Bench to Bedside

December 2016/January 2017

A Look Back at 2016: A Banner Year for Pediatric Research at CHOP


As we look ahead to a new year and new opportunities in 2017, it’s worth first taking a look back at how much we achieved in 2016. It was a busy year, and investigators from Children’s Hospital of Philadelphia made remarkable progress and reached major milestones of discovery to aid children’s health. These achievements are too numerous to mention in a single roundup, so this review will not be comprehensive. Even so, these highlights of stories we brought you over the course of 2016 in Bench to Bedside should give a good taste of where we’ve been — and whet your appetites for what’s to come.

New Research Initiatives Launch

The past year saw the launch of several new research initiatives at CHOP. In January, the Center for Data-Driven Discovery in Biomedicine was announced, and in November we brought you the latest updates on its major data-sharing platform, CAVATICA. Two new Research Affinity Groups also began, focused on mHealth (health research using mobile and digital tools) and on global health. And in the fall, CHOP announced the new $50 million Roberts Collaborative for Genetics and Individualized Medicine.

Childhood Cancer Advances

Childhood cancers are among the most critical and devastating diseases that researchers at CHOP are working to address. CHOP was honored to host the Coalition Against Childhood Cancer for its summit and annual meeting over the summer, while CHOP researchers have worked on continuous advances against cancer at multiple levels including molecular, technological, and national leadership. Some of the highlights include:

Molecular Secrets Unlocked

The “bench” aspect of bench-to-bedside research is critically important for understanding mechanisms of disease at a molecular level and pioneering new treatments that act on these mechanisms. CHOP had more than its fair share of exciting basic science discoveries in 2016 — and many had clear connections to future translation into therapies. CHOP researchers found an epigenetic mechanism that connects cancer cell growth with cancer cell proliferation that might apply to numerous cancer cell types. Another team discovered an RNA molecule that might play a critical role in the body’s inflammatory responses in multiple diseases. Other immunology research uncovered a mechanism by which viruses can trick the immune system and take over its machinery, which gives new insight into how that machinery works in the first place. And another team described an important molecular interaction in the braking system that the body uses to prevent an allergic or autoimmune response.

Enhancing Child, Caregiver, and Pediatrician Relationships

At the opposite end of the bench-to-bedside research spectrum, investigators at CHOP are also deeply invested in improving care for patients. Several studies in 2016 show how CHOP researchers and clinicians are doing so by enhancing relationships among pediatric providers, parents or other caregivers, and children, to support children’s health together. Some highlights:

Research Partnerships with Education, Government, and Large Systems

To truly make an impact on children’s health, no single pediatric hospital can go it alone. Researchers at CHOP have embraced this principle and worked in partnership with schools, government agencies, and large healthcare systems to reach children wherever they may need assistance to thrive in full health.

In schools, for example, CHOP researchers are partnering to improve behavioral health services for students and to collaboratively create and test behavioral interventions to help children develop healthy relationship skills. They are also partnering with teachers and students on creative ways to improve education in cardiopulmonary resuscitation (CPR) to improve the health of entire communities.

Other CHOP research on CPR in the hospital is tied into larger healthcare systems so that innovations developed in critical care here can save lives across the country and around the world. Likewise, innovative projects in pediatric medical trauma systems and in psychological trauma-informed care are connected to opportunities for systemic improvement through the healthcare system and through policymakers. CHOP researchers also worked closely with policymakers to improve care for vulnerable children who receive prescriptions for psychotropic medications that may be unwarranted.

Mitochondrial Mysteries

CHOP is a powerful hub for research on the power plants of the cell, the mitochondria. Efforts in the Center for Mitochondrial and Epigenomic Medicine, the Mitochondrial Disease Clinical Center, and across other collaborating specialties, are converging on deeper understanding of how energy production in mitochondria relates to a wide range of diseases and conditions, as well as new approaches to treating conditions known to be connected to mitochondria. Among the highlights this year:

Technological Innovation

The promise of new technology is often exciting, and it is certainly so in pediatric research. In addition to highlighting the launch of the CHOP mHealth Research Affinity Group, we also shared several stories about mHealth projects in a June special issue of Bench to Bedside: CHOP researchers are using social media tools to improve HIV prevention and care for teenagers, using a mobile app to collect data about babies’ sleep patterns, connecting young adult survivors of childhood cancer with continued care, and using digital tools with incentives to improve teens’ control over diabetes.

New technologies infuse many other areas of research, too. The advent of lymphatic imaging technologies is one area where CHOP and Penn researchers have made remarkable strides in both understanding and treating an underappreciated organ system. Elsewhere at CHOP, researchers are developing imaging tools to understand the placenta, testing ways to speed blood vessel repair with magnetism, developing computer-vision technologies to quantify dermatological assessments, and creating tools to link pediatric care with public health systems.

What innovations will 2017 bring? Stay with us to find out, and get a preview in our look ahead!

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Looking Ahead to Having a Bigger Impact and Greater Success


The new year often brings great optimism and hope, as we look to the promise and potential it brings, and to the experiences that will shape us individually, professionally, and organizationally.

Reflecting upon the past year at the CHOP Research Institute, and looking at our plans moving forward, we have a lot to be excited about. The Institute recently completed its strategic planning process, which involved taking a close look at the Institute’s programs and organizational strengths, and making honest assessments of what to improve upon, where the Institute wants to go, and how to get there.

What will guide CHOP Research in the coming year and beyond? What is the direction of the institution, and what are the driving factors and goals that will move the CHOP Research Institute forward?

It comes down to two things: impact and success.

On the surface, that may seem simple and obvious. After all, the Institute’s mission has always been one that focuses on making an impact on the health and well-being of children, and that has historically been used as a marker of our success. But here those terms refer to something deeper — a concentrated effort to support research programs and initiatives that have both a high impact on the lives of children as well as a high likelihood of success.

The Institute’s experiences with its gene therapy, genomics, and cancer programs are examples of those with a high impact and — as demonstrated over time — a high likelihood of success.

Looking ahead, the Institute has identified four big areas of research that have the potential to have a similar trajectory in terms of impact and success. Those four areas aim to:

The Institute’s effort with respect to rare and complex diseases will focus on developing a basic science recruitment program for the next several years in the targeted areas of computational and quantitative biology, epigenetics/epigenomics, bioenergetics, metabolomics, and developmental and cell biology. CHOP Research will also fortify its partnership with Penn’s basic science departments and invest in our “big data” and quantitative capabilities.

Novel therapeutics present several exciting opportunities for the CHOP Research Institute. The most promising area of potential therapies to emerge from CHOP rests with biologic therapeutics, and our future success will focus on more basic science capabilities. The Institute will also place heavy emphasis on entrepreneurship and the development of pediatric drugs and devices. CHOP is positioned to become the international leader in pediatric devices and can serve as the place for first-in-pediatrics drug trials.

The Institute’s lifespan research initiative will have a multidisciplinary research approach that links pediatric and adult groups through clinical partnerships. This will involve working with various institutions, which will have designated approaches to developing and integrating various diverse datasets that span the continuum from genetic to healthcare utilization. The aim with this program is to prevent adult diseases through interventions in children and younger adolescents before symptoms emerge. In addition, the lifespan research will aim to provide better long-term outcomes for children living with disease.

The fourth aim, on precision health, focuses on getting the right preventive or therapeutic intervention to the right person at the right place and time. To achieve this, the CHOP Research Institute will partner with economists, engineers, public health researchers, and mathematicians. It will also develop a pediatric knowledge network to support lifespan and precision health studies and trials, and establish new core programs — like community-based research and mHealth/digital health research — to support precision health research.

Looking at these ambitious goals, it is clear the CHOP Research Institute is ushering in a new era of research, one that facilitates research and innovation breakthroughs while enabling CHOP to function as a high-performing Research Institute. With continued support and dedication from our investigators, staff and colleagues, the CHOP Research Institute will help solve the most challenging problems in child health — and what tremendous hope and excitement that brings!

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Michael Marks, PhD, Named AAAS Fellow for Organelle Assembly Insights


A brightly painted ceramic cat waving a paw, a delicate starfish, and even a shrunken head are among an eclectic collection that fills the bookshelves of cell biologist Michael Marks, PhD. Many are souvenirs given to him by postdoc and undergrad researchers who have come from all over the world to work in his lab at Children’s Hospital of Philadelphia and appreciate his boundless curiosity and sense of humor.

Dr. Marks will soon have a new treasure to display: a rosette pin awarded to him by the American Association for the Advancement of Science (AAAS) in honor of his efforts to advance science and achievement of the AAAS Fellow distinction. The AAAS is the world’s largest general scientific society, including nearly 250 affiliated societies and academies of science.

Dr. Marks, who also is a professor in the department of Pathology and Lab Medicine, and the department of Physiology at the Perelman School of Medicine at the University of Pennsylvania, has devoted two decades to figuring out the intricacies of how melanosomes and other lysosome-related organelles form within cells.

“Broadly, I am interested in how stuff moves about inside of cells and how intracellular organelles actually form,” Dr. Marks said. “I study these strange, specific organelles that are only found in certain cell types in our bodies.”

These include melanosomes that make pigment in our skin, hair, and eyes; dense granules in platelets that regulate blood clot formation; organelles within dendritic cells that regulate our immune response; and lamellar bodies in lung epithelial type II cells that release pulmonary surfactant. Failure of some or all of these four organelles’ mechanisms due to genetic defects can lead to a rare disease called Hermanksy-Pudlak syndrome (HPS) that affects one in 500,000 to 1,000,000 individuals worldwide, especially in Puerto Rico. Dr. Marks and his collaborators have gained novel understanding about how each of these cell types is affected by the gene mutations that scientists have identified as causing 10 forms of HPS.

People with HPS have problems with blood clotting and abnormally light coloring of skin, hair, and eyes. Some forms of HPS also cause scar tissue formation in the lungs, called pulmonary fibrosis, which lead to breathing problems that can contribute to individuals’ shortened lifespans of about 40 to 50 years.

Dr. Marks’ lab so far has been most successful in establishing how melanosomes are built, and it is this work that the AAAS cited as worthy of the esteemed recognition of Fellow by his peers. The 10 genes that go awry in HPS encode subunits of four protein complexes, Dr. Marks explained. Three of those complexes take proteins from endosomes, which are intermediate compartments that act as sorting stations for proteins in cells, and then target them specifically for melanosomes.

The fourth complex, as Dr. Marks and his co-authors described in an August paper published in The Journal of Cell Biology, retrieves certain proteins back from the melanosomes and returns them to the endosomes that are then required again in that forward pathway. All four of these protein complexes function in a loop between early endosomes and melanosomes to deliver cargo to and from these maturing organelles. Dr. Marks’ dissection of how melanosomes are assembled within cells is valuable knowledge that can be applied to several fronts, from basic science to potential therapies.

“First of all, we might be able to figure out through interactions between these complexes and other proteins what other diseases may be caused by the same type of problem,” Dr. Marks said. “From a biological perspective, understanding how these organelles are adapted within these different cell types for these specialized functions is also important to understand. And eventually, knowing more about how they actually function will perhaps allow us to generate some kind of drugs to fix the basic problems these kids have. It would be a big deal if we could find a way to prolong their lives.”

The next step for Dr. Marks’ lab is to better understand how well the melanocyte system models the other organelle systems involved with the manifestations of HPS. Taking a closer look at the comparative anatomy of these different organelle systems in different cell types may help to potentially explain why some HPS patients get certain types of symptoms and not others.

Dr. Marks is quick to point out that his research projects are absolutely dependent on the connections he has made with collaborators at CHOP and elsewhere. “I don’t know how to do anything in my lab anymore,” he joked, giving credit to Mortimer Poncz, MD, chief of the Division of Hematology at CHOP, for his platelet expertise; Susan Guttentag, MD, formerly of CHOP who is now at Vanderbilt University School of Medicine, for her insights into lung epithelial cells; and Graca Raposo at the Institut Curie in Paris for her electron microscopy skills, to only name a few.

Another collaborator, Edward Behrens, MD, chief of the division of Rheumatology at CHOP, brought an intriguing question to Dr. Marks in 2015. They began talking about a symptom of HPS 1 (the most common form of HPS) that has received little attention: inflammatory bowel disease (IBD).

“It turns out that no one has ever studied it, although about one-third of this subset of patients get what some papers occasionally refer to as granulomatous colitis symptoms, and nobody has any clue why that happens,” Dr. Marks said. “But I had some insight from the work that we’ve been doing in dendritic cells in one of the other HPS forms that might be relevant toward this disorder.”

Drs. Marks and Behrens came up with a plan to test patients’ dendritic cells for their response to different stimulants in terms of the innate immune functions that they illicit. Their preliminary screen of patient sera seems to support the idea that some HPS patients overproduce certain inflammatory molecules. If the researchers are on the right track, then their findings could possibly lead to adapting treatments already being used in clinical trials for other disorders to better regulate this immune response.

Finding a way to reduce the IBD symptoms that children with HPS 1 experience could be one way to help improve their quality of life. Dr. Marks shared the example of family he was introduced to at a conference of the Hermansky-Pudlak Syndrome Network (HPS Network). Their child spent more than half her life — from the age of six months to 12 years — in and out of hospitals dealing with IBD and other complications related to HPS. The HPS Network awarded Drs. Mark and Behrens a grant this summer to develop a mouse model of HPS to further investigate immune function in the gut.

“I didn’t realize how bad inflammatory bowel symptoms could be for some HPS patients,” Dr. Marks said. “When you see kids who have these problems, it really motivates you to try to come up with something that is going to help them.”

The tradition of AAAS Fellows began in 1874, according to a press release in November announcing the 391 members who were awarded this honor in 2016. Dr. Marks will join this prestigious group at the AAAS Annual Meeting in Boston that will be held in February. They will be presented with official certificates and gold and blue rosette pins, representing science and engineering, respectively.

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Researchers Find Compelling Preclinical Evidence for High-Risk Leukemia Therapies


If there is some small bright spot in getting a diagnosis of childhood cancer, it is that acute lymphoblastic leukemia (ALL), the most common form of leukemia in children, has one of the highest cure rates of all childhood cancers. Yet families may struggle to see that bright spot if they learn that their child’s ALL is in the genetic subgroup known as Philadelphia-like B-cell lymphoblastic leukemia (Ph-like ALL), named for the disease biology’s similarity to cancers caused by the famous Philadelphia chromosome mutation.

Although Ph-like ALL was first thought to be a rare subset of ALL cases, it is now known to account for 10 to 20 percent of B-cell ALL occurring in children and adolescents and nearly 30 percent of the disease in young adults. Patients with Ph-like ALL have a very high risk of relapse and poor responses to usual chemotherapy. After relapse, the prognosis for ALL is dim.

Researchers at Children’s Hospital of Philadelphia are working to restore the bright light of hope for children, adolescents, and young adults with Ph-like ALL by evaluating new drugs that could potentially better target leukemia cells than existing therapies. CHOP pediatric oncologist Sarah Tasian, MD, recently led a study of several drug compounds that act on signaling pathways previously implicated in Ph-like ALL. The results, published in the journal Blood, showed potent efficacy of one individual drug and several drug combinations in preclinical models of the disease.

“There’s been a tremendous amount of work across the world defining the genetics of Ph-like ALL, but fewer studies have focused on the functional implications of those genetic mutations,” said Dr. Tasian, who is also an assistant professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania. Her lab has long been interested in both understanding and therapeutically targeting the functional changes in leukemia cells caused by Ph-like ALL mutations.

Several years ago, Dr. Tasian and her collaborators at CHOP and other institutions identified molecular signaling pathways that were activated in Ph-like ALL. They have recently turned their attention to further experiments with one of these, the PI3 kinase pathway, because newer drugs have emerged to target this pathway.

“There has been a lot of progress with ‘next-gen’ PI3 kinase pathway inhibitors that have only been explored in adult cancers, primarily solid tumors,” Dr. Tasian said. “They haven’t really been tested in leukemia and particularly not in pediatrics.”

The study used immune system-deficient mice engrafted with leukemia cells from children with Ph-like ALL to measure individual effects of four drugs that target different steps of the PI3 kinase pathway. One drug, gedatolisib, has a dual mechanism of action and simultaneously targets both PI3 kinase and mTOR proteins in the pathway. The researchers also tested the PI3 kinase inhibitors in combination with drugs that target other signaling pathways.

Dr. Tasian was surprised to find that gedatolisib worked particularly well, even without being combined with other drugs. It not only shut down leukemia proliferation, but it also actively killed the cancer cells. This single drug nearly cured the mice of their leukemias, which was an unexpected result; in patients, multiple types of chemotherapy drugs are usually combined to achieve cure. In addition, the mice appeared to tolerate the drug well without losing weight or having changes in their healthy blood cell levels. These were particularly favorable results because adult patients treated with PI3 kinase inhibitors for other types of cancer have reported numerous toxic side effects.

“We were really excited to see the efficacy, but we’re also very happy to see the tolerability,” Dr. Tasian said. “You want a cure, but not at a great cost.”

In addition to these findings about gedatolisib as a stand-alone therapy, Dr. Tasian and colleagues found that combining drugs to target the PI3 kinase pathway and another signaling pathway simultaneously even more potently inhibited leukemia growth in the mice and was superior to the effects of individual drugs.

Because this was the first time researchers have evaluated “next-gen” PI3 kinase inhibitors in depth for pediatric Ph-like ALL, more preclinical data will be necessary before these drugs can be considered for clinical testing in patients. Even though many important steps remain before patients can potentially see an impact of this work, Dr. Tasian describes the initial findings as “compelling.”

“We need very robust preclinical data to convince investigators and our industry colleagues to take the leap to clinical trials, particularly in children,” she said. “A constant challenge for us in pediatric oncology is access to cutting-edge cancer drugs and the ability to partner with pharmaceutical companies for testing in children.”

Fortunately, Dr. Tasian and her colleagues in the Children’s Oncology Group (COG) have a strong track record of making that happen. At the same time that they identified activation of PI3 kinase signaling in Ph-like ALL a few years ago, they also found that the JAK signaling pathway is similarly hyperactivated. They previously tested a JAK inhibitor in a COG Phase 1 clinical trial and found it to be well tolerated in children with relapsed cancers. Dr. Tasian now chairs a COG Phase 2 clinical trial testing a JAK inhibitor and chemotherapy specifically in children with Ph-like ALL.

The speed of discoveries along this road, from genetic discoveries to mechanistic molecular understanding, to preclinical and ultimately clinical testing, has been fueled by the dual supports of funding and scientific collaboration. Early support from childhood cancer foundations including the Alex’s Lemonade Stand Foundation and the Rally Foundation allowed Dr. Tasian to generate pilot data and secure additional funding from the National Institutes of Health to expand and bring the ideas to fruition. At the same time, collaboration and mentorship through COG and at CHOP have been vital, including the input of co-authors of the new study who include Stephan Grupp, MD, PhD; David Teachey, MD; and Stephen Hunger, MD. In this environment, Dr. Tasian has taken findings first identified during her post-doctoral fellowship at the University of California, San Francisco with Mignon Loh, MD, through to chairing a major COG clinical trial in less than a decade.

“My goal with all of this work is to get innovative therapies to children as quickly as we possibly can,” Dr. Tasian said. “Being at CHOP in such a translational environment is amazing in its emphasis not just upon scientific discovery, but also asking, ‘How do we move that along?’ We are unequivocally committed to providing the best possible care for children.”

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Trio of Studies Shows Oral Antibiotics Are As Good As IV Antibiotics After Discharge


When a family takes a child home from the hospital after treatment for a serious infection, some worrying about how well and how quickly the child will recover may be inevitable. But families may find some reassurance in the new knowledge that if their child has a prescription for oral antibiotics, they are receiving sound medicine.

For the most serious infections, instead of oral antibiotics, which are easy and safe to take at home, some doctors prefer to prescribe continued intravenous (IV) antibiotics inserted through a peripherally inserted central catheter (PICC) line. Because they tap directly into the circulatory system, PICC lines offer maximum drug delivery. But they also require significant maintenance by caregivers and come with risks of dangerous complications, like infection, clotting, and dislodgement. Many clinicians have wondered but for a long time lacked a solid answer to the question: Are IV or oral antibiotics better for use at home?

“Oral antibiotics work just as well and have far fewer complications. You get a lot of complications from the PICC line,” said Ron Keren, MD, MPH, an attending physician, vice president of quality, and chief quality officer at Children’s Hospital of Philadelphia.

That is the bottom line from a set of three large, rigorous, multi-institution studies for which Dr. Keren was principal investigator. The research, funded by the Patient-Centered Outcomes Research Institute (PCORI), included data about children hospitalized for each of three types of serious infections that require a long-term course of antibiotics. Data came from the Pediatric Health Information System® database of the Children’s Hospital Association. The research team, including co-leaders Samir Shah, MD, MSCE, director of the division of Hospital Medicine at Cincinnati Children’s Hospital; Shawn Rangel, MD, MSCE, a surgeon at Boston Children’s Hospital; and Rajendu Srivastava, MD, MPH, assistant  vice president of research at Intermountain Healthcare; organized a massive chart review across the 36 hospitals whose records were included. The review ensured that their database accurately and consistently captured essential details about the antibiotic delivery method each child received and key variables such as complications for children who had all three types of infections they studied.

Dr. Keren’s analysis of bone infections, called acute osteomyelitis, was completed first and published in JAMA Pediatrics in 2015. Dr. Rangel’s analysis of complicated appendicitis — cases with a perforated appendix, not just routine appendectomies — was published in Annals of Surgery in July 2016. And the final word on this set of studies came with Dr. Shah’s analysis of complicated pneumonia, published early online in Pediatrics in November 2016. The three analyses consistently showed that oral antibiotics were safe and effective, while IV antibiotics came with risks of complications.

“That’s what we hypothesized, but we needed to do a study to prove to physicians that they should feel comfortable transitioning kids to oral antibiotics after they’ve received an adequate IV course in the hospital,” said Dr. Keren, who is also a professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania.

Parents should also know that they have other options that are effective and less risky than IV antibiotics at home, and discuss those options with their child’s doctor.

Fortunately, the researchers found that in the case of complicated appendicitis, prescribing IV antibiotics had already declined to a low rate by the time of the study. However, PICC lines remained common in acute osteomyelitis and moderately so in complicated pneumonia.

The researchers found that there was a high degree of variability between hospitals in the type of antibiotics they prescribed at discharge for children with serious infections, though not much variability within each hospital. Some hospitals regularly prescribed IV antibiotics at high rates, while some rarely did so, and others fell somewhere in the middle. Dr. Keren said that he and his collaborators hope to work with the Children’s Hospital Association to share data through their quality and safety programs. When hospitals and physicians see their performance and how their prescribing rate compares to recommended practices, they are likely to change their practices. Dr. Keren was part of a team at CHOP that demonstrated this principle in improving antibiotic prescribing for common infections in primary care.

As for why physicians might have missed the non-trivial rate of complications among their patients who received IV antibiotics at home after discharge, the structure of the healthcare system may be to blame.

“This is likely a symptom of the fragmentation of the way that healthcare is delivered,” Dr. Keren said. “If the same doctor who put in the PICC lines, with the best of intentions thinking it’s the best way to get antibiotics to kids with serious infections, then kept seeing that 16 percent of the time the same patients kept coming back with problems, they would notice. But there is often no feedback loop to that provider if parents show up in the emergency department or kids are readmitted to the hospital weeks later.”

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Neuroblastoma Genetics Study Seeks to Spell Out Structural Errors


As scientists learn to decipher and read more of the recipe book of life — our genomes — they have found many insights and opportunities about diseases in the errors in that text. Among those insights are numerous indicators that adult cancers often happen because of point mutations, a fairly easy-to-spot category of genetic mistake, like a one-letter typo that changes “well” to “hell.”

“In some childhood cancers we see far fewer point mutations in coding regions than in adult cancers — sometimes orders of magnitude less,” said Sharon Diskin, PhD, a cancer researcher at Children’s Hospital of Philadelphia and assistant professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania.

Because a simple spell-check of the genome has not turned up many of these simple explanations for childhood cancers, Dr. Diskin is taking a closer read of the genetics of the deadly childhood cancer neuroblastoma. She hopes to find better indicators of disease risk and targets for future treatments through her project that was recently awarded funding from the National Cancer Institute (NCI).

Dr. Diskin’s three-pronged approach looks at the genomes of children with the disease, from their healthy cells, tumors at diagnosis, and tumors after relapse. Her focus is on structural variations in the genetics of this disease, which include many different types of changes in chromosomes, such as rearrangements of letters in a word (changing “Santa” to “Satan”) or words in a sentence, or repeated instances of a word (the difference in meaning between “well” and “well, well, well”), and even small deletions of words or passages.

One hypothesis she is pursuing is that the disease risk for neuroblastoma and potentially other childhood cancers can be read from the genome in the non-cancerous cells of affected children. This potential germline component to cancer could come from inherited risk genes that families carry or novel mutations arising at the child’s conception. She is analyzing genome sequences from the TARGET program, a collaborative effort of the NCI, Children’s Oncology Group (COG), and a network of investigators to intensively investigate the genetic drivers of childhood cancers. She will later analyze additional sequencing data to assess heritability in partnership with the labs of John Maris, MD, a world-recognized neuroblastoma specialist at CHOP and professor of Pediatrics at the Perelman School of Medicine, and Andrew Olshan, PhD, professor of Cancer Epidemiology and chair of the department of Epidemiology at the University of North Carolina.

In preliminary work on this project, the team has identified rare germline deletions on chromosome 16 that are enriched in neuroblastoma. With the new grant, they aim to replicate and build on these findings and probe into an intriguing finding of germline deletions in neuroblastoma that were previously linked to diverse phenotypes, including neurological disorders.

“These are rare events but have much larger effect size than typically seen by GWAS [genome-wide association study] approaches, and we think we’ll be able to uncover some of the ‘missing heritability’ in neuroblastoma,” Dr. Diskin said.

In parallel, the team is also looking at tumor DNA to seek disease-related structural variants, again using sequencing data from the TARGET project. In preliminary stages of this work, Dr. Diskin and colleagues have identified recurrent disruptions of a gene on chromosome 11 that may be a tumor suppressor gene. They have also identified structural variants affecting the promoter region for the TERT gene, which plays an important role in protecting DNA from damage. Dr. Diskin plans to continue investigating how this promoter region might shape neuroblastoma risk, and to extend the work to find more clues in other noncoding regions of the genome. Any such variants that turn out to be critical could be useful indicators of disease risk in newly diagnosed tumors, or even new molecular targets for future therapies.

The study’s third component focuses on the most critical and deadly cases: relapsed neuroblastoma. Little published genetic sequencing research on relapsed neuroblastoma tumors is yet available, in part because biological samples from across the evolution of this rare disease are hard to come by. To identify the mutations associated with relapse, neuroblastoma researchers need to have a series of samples from individual patients taken from their non-cancerous cells, from their tumor at the time of diagnosis, and from their tumor after relapse. For her study of structural variants in these relapsed tumors, Dr. Diskin is partnering with the lab of Yael Mossé, MD, a CHOP pediatric oncologist and associate professor of Pediatrics at the Perelman School of Medicine, who is leading a precision-medicine clinical trial for relapsed neuroblastoma that involves sequencing the relapsed tumor samples. Dr. Diskin will also partner with St. Jude Children’s Research Hospital and with the COG to work with additional samples and sequences, comparing relapsed tumors to tumors at diagnosis. These combined efforts could yield new insights about how the relapsed genome differs from primary tumors and point to potential targets for therapy.

Even beyond finding structural variants with the three-pronged approach to studying germline, diagnostic tumor, and relapsed tumor DNA, Dr. Diskin will look deeper into the biological mechanisms connecting these variants to disease. Already, she has begun bench work with two of the variations identified in her preliminary work — carrying out the error-laden recipes, not just marking up the problems in the text. This work could give a flavor of what new therapies might need to do to counteract the mutations’ effects.

“Now that we have all this sequencing data, I think this unexplored area could lead us to new insights,” Dr. Diskin said.

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Physician-Scientists Explore New Ways to Quell Cytokine Storm Syndromes


Imagine that every household in a large city turned on every light switch in their houses at the same time, overloading the electrical power plant. The same kind of scenario happens in cytokine storm syndromes, when overactivation of the immune system produces too many inflammatory molecules (hypercytokinemia) that damage healthy organs. Researchers at Children’s Hospital of Philadelphia are studying small signaling proteins in the IL-1 cytokine family that potentially could be targeted as circuit breakers by drugs used to block the overwhelming immune response.

As a rheumatologist, Edward Behrens, MD, chief of the Division of Rheumatology and the Joseph Lee Hollander Chair in Pediatric Rheumatology at CHOP, encounters a cytokine storm syndrome, also known as macrophage activation syndrome (MAS), in some young patients with systemic juvenile idiopathic arthritis, an autoimmune disorder. The signs and symptoms of MAS are similar to those seen in patients with sepsis who have serious infections usually caused by some type of bacteria or virus; however, in MAS, bacteria aren’t inciting the systemic inflammation. The body’s immune system mistakenly attacks some of its own healthy cells and tissues. They experience massive inflammation that progresses to multiorgan failure. A strikingly similar disease process occurs in another disorder called hemophagocytic lymphohistiocytosis (HLH), a rare condition in which the body makes too many immune cells. While the context of these cytokine storm syndromes may differ, all are life-threatening.

“The common theme seems to be that the immune system is spitting out too many cytokines,” said Dr. Behrens, who also is an associate professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania. “If we can figure out which cytokines are actually causing the disease, we could try to block them.”

Dr. Behrens received a new grant from the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, to investigate the cellular and molecular mechanisms that contribute to hypercytokinemia and subsequently find new treatment strategies that could potentially reduce these devastating complications. The project’s main focus is to study the cytokine IL-33, which will benefit from novel insights that Dr. Behrens has gained from tangential work with its sister cytokine, IL-18.

Important Preclinical IL-33 Groundwork

Dr. Behrens’ previous research in mice models of HLH suggests that blocking IL-33 helped the mice get better. IL-33 acts as an “alarmin” that signals increases in another cytokine called interferon-gamma that is produced by CD8 T cells. Interferon-gamma is critical for fighting off viral and bacterial infections, yet excessive amounts cause inflammation that can lead to organ failure in HLH. When the researchers blocked IL-33, interferon-gamma levels in the mice went down. The new grant will allow Dr. Behrens and his study team to take a closer look at how IL-33 could promote inflammatory diseases and under what circumstances.

“We think we have this great target,” Dr. Behrens said. “Now we need to understand in the most minute details how we could treat patients with IL-33 blocking antibodies to cure their HLH. It’s in the realm of possibility because a drug that blocks IL-13 already exists for a completely different purpose. This new grant will help us to provide the rationale and important preclinical data to one day open a clinical trial to see if blocking IL-33 is a safe and effective treatment for humans with HLH.”

This concept could be extended to other examples of conditions where CD8 T cells make too many cytokines. For example, the researchers’ preliminary results in mouse models suggest that blocking IL-33 could be beneficial in graft vs. host disease, which can occur after a bone marrow or stem cell transplant. The donated bone marrow/cells attack the recipient’s body because they view it as foreign. Taku Kambayashi, MD, PhD, an associate professor of Pathology and Laboratory Medicine at Penn Medicine, is working with Dr. Behrens to study how IL-33 works in HLH and graft-host disease animal models.

A Hunch Leads to Novel Intervention

Dr. Behrens’ research interests in the IL-1 family of cytokines enabled him to make a serendipitous connection between a seriously ill infant’s symptoms and a gene called NLRC4 that produces IL-18, another cytokine that contributes to inflammatory processes. He described the life-threatening case of infantile-onset MAS and enterocolitis in an article published early online in the Journal of Allergy and Clinical Immunology.

The six-week-old patient was admitted to CHOP’s pediatric intensive care unit on a weekend that Dr. Behrens was on call, and her condition was getting worse. Dr. Behrens suspected that she had MAS, but one of her symptoms — severe diarrhea — isn’t usually a part of that diagnosis.

Then he recalled a paper that a former CHOP rheumatology fellow who he had trained, Scott Canna, MD, had published in Nature Genetics about a family who had a mutation in NLRC4 that caused the gene to be “turned on” all the time, and as a result they developed MAS and diarrhea. A new attending physician in Immunology at CHOP, Neil Romberg, MD, had independently discovered that the exact same gene caused the exact same syndrome in another family. He reported his findings in the same issue. Dr. Behrens and his colleagues wondered that it would be a massive coincidence if the sick baby under their care could be the fifth person in the world to be diagnosed with NLRC4-MAS.

Their hunch was right. Whole exome testing, a technique for sequencing all the expressed genes in a genome, revealed that the baby had the NLRC4 mutation.

“We knew she had the disease, but the problem was no one knew what to do about it,” Dr. Behrens said. “We tried all of our usual things, and for four months she was in the hospital and was not getting better. Everyone was concerned that if we didn’t try something drastic, this case would not have a good outcome.”

One option was to try a way block IL-18, but finding the right drug wasn’t an easy task. Fortunately, Dr. Behrens discovered a company Switzerland that was testing an IL-18 blocking drug in small clinical trials for some other indications. When Dr. Behrens explained that the drug potentially could help a gravely ill six-month-old who had a gene mutation that caused her to produce too much IL-18, the company and the U.S. Food and Drug Administration agreed that it could be used as an emergency investigational new drug.

“After two doses, it seemed she was cured,” Dr. Behrens said. “She sat up and smiled. The inflammation melted away. Today, you would never know she had been so sick.”

In this breakthrough case, blocking IL-18 indeed was like finding a central circuit breaker to turn off the immune system’s overpowering response. Dr. Behrens and his team are hoping that the same concept holds true for IL-33 and HLH.

“Even if the results aren’t as dramatic, we could have another tool that will help,” Dr. Behrens said. “This patient story demonstrates how our work goes from the bedside, to the bench, to the bedside. What you do in the lab is hopefully motivated by what you see in the clinic. And then, what you do in the clinic will be motivated by what you do in the lab. It’s not one directional, and you need to have the infrastructure that allows you to bridge all those gaps. CHOP is one of the few places in the world where you can do that.”

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Transformational Science: Q&A with Douglas Wallace, PhD, Winner of Franklin Medal


Douglas Wallace, PhD, does not shy away from controversial ideas in science. But unlike your kooky uncle who proselytizes about grand theories of the universe, Dr. Wallace has pushed forward several ideas that have turned out to be correct, overturning longstanding assumptions about biology, medicine, and even human history along the way. Dr. Wallace, founding director of the Center for Mitochondrial and Epigenomic Medicine at Children’s Hospital of Philadelphia, is a world-renowned expert in the genetic study of mitochondria, the organelles inside cells that produce most of the body’s energy and that have several of their own genes distinct from the genes in a cell’s nucleus. Through the study of these mitochondrial DNA genes, Dr. Wallace founded the field of mitochondrial genetic medicine.

In recognition of his achievements, Dr. Wallace was recently named the 2017 recipient of the Franklin Medal in Life Sciences from the Franklin Institute. The Franklin Medal, a highly esteemed international award established in 1824, has been awarded to scientific superstars including Albert Einstein, Thomas Edison, Stephen Hawking, Marie Curie, Nikola Tesla, Niels Bohr, Bill Gates, and Max Planck. More than 100 Franklin Medal laureates also have received Nobel Prizes.

On the occasion of this honor, we sat down with Dr. Wallace to reflect on his career, his noteworthy discoveries, his unflagging support of patients with mitochondrial diseases, and the audacious ideas that he believes could result in a major medical transformation. The edited conversation follows below.

You’ve made so many noteworthy discoveries over the course of your career, it will be hard to cover them all. So let’s narrow down. Can you describe what you think of as the top four?

Sure. Number one, in the early 1970s, we showed in cultured cells that the mitochondrial DNA (mtDNA) had heritable traits. We did this by inventing the “cybrid transfer technique.” In cybrid analysis, we start with a cell that has a particular trait; we first used cells resistant to the drug chloramphenicol which inhibits mitochondrial protein synthesis. We physically remove the nucleus for the mutant cell so we just have the cytoplasm with the mitochondria and mtDNA. We then fuse this “cytoplast” to a cell that doesn’t have that genetic trait. The resulting cybrid cell will then have the non-mutant cell nucleus, but the mutant cell mtDNA. If the mutant trait is transferred with the mitochondrion, it must be coded in the mtDNA. By this approach, we showed that chloramphenicol resistance is coded in the mtDNA, providing the first proof that the mtDNA could code for heritable traits. These studies were done in the early 1970s. Yet our cybrid technique is still the standard method for determining if a genetic trait is mtDNA encoded.

Number two, in the late ‘70s, we found that the human mtDNA had a high degree of sequence variation. We then used these sequence polymorphisms as genetic markers to follow the inheritance of the mtDNA in families: mother, father, and children. This showed that in every case the mtDNA came only from the mother, not from the father. Hence, the human mtDNA is exclusively maternally inherited.

Number three, we decided to survey the mtDNA variation between different aboriginal populations around the world. This revealed that every regional population had its own unique mtDNA polymorphisms. Since the mtDNA is exclusively maternally inherited, then the only way that the mtDNA sequence can change is by the sequential accumulation of mutations along radiating maternal lineages. Therefore, the number of mtDNA variants between any two individuals is proportional to that time that they shared a common mother. By comparing the number of mutations between aboriginal populations, we could reconstruct the genetic relationship between them and even calculate the time since they separated. By correlating this with their geographic locations, we were able to reconstruct the history of women.

Number four, since we found that mtDNA variation was much higher than nuclear DNA variation, then mtDNA mutations must be very common, and some of these mutations must damage critical mtDNA energy genes and cause disease. Our search for these mtDNA diseases culminated in 1988 in our reporting two maternally inherited diseases, Leber Hereditary Optic Neuropathy, and Myoclonic Epilepsy and Ragged Red Fiber Disease, thus creating the field of mitochondrial medical genetics.

All of those achievements are transformative ideas in biology. You’re now a proponent of a newer controversial idea about mitochondria, that medical models of disease based on the anatomy of organs should give way to a disease model based on bioenergetics. Do you encounter a fair amount of skepticism about that?

That’s putting it mildly. Any new idea is going to be challenged. That’s how science works. Those ideas that lead to a new way of looking at the disease process and ultimately benefit patients and society will survive. Since our demonstration that mtDNA mutations can cause diseases, defects in the mtDNA bioenergetic genes have been associated with forms of blindness, deafness, epilepsy, dementia, heart disease, renal problems, chronic fatigue, diabetes, various cancers, and aging. Since the mtDNA only codes for bioenergetic genes, it follows that these clinical manifestations can be caused by chronic systemic energy deficiency. The organs with the highest energy demand are the brain, heart, muscle, renal, and endocrine system, so it follows that partial systemic energy deficiency could be responsible for the symptoms commonly observed in these organs.

Therefore, the common “complex” diseases, such as diabetes, neuropsychiatric disorders, cardiac and renal problems might all be the result of mitochondrial energy defects. If so, then by classifying diseases by the organ of the primary symptom, we may have completely missed the underlying bioenergetic etiology of these diseases.

If correct, this would be transformative. We would need to stop focusing on treating organ specific symptoms and start treating the underlying systemic mitochondrial energy deficiency.

But that would require changing the way medicine is organized, because for the past half a millennium Western medicine has been organized around anatomy from divisions, to departments, to NIH Institutes.

While the bioenergetic basis of common disease is a hypothesis, every experiment that we have done to date has validated it. Still, the idea that systemic energy defects could cause organ-specific symptoms is very different from the way medicine has been taught. That this idea would be recognized by the Franklin Institute is quite exciting since it indicates that the bioenergetics hypothesis of diseases is finally getting traction.

For trainees starting out now, what do you think mitochondrial science and medicine might look like in the next 40 or 45 years?

Keep in mind you’re talking to the world’s most extreme “mitochondriac.” Still, I believe that bioenergetic deficiency is a major factor in the etiology of the common complex diseases. If so, there will come a time when medicine will emphasize bioenergetics in the etiology of disease, and this will not just be a subspecialty, but a main trust of medicine. If so, this may permit us to understand and treat many clinical problems that are currently difficult to manage.

A new approach is badly needed, since the number of patients who cannot be successfully cured is legion. Unfortunately, these patients are frequently left outside “modern” medicine because they don’t fit into the current diagnostic paradigms. As an example, I saw a woman in the clinic at Emory 30 years ago. She was 23 or 24 at the time, was obviously very weak, had myoclonus, progressive memory loss, and a large cervical fat pad (cervical lipoma) on her back. But for much of her life, she had been referred to psychiatrists. You only needed to examine her back to know this was not a psychiatric problem. We ultimately showed that she harbored a mtDNA mutation. Referral to the psychiatrist had often been the fate of such patients.

How did you become a leading authority on mitochondria, when it wasn’t something people thought about as significant to medicine when you started out?

I’ve always been interested in science. There are three things that I’ve always wanted to know: Who am I? Where did I come from? And why do I feel bad? Those have really been, ever since I could think, what I wanted to know about. After a lot of searching, I realized that science had the most meaningful answers for me.

I was very fortunate be able go to Cornell University quite inexpensively because I was a resident of the state of New York. I will always be grateful to New York for their support. At Cornell, I had the opportunity of having a very open curriculum, so I took a lot of science because that is what I was interested in. I was particularly interested at that time in physics. This was, ’64 to ’68, and people were just working out the elementary particles of matter. Cornell was strong in that area, so I was fortunate to be exposed.

After two years in the public health service, I went to Yale. At Yale, I decided to study human mitochondrial genetics. I rationalized that since the mitochondria produce 90 percent of our energy, they could not be trivial, and since the mitochondria had its own DNA, that DNA must mutate and cause disease. Moreover, it had just been reported by Vinograd at Caltech that the mtDNA could be purified from human cells. Hence, for the first time, we could do molecular biology on a specific human DNA sequence.

Yale was perfect for me since one of the professors was Harold Morowitz, a physicist who was thinking about physical principles in biology. Also, Yale had just decided to form a human genetics department out of microbiology and parts of medicine. Thus, I spent one day a week with Harold Morowitz thinking about the physics of biology and six days a week studying mitochondrial bioenergetics and determining if the mtDNA was relevant to medical genetics.

By developing the cybrid system, we showed there was a mtDNA genetics, resulting in my PhD. The problem was, where should I go after graduation since no one else in the world was doing what I was. So I wrote to NIH and said, “There is no other laboratory that does what I do, so I want to stay where I am and continue this work.” NIH said OK, so I just stayed at Yale in my back corner lab and continued to do mitochondrial genetics work until I got an assistant professorship at Stanford.

Even up until the late 1980s, it was very difficult to get funded to study the clinical biology of the mitochondria. Mitochondrial disease had not been discovered yet. I was told, “Mitochondria has nothing to do with medicine,” or, “There is no such thing as a mitochondrial disease.” How could I say that I was going to study mitochondrial disease if it didn’t exist?

So finding resistance to your controversial ideas is familiar territory.

Absolutely. However, it was clear to me that humans were the most animated objects in my environment, and Newton had shown that mass does not move unless acted on by energy. Therefore, energy must be critical to being human. The cadaver has excellent anatomy. It is just dead. What it lacks is energy. This seems so compelling to me that it doesn’t matter how many times I’m told, “No.” I remain determined to pursue the bioenergetic perspective of health and disease.

After my discoveries at Stanford of the maternal inheritance of human mtDNA and the high mtDNA sequence variability, I was approached by the Division of Medical Genetics at Emory Medical School. Emory appreciated the potential clinical implications of our discoveries. Accordingly, Emory offered me a professorship, so I moved to Emory advancing from assistant professor to full professor, bypassing associate professor.

Tell us about some of the other milestones in mitochondrial medicine that you went on to achieve.

At Emory, we continued to define the principles of mtDNA genetics, were among the first to clone nuclear DNA-coded mitochondrial genes, used mtDNA variation to reconstruct the origins and ancient migrations of women, identified the first maternally inherited diseases, showed that mtDNA mutations accumulate with age creating the aging clock, identified the first mtDNA variants that predispose to Alzheimer’s and Parkinson’s disease, and created the first mouse models of mitochondrial disease. Based on this work, we set up a combined mitochondrial medicine clinical-diagnostic-research program at the beginning of the 1990s, which provided truly integrated care for mitochondrial disease patients. As a result, we had referrals from all over the world.

Even today, I’m still working on some of the Emory cases. My pact with my patients and their families is that I will not give up until I either find the answer to their problem or die. Unfortunately, many of the patients have died. However, we are still working on their problems because the answers may save the lives of others.

After being at Emory for 19 years, I was recruited to the University of California, Irvine (UCI) to build an integrated mitochondrial medicine program for the California university system. This we did, expanding our clinical research, maintaining a cutting-edge mitochondrial genetic diagnostics program, developing powerful new mouse models of mtDNA disease, and moving strongly into studies of the role of mitochondrial dysfunction on common diseases such as Alzheimer’s and Parkinson’s disease, diabetes, and cardiac disease.

In 2009, I was invited by Dr. Marni Falk to present a lecture at CHOP on our mitochondrial medicine work. Over dinner that evening, Drs. Falk and Marc Yudkoff indicated that CHOP wished to expand its mitochondrial medicine program and was looking to recruit a leader in that field. CHOP already had outstanding clinical investigators such as Drs. Falk and Yudkoff plus a 50- year tradition of research in bioenergetics under the leadership of Dr. Britten Chance. I saw this as an outstanding opportunity to advance patient care in mitochondrial medicine. So I signed up to form the Center for Mitochondrial and Epigenomic Medicine, where we are applying our novel concepts to find powerful new approaches to understand and treat both rare and common diseases.

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Produced by The Children’s Hospital of Philadelphia Research Institute.

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