Johns Hopkins Kimmel Cancer Center investigators report they have designed a blood test that accurately detects the presence of advanced breast cancer and also holds promise for precisely monitoring response to cancer treatment.
The test, called the cMethDNA assay, accurately detected the presence of cancer DNA in the blood of patients with metastatic breast cancers up to 95 percent of the time in laboratory studies.
Currently, there is no useful laboratory test to monitor patients with early stage breast cancer who are doing well, but could have an asymptomatic recurrence, says Saraswati Sukumar, Ph.D., who is the Barbara B. Rubenstein Professor of Oncology and co-director of the Breast Cancer Program at the Johns Hopkins Kimmel Cancer Center. Generally, radiologic scans and standard blood tests are indicated only if a woman complains of symptoms, such as bone aches, shortness of breath, pain, or worrisome clinical exam findings. Otherwise, routine blood tests or scans in asymptomatic patients often produce false positives, leading to additional unnecessary tests and biopsies, and have not been shown to improve survival outcomes in patients with early stage breast cancer who develop a recurrence.
Sukumar, also a professor of pathology at Johns Hopkins, says that the current approach to monitoring for recurrence is not ideal, and that ‘the goal is to develop a test that could be administered routinely to alert the physician and patient as soon as possible of a return of the original cancer in a distant spot. With the development of cMethDNA, we’ve taken a first big step toward achieving this goal.’
To design the test, Sukumar and her team scanned the genomes of primary breast cancer patients, as well as DNA from the blood of metastatic cancer patients. They selected 10 genes specifically altered in breast cancers, including newly identified genetic markers AKR1B1, COL6A2, GPX7, HIST1H3C, HOX B4, RASGRF2, as well as TM6SF1, RASSF1, ARHGEF7, and TMEFF2, which Sukumar’s team had previously linked to primary breast cancer.
The test, developed by Sukumar, collaborator Mary Jo Fackler, Ph.D., and other scientists, detects so-called hypermethyation, a type of chemical tag in one or more of the breast cancer-specific genes present in tumour DNA and detectable in cancer patients’ blood samples. Hypermethylation often silences genes that keep runaway cell growth in check, and its appearance in the DNA of breast cancer-related genes shed into the blood indicates that cancer has returned or spread.
In one set of experiments, the researchers tested the assay’s ability to detect methylated tumor DNA in 52 blood samples – 24 from patients with recurrent stage IV breast cancer and 28 from healthy women without breast cancer, and again in blood samples from 60 individuals – 33 from women with all stages of breast cancer and 27 from healthy women. In each case, the blood test was up to 95 percent accurate in distinguishing patients with metastatic breast cancer from healthy women.
The investigators also studied the assay’s potential to monitor response to chemotherapy. They evaluated 58 blood samples from 29 patients with metastatic breast cancer, some taken before the initiation of therapy and some taken 18 to 49 days after starting a new chemotherapy regimen. In as little as two weeks, they report, the test detected a significant decrease in DNA methylation in patients with stable disease or in those who responded to treatment; this decrease was not found in patients whose disease progressed or who did not respond to treatment.
‘Our assay shows great potential for development as a clinical laboratory test for monitoring therapy and disease progression and recurrence,’ Sukumar says. If it’s determined early that a treatment is not working, clinicians can save time and switch to a different therapy, she says.
In addition, the researchers tested the gene panel used in the cMethDNA assay against samples from The Cancer Genome Atlas, a catalog of genes in various cancer types, finding that the gene panel may also be useful in detecting recurrent lung or colorectal cancers but not as accurate in detecting recurrent ovarian, kidney or stomach cancers. Johns Hopkins Kimmel Cancer Center

For the first time, large-scale information on the biochemical makeup of small interfering RNA (siRNA) molecules is available publicly. These molecules are used in research to help scientists better understand how genes function in disease. Making these data accessible to researchers worldwide increases the potential of finding new treatments for patients.
NIH’s National Center for Advancing Translational Sciences (NCATS) collaborated with Life Technologies Corporation of Carlsbad, Calif., which owns the siRNA information, to make it available to all researchers.
RNA interference(RNAi), a cellular process that can stop specific proteins from being coded by silencing the genes that produce them.
The siRNA molecules, which can selectively inhibit the activity of genes, are used in RNA interference (RNAi) research. RNAi is a natural process that cells use to control the activity of specific genes. Its discovery led to the 2006 Nobel Prize in Physiology or Medicine.
Last month, a team of NIH scientists, led by Richard Youle, Ph.D., at the National Institute of Neurological Disorders and Stroke (NINDS), and Scott Martin, Ph.D., at NCATS, used RNAi to find genes that linked to Parkinson’s disease, a devastating movement disorder. The new genes may represent new starting points for developing treatments.
Scientists have harnessed the power of RNAi to study the function of many individual genes by reducing their activity levels, or silencing them. This process enables researchers to identify genes and molecules that are linked to particular diseases. To do this, researchers use siRNAs, which are RNA molecules that have a complementary chemical makeup, or sequence, to that of a targeted gene. While the gene is silenced, researchers look for changes in cell functions to gain insights about what it normally does. By silencing genes in the cell one at a time, scientists can explore and understand their complex relation to other genes in the context of disease.
Until now, a major limitation in the scientific community’s use of RNAi data has been the lack of a publicly available dataset, along with siRNA sequences directed against every human gene. Historically, providers have not allowed publishing of proprietary siRNA sequence information. To address this problem, NCATS and Life Technologies are providing all researchers with access to siRNA data from Life Technologies’ Silencer Select siRNA library, which includes 65,000 siRNA sequences targeting more than 20,000 human genes. Simultaneously, NCATS is releasing complementary data on the effects of each siRNA molecule on biological functions. All of this information is available to the public free-of-charge through NIH’s public database PubChem.
‘Producing and releasing these data demonstrate NCATS’ commitment to speeding the translational process for all diseases,’ said NCATS Director Christopher P. Austin, M.D. ‘The Human Genome Project showed that public data release is critical to scientific progress. Similarly, I believe that making RNAi data publicly available will revolutionise the study of biology and medicine.’
Experts from the NIH RNAi initiative, administered by NCATS’ Division of Pre-Clinical Innovation, conduct screens for NIH investigators. They will add new RNAi data into PubChem on an ongoing basis, making the database a growing resource for gene function studies.
‘By releasing all our siRNA sequences, we are enabling novel strategies to advance fundamental understanding of biology and discovery of new potential drug targets,’ said Mark Stevenson, president and chief operating officer of Life Technologies.
NIH invites other companies that sell siRNA libraries and researchers who conduct genome-wide RNAi screens with the Life Technologies library to deposit sequence data and biological activity information into PubChem. For assistance with submitting data to PubChem, researchers may contact info@ncbi.nlm.nih.gov.
‘Translation of siRNA library screening results into impactful downstream experiments is the ultimate goal of scientists using our library,’ said Alan Sachs, M.D., Ph.D., head of global research and development for Life Technologies. ‘The availability of these sequence data should greatly facilitate this effort because scientists no longer will be blinded to the actual sequence they are targeting.’ National Institutes of Health

In most cases of amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, a toxin released by cells that normally nurture neurons in the brain and spinal cord can trigger loss of the nerve cells affected in the disease, Columbia researchers report.
The toxin is produced by star-shaped cells called astrocytes and kills nearby motor neurons. In ALS, the death of motor neurons causes a loss of control over muscles required for movement, breathing, and swallowing. Paralysis and death usually occur within 3 years of the appearance of first symptoms.

The report follows the researchers’ previous study, which found similar results in mice with a rare, genetic form of the disease, as well as in a separate study from another group that used astrocytes derived from patient neural progenitor cells. The current study shows that the toxins are also present in astrocytes taken directly from ALS patients.

‘I think this is probably the best evidence we can get that what we see in mouse models of the disease is also happening in human patients,’ said the study’s senior author, Serge Przedborski, MD, PhD, the Page and William Black Professor of Neurology (in Pathology and Cell Biology), Vice Chair for Research in the department of Neurology, and co-director of Columbia’s Motor Neuron Center.

The findings also are significant because they apply to the most common form of ALS, which affects about 90 percent of patients. Scientists do not know why ALS develops in these patients; the other 10 percent of patients carry one of 27 genes known to cause the disease.

‘Now that we know that the toxin is common to most patients, it gives us an impetus to track down this factor and learn how it kills the motor neurons,’ Dr. Przedborski said. ‘Its identification has the potential to reveal new ways to slow down or stop the destruction of the motor neurons.’

In the study, Dr. Przedborski and study co-authors Diane Re, PhD, and Virginia Le Verche, PhD, associate research scientists, removed astrocytes from the brain and spinal cords of six ALS patients shortly after death and placed the cells in petri dishes next to healthy motor neurons. Because motor neurons cannot be removed from human subjects, they had been generated from human embryonic stem cells in the Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, also at CUMC.

Within two weeks, many of the motor neurons had shrunk and their cell membranes had disintegrated; about half of the motor neurons in the dish had died. Astrocytes removed from people who died from causes other than ALS had no effect on the motor neurons. Nor did other types of cells taken from ALS patients.
The researchers confirmed that the cause of the motor neurons’ death was a toxin released into the environment by immersing healthy motor neurons in the astrocytes’ culture media. The presence of the media, even without astrocytes, killed the motor neurons.

The researchers have not yet identified the toxin released by the astrocytes. But they did discover the nature of the neuronal death process triggered by the toxin. The toxin triggers a biochemical cascade in the motor neurons that essentially causes them to undergo a controlled cellular explosion.

Drs. Przedborski, Re, and Le Verche found that they could prevent astrocyte-triggered motor neuron death by inhibiting one of the key components of this molecular cascade.

These findings may lead to a way to prevent motor neuron death in patients and potentially prolong life. But the therapeutic potential of such inhibition is far from clear. ‘For example, we don’t know if this would leave patients with living but dysfunctional neurons,’ Dr. Przedborski said. The researchers are now testing the idea of inhibition in animal models of ALS.
The development of new therapies for ALS has been disappointing, with more than 30 clinical trials ending with no new treatments since the 1995 FDA approval of riluzole.

The lack of progress may be partly because animal models used to study ALS do not completely recreate the human disease. The new all-human cell model of ALS created for the current study may improve scientists’ ability to identify useful drug targets, particularly for the most common form of the disease.

‘Although there are many neuro-degenerative disorders, only for a handful do we have access to a simplified model that is relevant to the disease and can therefore potentially be used for high-throughput drug screening. So this model is quite special,’ Dr. Przedborski said. ‘Here we have a spontaneous disease phenotype triggered by the relevant tissue that causes human illness. That’s one important thing. The other important thing is that this model is derived entirely from human elements. This is probably the closest, most natural model of human ALS that we can get in a dish.’ Columbia University Medical Center

Doctors commonly use magnetic resonance imaging (MRI) to diagnose tumours, damage from stroke, and many other medical conditions. Neuroscientists also rely on it as a research tool for identifying parts of the brain that carry out different cognitive functions.

Now, a team of biological engineers at MIT is trying to adapt MRI to a much smaller scale, allowing researchers to visualise gene activity inside the brains of living animals. Tracking these genes with MRI would enable scientists to learn more about how the genes control processes such as forming memories and learning new skills, says Alan Jasanoff, an MIT associate professor of biological engineering and leader of the research team.

‘The dream of molecular imaging is to provide information about the biology of intact organisms, at the molecule level,’ says Jasanoff, who is also an associate member of MIT’s McGovern Institute for Brain Research. ‘The goal is to not have to chop up the brain, but instead to actually see things that are happening inside.’

To help reach that goal, Jasanoff and colleagues have developed a new way to image a ‘reporter gene’ — an artificial gene that turns on or off to signal events in the body, much like an indicator light on a car’s dashboard. In the new study, the reporter gene encodes an enzyme that interacts with a magnetic contrast agent injected into the brain, making the agent visible with MRI. This approach allows researchers to determine when and where that reporter gene is turned on.
MRI uses magnetic fields and radio waves that interact with protons in the body to produce detailed images of the body’s interior. In brain studies, neuroscientists commonly use functional MRI to measure blood flow, which reveals which parts of the brain are active during a particular task. When scanning other organs, doctors sometimes use magnetic ‘contrast agents’ to boost the visibility of certain tissues.

The new MIT approach includes a contrast agent called a manganese porphyrin and the new reporter gene, which codes for a genetically engineered enzyme that alters the electric charge on the contrast agent. Jasanoff and colleagues designed the contrast agent so that it is soluble in water and readily eliminated from the body, making it difficult to detect by MRI. However, when the engineered enzyme, known as SEAP, slices phosphate molecules from the manganese porphyrin, the contrast agent becomes insoluble and starts to accumulate in brain tissues, allowing it to be seen.

The natural version of SEAP is found in the placenta, but not in other tissues. By injecting a virus carrying the SEAP gene into the brain cells of mice, the researchers were able to incorporate the gene into the cells’ own genome. Brain cells then started producing the SEAP protein, which is secreted from the cells and can be anchored to their outer surfaces. That’s important, Jasanoff says, because it means that the contrast agent doesn’t have to penetrate the cells to interact with the enzyme.

Researchers can then find out where SEAP is active by injecting the MRI contrast agent, which spreads throughout the brain but accumulates only near cells producing the SEAP protein.
In this study, which was designed to test this general approach, the detection system revealed only whether the SEAP gene had been successfully incorporated into brain cells. However, in future studies, the researchers intend to engineer the SEAP gene so it is only active when a particular gene of interest is turned on.

Jasanoff first plans to link the SEAP gene with so-called ‘early immediate genes,’ which are necessary for brain plasticity — the weakening and strengthening of connections between neurons, which is essential to learning and memory.

‘As people who are interested in brain function, the top questions we want to address are about how brain function changes patterns of gene expression in the brain,’ Jasanoff says. ‘We also imagine a future where we might turn the reporter enzyme on and off when it binds to neurotransmitters, so we can detect changes in neurotransmitter levels as well.’ MIT

For years, scientists have observed that tumour cells from certain breast cancer patients with aggressive forms of the disease contained low levels of mitochondrial DNA. But, until recently, no one was able to explain how this characteristic influenced disease progression.

Now, University of Pennsylvania researchers have revealed how a reduction in mitochondrial DNA content leads human breast cancer cells to take on aggressive, metastatic properties. The work breaks new ground in understanding why some cancers progress and spread faster than others and may offer clinicians a biomarker that would distinguish patients with particularly aggressive forms of disease, helping personalise treatment approaches.

The study was led by the Penn School of Veterinary Medicine’s Manti Guha, a senior research investigator, and Narayan Avadhani, Harriet Ellison Woodward Professor of Biochemistry in the Department of Animal Biology. Additional Penn Vet collaborators included Satish Srinivasan, Gordon Ruthel, Anna K. Kashina and Thomas Van Winkle. They teamed with Russ P. Carstens of Penn’s Perelman School of Medicine and Arnulfo Mendoza and Chand Khanna of the National Cancer Institute.

Mitochondria, the ‘powerhouses’ of mammalian cells, are also a signalling hub. They are heavily involved in cellular metabolism as well as in apoptosis, the process of programmed cell death by which potentially cancerous cells can be killed before they multiply and spread. In addition, mitochondria contain their own genomes, which code for specific proteins and are expressed in co-ordination with nuclear DNA to regulate the provision of energy to cells.

In mammals, each cell contains between 100 and 1,000 copies of mitochondrial DNA, but previous research had found that as many as 80 percent of people with breast cancer have low mitochondrial DNA, or mtDNA, content.

To gain an understanding of the mechanism that connects low mtDNA levels with a cellular change that leads to cancer and metastasis, Guha, Avadhani and their colleagues set up two systems by which they could purposefully reduce the amount of mtDNA in a cell. One used a chemical to deplete the DNA content, and another altered mtDNA levels genetically. They compared normal, non-cancer-forming human breast tissue cells with cancerous breast cells using both of these treatments, contrasting them with cells with unmanipulated mtDNA.

The differences between cells with unmodified and reduced mtDNA levels were striking, the researchers found. The cells in which mtDNA was reduced had altered metabolism and their structure appeared disorganised, more like that of a metastatic cancer cell. Even the non-tumour-forming breast cells became invasive and more closely resembled cancer cells. Significantly, cells with reduced mtDNA became self-renewing and expressed specific cell surface markers characteristic of breast cancer stem cells.

‘Reducing mitochondrial DNA makes mammary cells look like cancerous stem cells,’ Avadhani said. ‘These cells acquire the characteristics of stem cells, that is the ability to propagate and migrate, in order to begin the process of metastasis and move to distal sites in the body.’

‘Most patients who had low copy numbers of mitochondrial DNA have a poor disease prognosis,’ Guha said. ‘We’ve shown a causal role for this mitochondrial defect and identified a candidate biomarker for aggressive forms of the disease. In the future, mtDNA and the factors involved in mitochondrial signalling may serve as markers of metastatic potential and novel points for therapeutic intervention of cancer stem cells. Since the specific inducers of cancer stem cells, which are key drivers of metastasis, remain elusive, our current findings are a significant advancement in this area.’

No two breast cancers are exactly alike, so having a way to recognise patients who are at high-risk for developing particularly invasive and rapidly metastasising cancers could help physicians customise treatments. In addition, researchers are currently filling in the unknown components of the signalling pathway linking a cell’s mitochondrial DNA levels and its involvement in metastatic disease. University of Pennsylvania