Researchers develop single blood test for 8 cancersResearchers develop single blood test for 8 cancers

Researchers at Johns Hopkins Kimmel Cancer Center have developed a single blood test that screens for eight common cancer types and helps identify the location of the cancer. Five of the cancers covered by the test currently have no screening test.

The test, called CancerSEEK, is a unique noninvasive, multianalyte test that simultaneously evaluates levels of eight cancer proteins and the presence of cancer gene mutations from circulating DNA in the blood.

“The use of a combination of selected biomarkers for early detection has the potential to change the way we screen for cancer, and it is based on the same rationale for using combinations of drugs to treat cancers,” says Nickolas Papadopoulos, Ph.D., senior author and professor of oncology and pathology.

The findings were published online by Science on 18 January 2018.

“Circulating tumour DNA mutations can be highly specific markers for cancer. To capitalize on this inherent specificity, we sought to develop a small yet robust panel that could detect at least one mutation in the vast majority of cancers,” says Joshua Cohen, an M.D.-Ph.D. student at the Johns Hopkins University School of Medicine and the paper’s first author. “In fact, keeping the mutation panel small is essential to minimize false-positive results and keep such screening tests affordable.”

The investigators initially explored several hundred genes and 40 protein markers, whittling the number down to segments of 16 genes and eight proteins. They point out that this molecular test is solely aimed at cancer screening and, therefore, is different from other molecular tests, which rely on analyzing large numbers of cancer-driving genes to identify therapeutically actionable targets.

In this study, the test had greater than 99% specificity for cancer. “Very high specificity was essential because false-positive results can subject patients to unnecessary invasive follow-up tests and procedures to confirm the presence of cancer,” says Kenneth Kinzler, Ph.D., professor of oncology and co-director of the Ludwig Center. The test was used on 812 healthy controls and produced only seven false-positive results.

The test was evaluated on 1,005 patients with nonmetastatic, stages I to III cancers of the ovary, liver, stomach, pancreas, oesophagus, colorectum, lung or breast. The median overall sensitivity, or the ability to find cancer, was 70% and ranged from a high of 98% for ovarian cancer to a low of 33% for breast cancer. For the five cancers that have no screening tests – ovarian, liver, stomach, pancreatic and oesophageal cancers – sensitivity ranged from 69% to 98%.

“A novelty of our classification method is that it combines the probability of observing various DNA mutations together with the levels of several proteins in order to make the final call,” says Cristian Tomasetti, Ph.D., associate professor of oncology and biostatistics, who developed the algorithm. “Another new aspect of our approach is that it uses machine learning to enable the test to accurately determine the location of a tumour down to a small number of anatomic sites in 83% of patients.”

Although the current test does not pick up every cancer, it identifies many cancers that would likely otherwise go undetected.

“Many of the most promising cancer treatments we have today only benefit a small minority of cancer patients, and we consider them major breakthroughs. If we are going to make progress in early cancer detection, we have to begin looking at it in a more realistic way, recognizing that no test will detect all cancers,” says Bert Vogelstein, M.D., co-director of the Ludwig Center, Clayton Professor of Oncology and Howard Hughes Medical Institute investigator.

To zero in on the analytes they included in their CancerSEEK test, the research team pulled data from more than three decades of cancer genetics research generated at their Ludwig Center at Johns Hopkins, where the first genetic blueprints for cancer were created, as well as data from many other institutions.

To precisely determine the optimal number of DNA bases to assess in the CancerSEEK test, the researchers used a method based on diminishing returns. “The more DNA bases you assay, the more mutations you are capable of finding, but eventually you reach a point of diminishing returns,” explains Cohen. “We designed our test to reflect this point of diminishing returns, including the DNA markers that were useful to detecting the cancers and eliminating those that did not add benefit.” The result was a relatively small panel of highly selective DNA markers.

“This test represents the next step in changing the focus of cancer research from late-stage disease to early disease, which I believe will be critical to reducing cancer deaths in the long term,” says Vogelstein.

CancerSEEK is noninvasive and can, in principle, be administered by primary care providers at the time of other routine blood work. “This has the potential to substantially impact patients. Earlier detection provides many ways to improve outcomes for patients. Optimally, cancers would be detected early enough that they could be cured by surgery alone, but even cancers that are not curable by surgery alone will respond better to systemic therapies when there is less advanced disease,” says Anne Marie Lennon, M.D., Ph.D., associate professor of medicine, surgery and radiology, clinical director of gastroenterology anddirector of the Multidisciplinary Pancreatic Cyst Program.

The investigators feel that a test that will be used routinely for cancer screening must have a cost in line with or less than other currently available screening tests for single cancers, such as colonoscopy. They envision that the CancerSEEK test will eventually cost less than $500.

Larger studies of the test are currently under way.


New research adds to evidence linking gut bacteria and obesity

A new Johns Hopkins study of mice with the rodent equivalent of metabolic syndrome has added to evidence that the intestinal microbiome – a “garden” of bacterial, viral and fungal genes – plays a substantial role in the development of obesity and insulin resistance in mammals, including humans.

A report of the findings, published 24 January 2018 in Mucosal Immunology, highlights the potential to prevent obesity and diabetes by manipulating levels and ratios of gut bacteria, and/or modifying the chemical and biological pathways for metabolism-activating genes.

“This study adds to our understanding of how bacteria may cause obesity, and we found particular types of bacteria in mice that were strongly linked to metabolic syndrome,” says David Hackam, M.D., Ph.D., surgeon-in-chief and co-director of Johns Hopkins Children’s Center and the study’s senior author. “With this new knowledge we can look for ways to control the responsible bacteria or related genes and hopefully prevent obesity in children and adults.”

Metabolic syndrome, a cluster of conditions including obesity around the waist, high blood sugar and increased blood pressure, is a risk factor for heart disease, stroke and diabetes. While no precise cause for metabolic syndrome is known, previous studies of Toll-like receptor 4 (TLR4), a protein that receives chemical signals to activate inflammation, have suggested that TLR4 may be responsible in part for its development.

How and why TLR4 may be responsible for metabolic syndrome, however, has been unclear, says Hackam. Perhaps, the research team thought, TLR4 signalling in different cells and their association with the bacterial environment could result in different effects on the development of metabolic syndrome. To first determine whether TLR4 specifically in the intestinal epithelium (layer of cells that line the small and large intestines) would cause the development of metabolic syndrome, the research team ran a series of experiments on both normal mice and mice genetically modified to lack TLR4 in their intestinal epithelium.

The researchers fed both groups of mice “standard chow,” or food with 22% fat calories, for 21 weeks.

Compared to normal mice, those lacking TLR4 showed a series of symptoms consistent with metabolic syndrome, such as significant weight gain, increased body and liver fat, and insulin resistance.

The researchers then fed both groups of mice a high-fat diet comprised of 60% fat calories for 21 weeks to find out whether diet would affect the development of metabolic syndrome. Again, the genetically modified mice gained significantly more in weight and had greater body and liver fat than the normal mice.

To confirm the role of TLR4 expression in the intestinal epithelium, the researchers genetically modified three more groups of mice: one group expressed TLR4 only in the intestinal epithelium, another group lacked TLR4 in all body cells and the third group lacked TLR4 only in white blood cells.

All groups ate standard chow, and all groups had similar body weight, body and liver fat, and glucose tolerance compared to normal mice. Compared with normal mice, belly and small intestine fat was higher in mice lacking TLR4 only in the intestinal epithelium. This, the researchers say, provides further evidence that deleting TLR4 specifically from the intestinal epithelium is required for developing metabolic syndrome.

To investigate the role the bacterial makeup of the gut had on the mice, Hackam and his team then administered antibiotics to the normal and TLR4 intestinal epithelium- deficient mice. Antibiotics significantly reduced the amount of bacteria in the intestinal tract and prevented all symptoms of metabolic syndrome in the mice that lacked TLR4 in their intestinal epitheliums.

This demonstrates, the researchers say, that bacterial levels can be manipulated to prevent the development of metabolic syndrome.

To further explore the role of intestinal epithelial TLR4 on the development of metabolic syndrome, the research team analysed faecal samples from the TLR4 intestinal epithelium-deficient and normal mice. The team found that specific clusters of bacteria that contribute to the development of metabolic syndrome were expressed differently in the deficient mice than in normal mice. They also determined that the bacteria expressed genes that made them “less hungry” and thus less able to digest the nutrients present in the mouse chow. This resulted in a greater abundance of food for the mouse to absorb, which contributed to obesity.

The researchers then analysed the genes expressed in the lining of the intestinal mucosa – the site at which food absorption occurs – in normal and TLR4 intestinal epithelium-deficient mice. Of note, the team determined that important genes in the perixisome proliferator-activated receptor (PPAR) metabolic pathway were significantly suppressed in the deficient mice. Administering antibiotics prevented the differences in gene regulation between the two groups of mice, as did administering drugs to activate the PPAR signalling pathway, further explaining the reasons for which obesity developed.

“All of our experiments imply that the bacterial sensor TLR4 regulates both host and bacterial genes that play previously unrecognized roles in energy metabolism leading to the development of metabolic syndrome in mice,” says Hackam.

• doi: 10.1038/mi.2017.114


MERS antibodies shown to be safe in Phase 1 trial

An experimental treatment developed from cattle plasma for Middle East Respiratory Syndrome (MERS) coronavirus infectionshows broad potential, according to a small clinical trial led by US National Institutes of Health (NIH) scientists and their colleagues. The treatment, SAB-301, was safe and well tolerated by healthy volunteers, with only minor reactions documented.

The first confirmed case of MERS was reported in Saudi Arabia in 2012. Since then, the MERS coronavirus has spread to 27 countries and sickened more than 2,000 people, of whom about 35% have died, according to the World Health Organization. There are no licensed treatments for MERS.

SAB-301 was developed by SAB Biotherapeutics of Sioux Falls, South Dakota, and has been successfully tested in mice. The treatment comes from so-called “transchromosomic cattle”. These cattle have genes that have been slightly altered to enable them to produce fully human antibodies instead of cow antibodies against killed microbes with which they have been vaccinated – in this case the MERS virus. The clinical trial, conducted by the NIH’s National Institute of Allergy and Infectious Diseases, took place at the NIH Clinical Center.

In the study, 28 healthy volunteers were treated with SAB-301 and 10 received a placebo. Six groups of volunteers given different intravenous doses were assessed six times over 90 days. Complaints among the treatment and placebo groups – such as headache and common cold symptoms – were similar and generally mild.

The researchers believe they may be able to use transchromosomic cattle to rapidly produce human antibodies against other human pathogens as well, in as few as three months. This means they could conceivably develop antibody treatments against a variety of infectious diseases in a much faster timeframe and in much greater volume than currently possible.

SAB Biotherapeutics is planning a larger study of SAB-301 in patients infected with MERS coronavirus.

• doi: 10.1016/S1473-3099(18)30002- 1 (2018).


Brain-scan guided emergency stroke treatment breakthrough

Advances in brain imaging can identify a greater number of stroke patients who can receive therapy later than previously believed, according to a new study. The results of the Endovascular Therapy Following Imaging Evaluation for the Ischemic Stroke (DEFUSE 3) trial, presented at the International Stroke Conference 2018 in Los Angeles and published on January 24 in the New England Journal of Medicine, demonstrated that physically removing brain clots up to 16 hours after symptom onset in selected patients led to improved outcomes compared to standard medical therapy. The study was funded by the National Institute of Neurological Disorders and Stroke (NINDS), part of the US National Institutes of Health.

“These striking results will have an immediate impact and save people from life-long disability or death,” said Walter Koroshetz, M.D., director NINDS. “I really cannot overstate the size of this effect. The study shows that one out of three stroke patients who present with at-risk brain tissue on their scans improve and some may walk out of the hospital saved from what would otherwise have been a devastating brain injury.”

DEFUSE 3 was a large, multi-site study supported by NINDS’ StrokeNet, which is a network of hospitals providing research infrastructure for multi-site clinical trials. This study was conducted at 38 centres across the United States and was led by Gregory W. Albers, M.D., professor of neurology and neurological sciences at Stanford University School of Medicine, in California, and director of the Stanford Stroke Center. The study was ended early by the NIH on recommendation of the independent Data and Safety and Monitoring Board because of overwhelming evidence of benefit from the clot removal procedure.

Ischemic stroke occurs when a cerebral blood vessel becomes blocked, cutting off the delivery of oxygen and nutrients to brain tissue. Brain tissue in the immediate area of the blockage, known as the core, cannot typically be saved from dying, and it can enlarge over time. However, it has long been thought that the area surrounding the core (known as the ischemic penumbra) has the potential to be saved based on how quickly the blood flow can be restored. Over the past two decades, scientists have been working to develop brain scanning methods, called perfusion imaging, that could identify patients with brain tissue that can still be salvaged by removing the blockage. In perfusion imaging, a standard dye is injected and scanned for a few minutes as it passes through the brain.

Using an automated software known as RAPID to analyze perfusion MRI or CT scans, the DEFUSE 3 researchers identified patients thought to have salvageable tissue up to 16 hours after stroke onset. The participants were randomized to either receive endovascular thrombectomy plus standard medical therapy or medical therapy alone.

Endovascular thrombectomy, or the physical removal of the blockage, is currently approved for use up to six hours following onset of stroke symptoms. Dr Albers and the DEFUSE 3 researchers discovered that this intervention can be effective up to 16 hours after symptoms begin in this select group of patients. The findings showed that patients in the thrombectomy group had substantially better outcomes 90 days after treatment compared to those in the control group. For example, 45% of the patients treated with the clot removal procedure achieved functional independence compared to 17% in the control group. In addition, thrombectomy was associated with improved survival. According to the results 14% of the treated group had died within 90 days of the study, compared to 26% in the control group.

“Although stroke is a medical emergency that should be treated as soon as possible, DEFUSE 3 opens the door to treatment even for some patients who wake up with a stroke or arrive at the hospital many hours after their initial symptoms,” said Dr Albers.

DEFUSE 3 builds on results from the two earlier DEFUSE studies as well as the industry-sponsored DAWN trial, which used perfusion imaging technology to identify patients most likely to benefit from interventions such as thrombectomy. Those studies suggested that the advanced brain imaging could identify which patients could benefit from restoring blood flow in an extended treatment window.

• doi: 10.1056/NEJMoa1713973


3D model of molecules in yeast linked to enzyme that lengthens telomeres

Through the haze of a sonogram screen, an expectant mother catches a glimpse of the growing baby within her. The outline of a nose, chin and head, instantly recognizable as a tiny human, brings to life what parents, until then, could only imagine. Biologists, too, aim to bring their scientific discoveries to life by creating three-dimensional models – at the atomic level – of the inner workings of cells.

“We need atomic-resolution 3-D images of molecular structures for many reasons. For example, these images can show us precisely how interacting molecules bind to each other in order to carry out critical cellular functions. This helps us develop therapeutic drugs that control the interactions, and therefore also the biochemical processes that they perform in cells,” says David Zappulla, Ph.D., a researcher in the department of molecular biology and genetics at the Johns Hopkins University School of Medicine.

Zappulla’s research focuses on an enzyme found in cells, called telomerase, which lengthens repetitive bits of DNA at the end of chromosomes. These endcaps, called telomeres, erode each time a cell divides, and without these protective tips, this erosion would chip away at the chromosomes – including crucial genetic information – and kill the cell.

Telomerase is present in foetal cells to keep DNA from getting too clipped as cells multiply rapidly during early development, but then the enzyme is turned off, and telomeres erode over time, as part of the natural aging process of cells. It’s well-known that older people tend to have shorter telomeres than younger people.

Cancer cells, on the other hand, hijack telomerase and re-express it to maintain telomere length, making them impervious to aging-related death. To kill cancer cells, scientists have long sought drugs that target telomerase’s ability to keep cells alive.

But to develop such drugs, scientists need a better understanding of how telomerase gets to and extends the chromosomes’ ends.

“There appear to be multiple regulatory steps that precisely control telomerase and recruit it to the shortest chromosome ends where and when it’s needed,” says Zappulla, who has worked to reveal these processes. He published research in 2015 showing how two proteins, Ku and Sir4, interact to lure telomerase near the tips of yeast chromosomes.

In experiments looking at telomerase in baker’s yeast, his lab showed that the Ku protein helps telomerase sense when a telomere is short. They showed that Ku binds to another protein, Sir4, and this connection is important for telomere lengthening. He believes that Sir4 acts as a landing pad to attract telomerase preferentially to short chromosome tips that need an extension.

To visualize these concepts in 3-D, Zappulla teamed up with Ming Lei, Ph.D., an expert in creating crystal structures, at the Shanghai Jiao Tong University. The two met during their postdoctoral training at the University of Colorado Boulder.

For the current research, published 11 January 2018 in Cell, Lei’s team crystallized the baker’s yeast versions of key telomerase-recruiting proteins, as well as a piece of the telomerase enzyme’s RNA. Then they shot X-rays through the crystals and inferred the 3-D shape of each molecule based on how the X-rays’ paths are redirected. Then several co-teams collaborated to validate the structures by introducing mutations in the genes encoding the proteins and testing the altered molecules’ functions in live yeast cells. These experiments led to new insights into how telomerase-recruiting proteins work and interrelate in time and space.

“It’s amazing how much precise detail you can get from crystallography studies,” says Zappulla.

When Zappulla first saw the results, he says that they immediately answered one of his questions about how telomerase interacted with Ku and Sir4 to attach to the chromosome end. “The crystal structures show how Ku binds to both the RNA in telomerase and the Sir4 protein on the chromosomes, as we had proposed in our 2015 study.”

Zappulla says that yeast telomerase and the way it works will certainly be different than the human version; however, insights from yeast should help scientists understand fundamental molecular and cellular features that are similar or even have been conserved over evolution.

Zappulla works in the department of molecular biology and genetics at Johns Hopkins, which is led by Carol Greider, Ph.D., who discovered telomerase in 1984 and shares the 2009 Nobel Prize for Physiology or Medicine with Elizabeth Blackburn and Jack W. Szostak for the finding.


Global study highlights problem of surgical site infections

Globally, approximately 12% of patients develop a surgical site infection within 30 days of gastrointestinal surgery, according to a prospective cohort study of more than 12,500 people in 66 countries, published in The Lancet Infectious Diseases journal.

The incidence of surgical site infection varied between countries depending on their development level, with patients in high-income countries being least at risk, and patients in low-income countries being most at risk.

The results also suggest that globally more than one in five (22%) surgical site infections were resistant to antibiotics given before surgery to prevent infections.

“These findings begin to characterise the relationship between surgical site infections and global antimicrobial resistance,” says Dr Ewen Harrison, NIHR Unit on Global Surgery at the University of Edinburgh, UK. “Worldwide, large amounts of antibiotics were consumed to prevent and treat surgical site infections, yet in a fifth of cases the causative microorganism was resistant to the pre-surgery antibiotics given, and this increased to one of three cases in low-income countries. This high prevalence illustrates a potentially important area for improvement worldwide, and reducing surgical site infections will help to ensure safe and essential surgery around the world.”

The study tracked 12,539 patients from 343 hospitals in 66 countries who were undergoing elective or emergency gastrointestinal surgery to see whether they developed a surgical site infection within 30 days.

Overall, 59% of patients (7339 people) were from 30 high-income countries, 31% (3918) of patients were from 18 middleincome countries, and 10% (1282) of patients were from 18 low-income countries. 1.9% (235/12,539) of all surgery patients died within 30 days of their operation, with the highest incidence in low-income countries (4.8%, 61/1282 patients died).

The most common types of surgery were the removal of the gall bladder or appendix, and half of the patients (49%) had emergency surgery.

Of all patients in the study, 12% (1538/12539) developed a surgical site infection within 30 days of surgery. However, incidence varied depending on a country’s income level – with 9% (691/7339) of patients in high-income countries, 14% (549/3918) of patients in middle-income countries, and 23% (298/1282) patients in low-income countries developing surgical site infections.

The pattern remained even when taking into account the different patient characteristics, diseases, contamination levels, procedures, and hospitals in low-income countries.

Patients with a surgical site infection were more likely to die than patients without an infection (1.5% [162/11,001] of patients with no infection died, compared with 4.7% [73/1538] of patients with a surgical site infection), and were more likely to have another infection and further surgery. In addition, hospital stays for patients with surgical site infections were three times longer on average than people without infections (7 days vs 2 days).

The authors also analysed how common antibiotic-resistant infections were. Microbiology results were available for 610 patients with a surgical site infection, and 22% (132 people) of cases were resistant to the antibiotics given before surgery to prevent infections. Again, incidence varied depending on a country’s income level – with patients in low-income countries most at risk of antibiotic-resistant surgical site infections (36%, 46/128), and high income countries least at risk (17%, 49/295).

Looking into the causes of this difference, the authors found signs of overuse of antibiotics in low-income countries. Patients in low-income countries were more likely to receive antibiotics before and after surgery than patients in middle- and high-income countries (before surgery antibiotic use occurred in 96% of patients in low-income countries, 87% of patients in middleincome countries, and 88% of patients in high-income countries, after surgery these figures were 86%, 80% and 46%, respectively). This trend remained even when the higher levels of surgery contamination in low-income countries were controlled for.

The authors note some limitations, including that it was not possible to follow up all patients to 30 days after surgery, particularly in resource-limited settings. It is therefore possible that some cases were missed; however, they note that the size of the study limits the likelihood of bias.

Writing in a linked Comment, Dr Robert Sawyer, Western Michigan University Homer Stryker M D School of Medicine, USA, says: “To document surgical site infections in 343 centres across 66 countries in Africa, Asia, Europe, North America, Oceania, and South America is an impressive undertaking that substantially adds to our understanding of the global problem of post-operative infections and their associated morbidity and mortality… The GlobalSurg Collaborative clearly describe the magnitude of the problem of surgical site infections in all care settings but particularly in resource-stressed environments. Although the idea that a surgical site infection is just a surgical site infection is prevalent, it is now well known that the cost of a surgical site infection in terms of mortality, morbidity, healthcare costs, and loss of productivity is enormous.”

• doi: 10.1016/S1473-3099(18)30101-4


Chemical from cactus shows promise as new analgesic

A promising approach to post-operative incision-site pain control uses a naturally occurring plant molecule called resiniferatoxin (RTX). RTX is found in Euphorbia resinifera, a cactus-like plant native to Morocco, which is 500 times more potent than the chemical that produces heat in hot peppers, and may help limit the use of opioid medication while in the hospital and during home recovery.

In a paper published online in Anesthesiology, the peer-reviewed medical journal of the American Society of Anesthesiologists, researchers found that RTX could be used to block postoperative incisional pain in an animal model. Many medical providers turn to opioids, such as morphine or fentanyl, for moderate to severe postoperative pain relief, but these often come with side effects that can interfere with recovery, including respiratory depression, inhibition of gut motility and constipation, nausea and vomiting. Prolonged use of opioids can produce tolerance and introduces the risk of misuse. RTX is not an opioid and does not act in the brain but rather on the nerve endings in the skin. Scientists found that it can be used to block pain from the surgical incision selectively for approximately 10 days.

In the study, researchers pre-treated the skin incision site with RTX to render the nerve endings in the skin and subcutaneous tissue along the incision path selectively insensitive to pain. Unlike local anaesthetics, which block all nerve activity including motor axons, RTX allows many sensations, like touch and vibration, as well as muscle function, to be preserved. Long after the surgery, and towards the end of healing of an incision wound, the nerve endings eventually grow back. Thus, pain from the skin incision is reduced during the recovery period.

RTX has been found to be a highly effective blocker of pain in multiple other preclinical animal models and is in a Phase I clinical trial at the National Institutes of Health Clinical Center for patients with severe pain associated with advanced cancer.

• doi: 10.1097/ALN.000 000 000 000 2006

Date of upload: 24th Mar 2018

                                  
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