Bench to Bedside

November 2015

Researchers Reveal How B-ALL Leukemia Tumors Resist Immunotherapy


Leukemia tumors are cleverly deceptive, but researchers at The Children’s Hospital of Philadelphia have figured out one of their tricks that allows cancer cells to be invisible to chimeric antigen receptor-armed T cells (CTL019), a new form of investigational immunotherapy.

In recent years, CTL019 therapy has gained attention as an investigational therapy to treat high-risk B cell acute lymphoblastic leukemia (B-ALL) that resists standard chemotherapy, radiation, and stem cell transplants. Researchers at CHOP and the University of Pennsylvania genetically reprogrammed T cells to potentially seek and destroy B-cells, including tumor cells, that express the antigen CD19, a protein essential to cell processes that appears on the cells’ surface.

Earlier this year, the CHOP/Penn team presented data at the annual meeting of the American Society of Pediatric Hematology/Oncology, showing that 78 percent of children who received CTL019 treatment were still surviving at one year, yet there are relapses after CTL019 therapy, according to Stephan Grupp, MD, PhD, a pediatric oncologist at CHOP and professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania.

Andrei Thomas-Tikhonenko, PhD, a cancer cell biologist, and Elena Sotillo, PhD, a senior scientist in his lab at CHOP, have revealed one of the reasons why these treatment failures may occur: alternative splicing.

During gene expression, splicing assembles building blocks called exons to create a mature template for protein synthesis known as messenger RNA. However, sometimes exons are left out. The result is the creation of splicing variants that can cause tumor growth, or in this case, resistance to CTL019 therapy.

“Our findings were extremely surprising, and they happened very quickly,” Dr. Sotillo said. “We found that CD19 is expressed, but the cancer cell is hiding exon 2 — the region that is required for CTL019 to find it and kill it.”

The way alternative splicing makes CD19 invisible to the immune system is like a magician who slides a card up his sleeve, Dr. Thomas-Tikhonenko explained. The card is still there without the audience realizing it. In the same way, the CD19 protein is made, but it is an isoform that is lacking a piece (exon 2), and it is not fully present on the tumors’ surface. To a CTL019 reprogrammed T cell, it would seem like CD19 had vanished.

The research team described how these mechanisms work in a paper published in Cancer Discovery. With the help of Kristen Lynch, PhD, a professor of biochemistry and biophysics at the University of Pennsylvania, they discovered a splicing factor called SRSF3 that binds to exon 2 and promotes its inclusion. Levels of SRSF3 were lower in the relapsed leukemia cases that the researchers studied, Dr. Sotillo said.

Understanding the nuances of this splicing machinery will be the focus of the team’s future research. Scientists in Dr. Thomas-Tikhonenko’s lab aim to identify drugs that could restore splicing and make the full length of the entire CD19 protein. In other words, perhaps they will find a way to bring the missing card back from the magician’s sleeve.

“Really good epigenetic drugs are in clinical trials or at least available for research use that we think might work by regulating splicing,” Dr. Thomas-Tikhonenko said. “That is a new concept we plan to pursue. Splicing can be manipulated, and this gives us an opportunity to come up with something that would force exon 2 to be included.”

Another approach is for researchers to design new chimeric antibodies that do not depend solely on exon 2 to work. Instead, they could target multiple building blocks of the CD19 protein, such as exons 3 or 4.

“That would prevent resistance from occurring,” Dr. Sotillo said. “The antibodies would recognize other regions on the same protein to function.”

In addition to suggesting new research and treatment avenues, greater appreciation of alternative splicing has broader implications for the development of immunotherapeutics, Dr. Thomas-Tikhonenko said. He pointed out that CD19 isoforms without exon 2 had been reported previously in research databases, but until now they have not received much attention.

“Down the road, people will do their research a little differently and consider what part of the protein is being targeted by a new therapy and whether it is prone to alternative splicing or skipping of exons,” said Dr. Thomas-Tikhonenko, who also is a professor of Pathology and Laboratory Medicine at UPenn. “They can go to the databases and figure out what gene this really is and its pre-existing isoforms because there could be a built-in resistance mechanism.”

When researchers identify these isoforms in patients’ tumors, they also could be used as a way to predict treatment success with CT019. Unfortunately, resistance based on the disappearance of antigen is known to be a problem with other types of cancer therapies, so the researchers are interested to see whether alternative splicing plays a major role in curtailing the effectiveness of those methods as well.

The entire research team is excited about their accomplishment and the new directions that it opens. Some key people who contributed their expertise to the project from CHOP included Dr. Grupp, David Barrett, MD, PhD; Kathryn Black, PhD; Asen Bagashev, PhD; and John Maris, MD. In addition to Dr. Lynch, at UPenn the investigators worked closely with Marco Ruella, MD, and Yoseph Barash, PhD.

“At the end of the day, it was a huge collaborative effort that turned out great,” Dr. Sotillo said.

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Drugs May Be What’s the Matter With White Matter in HIV


Some of the neurological and psychiatric complications associated with HIV may be side effects of the medications that control the virus, and not caused by the virus itself, according to a new study from researchers at The Children’s Hospital of Philadelphia and the University of Pennsylvania. Their pre-clinical findings were published in the Journal of Neuropathology and Experimental Neurology.

Certain antiretroviral drugs were associated with problems in developing myelin proteins in cell models and animal models, and the drugs were associated with reductions in white matter in autopsy brain samples from a cohort of individuals with HIV, reported the research team led by co-senior authors Judith Grinspan, PhD, research professor of Neurology at CHOP, and Kelly Jordan-Sciutto, PhD, chair and professor of Pathology at Penn’s School of Dental Medicine.

Both senior researchers emphasized that individuals with HIV should continue taking lifesaving antiretroviral drugs as prescribed. They hope their current and future findings can help researchers refine drug designs to reduce side effects, and help clinicians pursue prescribing practices that are risk-informed and tailored to the patient’s age and stage of brain development. These future changes could be particularly important for children with HIV whose brains are still developing.

For individuals with HIV, multi-drug regimens of antiretroviral treatments are lifesaving, but lifelong. These drugs have transformed the infection from a death sentence into a manageable chronic condition, and they are increasingly being prescribed to high-risk uninfected individuals as a prophylactic treatment.

Since these drugs have helped patients control their viral levels, the severe neurocognitive deficits that were often widespread among individuals with HIV have become far less common. Still, about half of HIV-infected individuals experience HIV-associated neurocognitive disorders (HAND), most of them with mild to moderate deficits.

“The question is, why were they still there when there was no obvious virus in the brain?” Dr. Grinspan said. “The antiretrovirals themselves became the suspects.”

In earlier studies, Dr. Jordan-Sciutto had found that one class of antiretroviral drugs, known as protease inhibitors, killed neurons in cell culture, and she showed the cause to be linked to drug-induced oxidative stress. The drugs had no effect on survival of astrocytes, a type of helper cell in the brain. She wondered, but was not equipped to test in her own lab, whether the drugs affected a third type of cell in the brain, oligodendrocytes.

Oligodendrocytes produce myelin, a lipid-based substance that coats and insulates neurons and constitutes the whiteness of the brain’s white matter. White matter was known to be diminished and abnormal in patients with HIV, so the role of these cells was an intriguing question.

Luckily, as Dr. Jordan-Sciutto was pondering this question a few years ago, she showed up early before delivering a talk to incoming Penn neuroscience graduate students and overheard Dr. Grinspan describing her own study of oxidative stress in oligodendrocytes. She realized that, even though Dr. Grinspan’s work had never before addressed HIV, together they could answer this question that neither of them could tackle alone. The pair successfully applied for NIH funding together, and co-advised their new paper’s first author, Brigid Jensen, PhD, during her doctoral studies.

Out of many types of antiretroviral drugs that patients receive, Dr. Jordan-Sciutto focused the team’s attention on two classes of drugs with known neurotoxicities that are most widely used around the world, and virtually universally in Africa where HIV infection is most prevalent. They tested one nucleoside reverse transcriptase inhibitor (the drug class that includes AZT) and two protease inhibitors.

In the cell-based portion of the team’s study, they applied the drugs to a model of the maturation process of oligodendrocytes from precursor cell types. They found that the protease inhibitors interfered with the cells’ maturation process. The nucleoside reverse transcriptase inhibitor had no effect.

They next treated mice to see if the drugs might affect these cells and their myelin production in a living animal.

“Myelin turns over, but slowly,” Dr. Grinspan said. “With just two weeks of these drugs we saw decreases in myelin proteins.”

Dr. Jordan-Sciutto added, “The rodent study is so important because it shows an animal without any virus had changes in myelin proteins, just from the drug treatment.”

The team was looking at adult mice, so these rapid changes in proteins reflect a potential shift in regular myelin maintenance in adult brains. Dr. Grinspan and Dr. Jordan-Sciutto pointed out that the drugs could potentially have more profound effects on brain development in immature individuals who are still developing their myelin coatings. They intend to address this question in a next phase of their research because, if the model holds for humans, the implications for children could be profound.

“The bulk of the myelin production in the human brain occurs during the first two years of life, but the process overall can take up to 20 years,” Dr. Grinspan said. “If indeed kids are exposed to drugs that stop them from myelinating, they’re going to be in trouble.”

Looking at autopsy samples of adult human brains, the researchers found further evidence of an association between antiretrovirals and loss of white matter: After controlling for factors including age, length of time on drugs, and length of time since death, HIV-infected individuals on antiretroviral therapy had alterations in several myelin proteins, especially decreases in the very important myelin basic protein, compared to uninfected individuals or compared to HIV-infected individuals who never took the drugs.

They have further work to do, both clarifying the mechanisms of how protease inhibitors affect the maturation of oligodendrocytes, and understanding the effects of the drugs on a maturing brain.

“A bonus for me as a basic developmental biologist is that in understanding the mechanisms of the drugs, we learn more about what controls oligodendrocyte differentiation,” Dr. Grinspan said. “That actually ends up being important for diseases like multiple sclerosis, which is a disease of myelin, and in cerebral palsy. Lessons we learn from HIV can affect many other conditions.”

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Targeted Gene Therapy Aimed at Devastating Childhood Disease


When something important is missing, we often search for a replacement. After many years of looking, a team of researchers at The Children’s Hospital of Philadelphia and the University of Missouri have found a way to substitute for a missing gene linked to a relentless childhood neurodegenerative disease.

Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL-Batten Disease) is a rare, inherited condition that erodes toddlers’ abilities to walk, talk, think, and see. Their development appears normal until debilitating symptoms and seizures arise between ages 2 to 4 and then gradually worsen until the children succumb to disease, generally by age 10.

LINCL-Batten is caused by mutations in the TPP1 gene, which encodes the lysosomal enzyme TPP1 that every brain cell needs in order to get rid of waste. When TPP1 is deficient, scientists using fluorescent microscopes can see glowing accumulations of storage material in cells, which is a hallmark of LINCL-Batten.

Beverly Davidson, PhD, director of the Raymond G. Perelman Center for Cellular and Molecular Therapeutics at CHOP and her research team have been trying to understand what is going wrong in the absence of these proteins and how they can develop therapies to treat children with LINCL-Batten.

In a new paper published in the journal Science Translational Medicine, they describe a novel approach using canine TPP1 gene transfer to treat dogs who have TPP1 deficiency and manifest the disease in ways comparable to humans. The lead co-authors included Luis Tecedor, PhD, and Yong Hong Chen, PhD, both from CHOP, and Martin Katz, PhD, of the University of Missouri.

During a 30-minute surgical procedure, a surgeon infuses the gene vector into the dog’s brain. The vector delivers its genetic material to a subset of cells that can then secrete the protein into the cerebral spinal fluid bathing the brain. Subsequent long-term and widespread distribution of TPP1 helps to correct the disease in the dog model.

A one-time infusion resulted in a “remarkable clinical benefit,” the study authors wrote. The treated dogs showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of lifespan.

“With gene therapy, we not only change when those symptoms occur, but we also spread them out,” Dr. Davidson said. “We give the dogs a profound improvement in their quality of life. It is really remarkable.”

Dr. Davidson envisions a similar short procedure could be used to target gene therapy in the brains of children with LINCL-Batten. Early diagnosis will be crucial so that the intervention could preserve as much of their functions as possible and prolong the onset of declines. It is unknown if TPP1 replacement could potentially alleviate any brain damage that already has occurred.

A “one and done” infusion would be a valuable alternative to another experimental therapy that is available called TPP1 enzyme replacement therapy (ERT). While the two approaches share the same basic concept, ERT does not rely on delivery of the TPP1 gene. Instead, it requires children to make biweekly visits to the intensive care unit to receive infusions of TPP1 through an indwelling device. Dr. Davidson anticipates her lab’s gene therapy research will be ready to translate to a human clinical trial soon.

“If the outcome in children with TPP1 deficiency is as profound as in the canine model, we would expect an enormous benefit to the quality of life of the affected children, and also their families,” Dr. Davidson said.

The National Institutes of Health, the Batten Disease Support and Research Association, Blake’s Purpose Foundation, the Roy J. Carver Trust, and the Children’s Hospital of Philadelphia Research Institute supported the canine investigations. The research team at the University of Missouri contributed to the study as well and provided exemplary collaboration in providing for the care and health of the dog colony.

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Girls in Families With Breast Cancer Risk Well-Adjusted


The thought of getting breast cancer can be a worrisome one, and it may be on the minds of young girls more than ever before, as knowledge about family and genetic risk has increased in the recent decade. The LEGACY Girls Study, a study taking place across five sites in North America, is the first to focus on preadolescent girls growing up in families with breast cancer risk. One of its many aims is to understand if they have poorer psychosocial adjustment and breast-cancer specific stress.

“We know that whether or not there is full disclosure about family breast cancer risk, that children pick up on things,” said Lisa Schwartz, PhD, a psychologist in the Division of Oncology at The Children’s Hospital of Pennsylvania and assistant professor at the Perelman School of Medicine at the University of Pennsylvania, who is a co-investigator for the LEGACY Girls Study. “We also know that it heightens anxiety of mothers, so we suspected that it could impact the family environment in some way, whether that be positive or negative.”

The study team, led by Angela Bradbury, MD, assistant professor in the Department of Medicine, Division of Hematology/Oncology, and the Department of Medical Ethics and Health Policy at the Perelman School of Medicine, between August 2011 and July 2013 enrolled 1,040 girls at ages 6 to 13. They analyzed behavioral surveys submitted by about 800 mother-daughter pairs. Half of the participants had a first or second-degree relative with breast cancer or a known BRCA1/2 mutation in the family, and the other half did not.

It is estimated that 5 to 10 percent of breast cancer cases result directly from high risk genes like BRCA1/2. As more women get access to genetic testing, it is important for clinicians to be aware of how this information could possibly influence daughters’ risk-taking or health-promoting behaviors, Dr. Schwartz said.

The study results, which were published in Pediatrics, did not reveal any stark differences in the general emotional well-being between the two groups. Girls from families with a history of breast cancer or risk seem to cope as well psychosocially as girls without that known risk. However, girls ages 10 to 13 did report higher breast-cancer specific distress if they came from a family at risk for breast cancer, and 12 percent had a distress level that would be considered clinically significant.

“This shows that these girls are aware that they are at higher risk for breast cancer than their peers, but it is not impacting them in a global way or permeating their whole psychological functioning,” Dr. Schwartz said. “Also, maternal anxiety was related to child anxiety, which is a very common finding. We can help mothers and daughters communicate with one another about the risk in a healthy way. We don’t want daughters to have a heightened sense of risk. We want them to have an accurate sense of risk and know that they have control over managing that risk and over their health.”

To that end, the LEGACY investigators are working on an internet-based intervention to educate adolescent girls about breast cancer risk and to create a platform for mothers to talk with daughters and relay accurate information in a developmentally sensitive way. The next step for the researchers is to test the intervention in a randomized trial with the same cohort of LEGACY Girls Study participants.

One of the messages the intervention will convey to girls is that it is highly unlikely for breast cancer to occur in the teenage years. It also will inform girls about how healthy choices during adolescence can minimize risk of adult cancer. And it will provide tips on how to handle everyday stressors that can make coping with an ambiguous health situation that has unknown outcomes difficult.

These are the types of important conversations that Norma Roth, a breast cancer survivor, has had with her children to reassure them that recent breast cancer advances have made it more curable than ever before. Her daughter, Marlena, is involved with the LEGACY Girls Study.

“Way before the BRCA genes were discovered, the women in my family truly thought something in their DNA was killing them, and they felt helpless,” Norma said in an interview that appeared in the Research Institute’s 2014 Annual Report. “Today we have better mammograms, we have MRIs, and we have genetic testing. All of these tools are very empowering for my generation. I think the more you know, the better you can control things and change the history in your life. That’s why I really wanted Marlena to be part of the LEGACY study.”

Teaching young girls how to prevent breast-cancer specific distress will become especially important, Dr. Schwartz pointed out, if there comes a time when experts determine that it makes sense to test for the genes for breast cancer in adolescence. Currently, professional societies do not recommend that children, even those with a family history of BRCA1/2 mutation, undergo genetic testing because no strategies are in place for how to respond to positive findings. Surveillance and imaging usually begins at age 25 for women who have a family history that suggests the presence of a harmful mutation.

The LEGACY Girls Study team is finishing up final assessments of data that they have gathered during the study period and plan to look at any changes in girls’ psychosocial adjustment over time. The project has been funded by the National Cancer Institute, and the investigators have submitted additional research grants with hopes of continuing to follow this unique cohort into young adulthood.

Read more about the latest study results in this CBS News report.

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Studying the Brain’s Fundamental Drum Beat to Understand Autism


A drum beat coordinating brain activity and thus organizing the music of life emerges from deep inside the human brain. This electromagnetic neural pulse —eight to 12 beats per second — is known as the resting-state alpha rhythm.

“Alpha rhythms may be the most fundamental brain rhythm, involved in coordinating brain processes from those as simple as hearing tones and those as complex as consciousness,” said  J. Christopher Edgar, PhD, a clinical neuropsychologist and brain imaging researcher in the Department of Radiology at The Children’s Hospital of Philadelphia.

Researchers have known for some time that electromagnetic (neural) brain activity is different in individuals on the autism spectrum. In a series of recent studies, Dr. Edgar and colleagues have shown that the resting-state alpha rhythm is stronger among individuals on the autism spectrum, and that stronger alpha rhythms are associated with more severe clinical symptoms.

With a new grant from the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH), Dr. Edgar will use state-of-the-art noninvasive brain imaging called magnetoencephalography (MEG) scanning to measure brain activities, including alpha rhythms, and magnetic resonance imaging to obtain structural brain measures in adolescents with and without autism spectrum disorder (ASD). He aims to find out why that metaphorical drum beat, setting the pace for the activities of other players or different parts of the brain, sounds different in children on the autism spectrum.

“The main goal of our study is to obtain measures of activity throughout the entire brain, rather than just on the surface of the head,” Dr. Edgar said. “We want to examine the association between the structure of the thalamus, pathways from the thalamus to the brain surface, and brain activity.”

Dr. Edgar suspects that timing abnormalities in the thalamus, a deep brain structure, may be the cause of surface-level brain alpha abnormalities in individuals with ASD. The thalamus acts as a central relay station, controlling the flow of information from the outside world into the brain. The thalamus is likely also the drummer that sets the pace of the alpha rhythm.

If Dr. Edgar’s findings bear out the hypothesis that the thalamus is associated with alpha-rhythm abnormalities in ASD (and perhaps also related to clinical symptoms), these findings would suggest new approaches to treating autism-related impairments via therapies that target the alpha-related activities. For instance, identifying possible abnormalities in the pathways between the thalamus and cortex, whose typical functioning is already known, could help to identify clear targets for treatments with medication or behavioral therapies.

“There are not yet any pharmaceutical treatments for the core symptoms of autism, and there are few promising treatments in the pipeline,” Dr. Edgar said. “A lack of effective treatment indicates the need to identify abnormal brain structure and function in ASD.”

Connecting the identification of brain abnormalities to treatment strategies will require many years of further work and may follow a long and winding path. Still, many researchers are committed to using brain imaging to guide the development of treatments for ASD and other neurodevelopmental disorders, as they consider this one of the most promising ways to make progress.

The example of the drug STX209 (arbaclofen) illustrates how brain-imaging research in ASD informs drug development, along the journey to potential future drug approval. This investigational drug targets a neurotransmitter involved in generating the brain’s gamma rhythm (30 to 50 beats per second), which is abnormal in several conditions including ASD and Fragile X syndrome.

A new MEG study at CHOP is being conducted to identify potential acute effects of STX209 on brain activity in adolescent boys with ASD. This study follows on past testing of STX209 in clinical trials, which did not achieve these trials’ intended clinical outcomes — a failure potentially attributable to variability between participants, as not all individuals with ASD have abnormal gamma rhythms.

The new, small study at CHOP addresses the hypothesis that the drug will have a brief “normalizing” effect on brain activity, potentially only in the subset of individuals with ASD who have abnormal gamma rhythms. Any positive finding could inform future clinical trials that may use brain imaging to identify individuals with the most potential to benefit based on their gamma rhythm abnormalities.

In the case of alpha rhythms, there is no drug yet known to directly target alpha rhythm abnormalities. As such, any discovery Dr. Edgar makes would be more foundational, proving an understanding of exactly how individuals on the autism spectrum march to a different beat.

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Educational Intervention Decreases Mean Girls’ Relational Aggression


The “Mean Girls” phenomenon is not just the subject of fiction. Relational aggression, such as using gossip and social exclusion to harm others, is all too common among preadolescent and adolescent girls. A new study from The Children’s Hospital of Philadelphia suggests that educational interventions including problem-solving skills and leadership opportunities can help, with lasting effects.

“As a psychologist and researcher with a particular interest in bullying, I am always interested in digging deeper into the ‘why,’” wrote Stephen Leff, PhD, in a blog post about the study, which he led. “Why is relational aggression — which involves the manipulation of social standing or reputations through gossip and social exclusion — so predominant among girls? Why is it associated with detrimental long-term outcomes for victims such as high levels of anxiety and depression? And, beyond the ‘why,’ how can we develop and test interventions that can combat this pervasive type of school violence, before it has a chance to become entrenched?”

The study, published in the journal Psychology of Violence, provides a partial answer to that last question. It is the first and only demonstration that a relational aggression intervention decreased these behaviors among urban minority girls for at least a year after the conclusion of the program. Specifically, in a randomized controlled trial, the Friend to Friend (F2F) aggression prevention program improved urban African-American relationally aggressive girls’ social problem-solving knowledge and decreased their levels of relational aggression.

“Including this type of positive skill development in urban school curricula is important because children attending inner-city, under-resourced schools are at high risk for emotional and behavioral problems,” said Dr. Leff, who is co-director of the Violence Prevention Initiative and a psychologist at CHOP. “There is evidence that having these skills and positive leadership opportunities increases their resilience and leads to better future social interactions. This is indicative of the positive approach taken by all of the school-based prevention programs that are part of our Violence Prevention Initiative at CHOP.”

The F2F program’s curricula and innovative teaching methods, including videos, cartoons, and role-plays, were developed and refined through more than a decade of committed research at CHOP in partnership with key community stakeholders. Students, teachers, and parents were all engaged as partners in the program’s design.

The current study involved 144 relationally aggressive girls in third to fifth grades from 44 classrooms across six School District of Philadelphia elementary schools. Participants were randomly assigned to either F2F or to a homework and study skills development program as a control group.

“Teachers were vital implementation partners for us, particularly in reinforcing newly learned pro-social skills and strategies outside of the structured sessions,” Dr. Leff said. “Having their buy-in and support was essential.”

To learn more about this study, view the CHOP press release and blog post from CHOP’s Center for Injury Research and Prevention.

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Study Gives New Insights Into Genetic Predisposition to Neuroblastoma


Pediatric oncology researchers have narrowed down a culprit in an aggressive form of the childhood cancer neuroblastoma that makes the disease progress once tumors form.

By mapping how DNA interacts with regulatory proteins that control transcription, part of the process by which DNA-encoded information carries out biological functions, the researchers pinpointed a precise variant in a single DNA base, guanine, that boosts LMO1 gene expression.

The change in the LMO1 gene results in a “super-enhancer” that causes tumors to arise and grow out of control in an aggressive subtype of neuroblastoma, a cancer of the peripheral nervous system that usually occurs as a solid tumor in a child’s chest or abdomen.

“Cancers in general, and neuroblastoma in particular, have complex origins,” said John M. Maris, MD, a pediatric oncologist at The Children’s Hospital of Philadelphia, who is senior author of the study reported online in Nature. “It’s not common to discover causal gene variants in cancer, especially in a single base within the DNA sequence such as this.”

A change in a single base of DNA is called a single-nucleotide polymorphisms, or SNP. Previous research by Dr. Maris’ team showed that common SNPs within the LMO1 gene drive neuroblastoma susceptibility and progression by abnormally altering gene transcription. This new discovery increases the researchers’ understanding of how the specific protein that functions abnormally in LMO1-driven neuroblastoma sets in motion the molecular mechanisms fueling this high-risk subtype. Components on this biological pathway may offer attractive treatment targets.

“Drugs that inhibit other parts of the gene transcription machinery may offer potential novel treatments for this aggressive subset of neuroblastoma,” Dr. Maris said.

The researchers also found that another genetic change has a beneficial effect: If the DNA base at the specified location is a different letter of the genetic alphabet — thymine instead of guanine — it protects against neuroblastoma. Gene studies in human populations suggest that this protective gene variant evolved after human ancestors migrated out of Africa, hundreds of thousands of years ago.

Finding Genetic Predisposition to Childhood Cancers Raises New Challenges

Why some children develop cancer and not others remains an enormous question that Dr. Maris tackled in a recent editorial in the New England Journal of Medicine, “Defining Why Cancer Develops in Children.” The editorial reflects on a major pediatric study of cancer predisposition genes in the same issue of the journal.

A team from St. Jude Children’s Research Hospital led by James R. Downing, MD, found that 8.5 percent of children and adolescents with cancer in their sample of 1,120 patients had mutations known to raise their risk of cancer. In the majority of cases, a child’s inherited cancer-predisposition mutation did not seem to lead to cancer in the parents or in other family members.

“More frequently than previously thought, children with cancer may have genes predisposing them to cancer, even when cancer doesn’t show up in the child’s family history,” said Dr. Maris, who also is the Giulio D’Angio Chair in Neuroblastoma Research at CHOP and a professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania. “We need to better understand how other genes may interact with the original mutation to cause cancer in the child.”

Dr. Maris discussed how advancements in genetic testing and genetic science have implications for counseling families as more become aware of these cancer-predisposition genes in both affected children and in their family members without cancer. In addition, because some cancer-related mutations may also increase the risk that a childhood-cancer survivor may later develop a second cancer, clinicians and cancer researchers will need to develop the most effective strategies for counseling patients from infancy through old age.

“Like all good research, this new study opens up more questions and areas for more investigation,” Dr. Maris said. “At the very least, this work should remind clinicians that we need to look beyond family history in treating and counseling pediatric cancer patients and their families.”

To read more of Dr. Maris’ comments, visit the CHOP press release and New England Journal of Medicine editorial.

To read more about genetic predispositions to neuroblastoma, visit the CHOP press release and the Nature article.

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Stroke Rehabilitation Research Connects Brain to Gait


If you have ever hit your stride on a moving walkway, the type commonly found in airports, consider how it felt when you stepped back onto solid ground. You may have felt a sudden but brief discombobulation while your brain worked to correct its temporary mismatch with your body’s sense of movement. Laura Prosser, PhD, PT, is trying to induce a similar reaction to rehabilitate children after stroke.

As a research scientist in the Division of Rehabilitation Medicine at The Children’s Hospital of Philadelphia, Dr. Prosser’s work is focused on how the brain and its connection to the body change after damage and during rehabilitation. Her focus on children addresses an under-researched area in rehabilitation.

“Understanding how rehabilitation can impact neuroplasticity is the most exciting aspect of this research to me,” Dr. Prosser said. “Not much of this work has been done in children. At CHOP we are in a unique position to understand how the brains of children respond differently to rehabilitation than the brains of adults who have had an injury.”

Dr. Prosser is now conducting a small pilot study testing physical therapy outcomes after pediatric stroke using high-tech tools including a split-belt treadmill and brain-stimulating technology called transcranial magnetic stimulation (TMS). She aims to learn which approaches seem most promising to pursue in future larger trials.

The split-belt treadmill is exactly what it sounds like: Instead of a single moving belt underfoot, under each foot there is a separate belt moving at its own speed. Imagine if you were walking with only one foot on the airport’s moving walkway and the other foot on the floor, forcing each leg to move at a different pace — it might feel strange at first, but Dr. Prosser said, healthy brains tend to adjust to the asymmetric motion quickly, and injured ones only slightly less so.

Using this treadmill for rehabilitation, physical therapists calibrate the speed of each belt separately according to the individual patient’s gait pattern. After a stroke, individuals often experience hemiplegia, which is difficulty moving one side of the body, and results in an asymmetric walking pattern. But instead of using the treadmill for children to practice walking with a corrected, more-symmetrical gait, the treadmill belts’ speeds are set to make the error worse.

“The idea with error augmentation therapy is to exaggerate the error and force the brain to perceive asymmetry in movement, which it has become accustomed to,” Dr. Prosser said. “The brain responds by working harder to correct this newly recognized asymmetry.”

In her study, half of participants are randomized to receive 24 sessions of this asymmetrical gait training, while the other half of participants receive standard rehabilitation for their injury.

The next trick is to try to understand neurological changes over the course of treatment. TMS is a method that uses a magnetic field over the surface of the head to direct a pulse to a targeted area of the brain. In brain-injury studies like Dr. Prosser’s, TMS is a tool to measure the strength of the brain’s connection to muscle groups in the body.

Sudha K. Kessler, MD, neurologist and director of the Transcranial Magnetic Stimulation Lab at CHOP, delivers each study participant TMS pulses to activate their weakened leg muscles from the injured side of the brain, and on the opposite side to get a comparison. The timing and strength of the muscle response is an indicator of the quality of the brain/body connection. The researchers are tracking these responses over time to measure potential improvements with therapy.

“We are among just a few people who are trying to understand the pathways to the leg muscles in children,” Dr. Prosser said. “More commonly, people are studying the pathways to the upper extremity muscles because based on the anatomy of the brain, those cortical areas are much easier to access with this type of technology.”

Dr. Prosser and Dr. Kessler are also considering the potential of TMS and other noninvasive cortical modulating treatments as an aid to rehabilitation, not just as measurement tools. For example, a repetitive TMS program delivered immediately prior to physical therapy may help prime the brain for learning, improving the effectiveness of rehabilitation sessions.

“One of the things we’re finding so far with the TMS data is that we’re seeing many different patterns of cortical mapping in the children,” Dr. Prosser said. “In adults, cortical remapping after a stroke is more predictable.”

The finding suggests brain-based treatments for children may need to be more individualized. Genetic data may inform those individual treatments in the future, too. As part of the pilot study, Dr. Prosser’s team is collecting blood samples from participants to determine the presence of two particular genetic polymorphisms related to neuroplastic potential. When these variants are present in adults who have had a stroke, they are associated with more severe outcomes. How these gene variants may interact with ongoing brain-development processes in children, and the potential associations between these genes and response to rehabilitation, are unknown.

The small pilot study is being conducted with Rebecca Ichord, MD, director of CHOP’s Pediatric Stroke Program, and Heather Atkinson, PT, NCS, clinical specialist in the Pediatric Stroke Program. Enrollment has nearly reached its target of 12 patients. When complete, Dr. Prosser will examine results from the TMS, genetic biomarker, and physical therapy outcomes data to see which interventions may benefit from further testing in a larger clinical trial — taking the process of pediatric stroke rehabilitation one step forward at a time.

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New CHOP Study to Understand Risk of Hospital Readmission


One in 20 infants is admitted to the hospital during the first year of life. As frightening as it may be for families to have a child whose health condition requires hospitalization, in too many cases the experience gets worse when a relapse or problem managing the condition after discharge means their child must be re-admitted later. Babies born prematurely are among the groups of children are at highest risk of hospital readmission.

With a new grant from the U.S. Department of Health and Human Services’ Agency for Healthcare Research and Quality (AHRQ), researchers at The Children’s Hospital of Philadelphia are working to reduce the need for pediatric readmissions by taking a population-level look at clinical factors, and an up-close look at familial and social factors, that send high-risk groups of children back to the hospital

“The long-term goal is to take this information and develop real-time predictions,” said study leader Scott Lorch, MD, MSCE, director of the Center for Perinatal and Pediatric Health Disparities Research at CHOP and associate professor of pediatrics at the Perelman School of Medicine at the University of Pennsylvania. “We are looking for things that can be fed back to the clinical team to say, based on these factors, this patient has a higher than usual risk of readmission.”

First, Dr. Lorch and his team will analyze population-based national pediatric healthcare data from a large number of hospitals and outpatient centers. They aim to determine what processes and structures of care may be associated with changing readmission risk in high-risk groups. In addition to premature infants, groups involved in the study may include children with asthma and children with complex or chronic medical conditions.

Notably, their analysis will cover both inpatient and outpatient data sources to consider the full picture of what care children receive after their hospital discharge and before potential readmission.

“Inpatient and outpatient data tend to be analyzed separately,” Dr. Lorch said. “That only gives you part of the story.”

Next, over a four-year period, Dr. Lorch’s team will survey families seen at CHOP to gain insights into factors such as familial stress, support, and other social factors that may affect the risk of hospital readmission. Families’ experience and preparation may make a big difference. Premature infants, for example, often remain in the hospital for weeks or months after birth.

“During that time, parents have more of a passive role,” Dr. Lorch said. “When they take the child home, the transition can be very difficult for families who have never done this in the past. Understanding how those factors relate to the risk of readmission allows us to understand the risk and to tailor interventions to mitigate it.”

Eventually these insights could lead to keeping clinicians better informed about which patients are at higher risk before children leave the hospital, and better equipped to support those high-risk families with resources to prevent the problems they experience today.

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