bypass, and the identification of markers that predict postoperative weight loss could provide important insight into those physiological mechanisms.”

The research team conducted genome-wide association studies of more than 1,000 patients who had bypass surgery at Mass General from 2000 to 2011, analyzing almost 2 million gene sites for associations between specific variants and the percentage of weight lost after surgery. One specific variant at a site on chromosome 15 was most closely associated with weight loss. Individuals with two copies of the beneficial version of the gene lost an average of almost 40 percent of their presurgical weight, while those with only one copy lost around 33 percent. The single individual in the study group who had no copies of the beneficial variant lost less than 30 percent of presurgical weight.

Expression of one of the genes closest to the site of this variant was also able to predict the percentage of weight lost. In addition, experiments in a mouse model of gastric bypass indicated that expression of the corresponding version of that human gene, as well as another gene adjacent to the variant site, was altered by bypass surgery. Additional gene variants not as strongly associated with the response to bypass surgery are candidates for further study in larger groups of patients.

Two predictive models developed by Kaplan and his team have had promising initial results. One of these combines the chromosome 15 genetic variant with clinical factors such as age, gender, the presence of diabetes and exercise behaviors to predict surgical outcomes; the other includes 12 additional gene variants the investigators are studying to determine their usefulness in treatment planning. 

Notably, none of the predictive gene sites identified in this study is involved in pathways previously known to influence the development of obesity, suggesting that different genes contribute to the benefits of bypass. Development of drugs that target the activity of those genes might produce some of the same benefits without the need for surgery, Kaplan said.

“The fact that genetics appears to play such an important role in how well bypass surgery works in an individual patient gives us even more evidence that obesity results from dysfunction of the biological mechanisms that regulate fat mass and body weight and not solely from aberrant behavior or limited willpower,” he adds. “Identifying the involved genes opens up the potential for new classes of antiobesity therapies that mimic or exploit the molecular mechanisms so effectively used by gastric bypass.”  

The study was supported by National Institutes of Health grants DK093257, DK088661 and DK090956, along with grants from Merck Research Laboratories and Ethicon Endo-Surgery.

Adapted from a Mass General news release.

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Clearing the Way for Alzheimer's Prevention

Clearing the Way for Alzheimer’s Prevention

Scientists find protective gene variant that removes toxic amyloid beta protein from the brain

By SUE McGREEVEY
Thu, 2013-04-25

Characteristics of Alzheimer’s disease. Image: National Institute on Aging

Harvard Medical School investigators at Massachusetts General Hospital have determined that one of the recently identified genes contributing to the risk of late-onset Alzheimer’s disease regulates the clearance of the toxic amyloid beta (A-beta) protein that accumulates in the brains of patients with the disease. 

In their report published in Neuron, the researchers describe a protective variant of the CD33 gene, which promotes clearance of A-beta from the brain. They also show that reducing expression of CD33 in immune cells called microglia enhances their ability to clear away A-beta protein, raising the possibility that blocking CD33 activity could help the brain’s immune system remove A-beta.

“Our findings show, for the first time, a ‘switch’ that controls how fast microglial cells can clear A-beta protein from the brain as we age. CD33 is the key,” said Rudolph Tanzi, Joseph P. and Rose F. Kennedy Professor of Child Neurology and Mental Retardation at Harvard Medical School and senior author of the Neuron paper. “If we can find a way of safely inactivating CD33 on microglia, we should be able to slow the accumulation of A-beta in aging brains and hopefully reduce risk for Alzheimer’s disease.”

In 2008, as part of the Alzheimer’s Genome Project, Tanzi, who is also director of the Genetics and Aging Unit in the Mass General Department of Neurology, and his team identified four novel genes containing variants that increased the risk of late-onset Alzheimer’s, the most common form of the devastating neurological disorder. One of these was CD33. The CD33 protein produced by the gene was known to play a role in regulation of the innate immune system—the body’s first line of defense against infection—but how it might function in the brain and possibly contribute to Alzheimer’s risk was not known. 

In the current study, the researchers first found that CD33 activity was significantly higher in microglia cells in brain samples from Alzheimer’s patients than in cells from non-demented controls. Moreover, they showed that the presence of a version of the gene that protected against Alzheimer’s disease reduced CD33 protein levels in the brain. Importantly, the same protective version of CD33 was found to reduce levels of A-beta 42—the primary constituent of the amyloid plaques that characterize the disease. Greater numbers of CD33-containing microglia also were associated with higher levels of A-beta 42 and more plaques overall.

In an Alzheimer’s mouse model, knocking out the CD33 gene improved the ability of microglia in the brain to clear away A-beta 42 and reduced the presence of amyloid plaques. Experiments with cultured microglia showed that increasing CD33 expression on the cells’ surface inhibited their ability to take up A-beta 42, while reducing CD33 activity led to greater clearance of A-beta 42.

“Collectively these experiments indicate that CD33 directly modulates the ability of microglial cells to clear A-beta 42 from the brain,” said Tanzi. “Our findings raise the possibility that inhibiting CD33 activity in the brain could represent a potentially powerful new approach to treating and possibly preventing Alzheimer’s disease.”

Primary support for the study includes grants from the Cure Alzheimer’s Fund and National Institutes of Health grants R37MH060009, P01AG15379, R01AG08487 and P50AG05134. 

Adapted from a Mass General news release.

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