CBTP Using High-Throughput Screening in Search for New DIPG Treatments

Uday Bhanu MaachaniDiffuse intrinsic pontine glioma (DIPG) is a complex and challenging tumor. Its location in the brain stem, combined with its diffuse nature, makes it inoperable. No chemotherapy drug has yet been effective against it. Radiation provides only temporary relief before the malignancy returns. And the scarcity of both tumor tissue and research funding has made it difficult for scientists to study it. Recent advances in the OR and in the lab, however, are starting to change the tumor’s grim profile.

For decades, neurosurgeons were reluctant to biopsy pontine gliomas for fear of inflicting devastating neurological harm on their young patients. Stereotactic needle biopsy now makes it possible to biopsy these tumors, not only confirming the diagnosis but also securing tissue samples for research.

At the Children’s Brain Tumor Project laboratory, we now have several distinct patient-derived DIPG cell lines growing in vitro. Using these cell lines, we can study the molecular alterations present in these tumors and select effective molecular targeted therapies to test. We are learning more every day about the genetic mutations, epigenetic alterations, and the activation of stem cell pathways that might be causing these malignancies. (We also have gliomatosis cerebri cell lines growing, but GC cells grow more slowly than DIPG cells do, so it will take a bit longer for them to be ready for testing.)
DIPG cell lines under the microscope

As we learn more about DIPG’s molecular characteristics, we can apply newly developed high-throughput screening (HTS) techniques to try to match those characteristics with existing FDA-approved drugs. Using HTS, we can examine millions of variables in already-approved oncology drugs in search of promising candidates to test against DIPG. These drugs are in clinical use today for other cancers, and we are testing them on our collection of DIPG cell lines. The goal of the initiative is to identify the most potentially effective drugs for DIPG, and then rapidly transition them to clinical trials.

Perhaps even more significantly, HTS allows us to predict which combinations of oncology drugs would make good candidates to use in synergy against DIPG based on the molecular profile of the tumor cells. (In other cancers, synergistic drug combinations have succeeded when single drugs have failed.) Synergistic drug pairs have special potential for success against chemo-resistant cancer cells. Drugs can be specifically paired to attack the cancer cells on parallel paths, so that when the first encounters drug resistance from the tumor, the second is able to continue on its mission. The combinations can also achieve a desired effect at a lower total dosage, usually with fewer side effects.uday maachani at microscope

The next step is to inject our DIPG cells into animal models, then use convection-enhanced delivery (CED, the technique being used in Dr. Souweidane’s clinical trial) to deliver specially selected drug pairs into the tumor. Since we’ll have very detailed information about the tumor cells, we’ll be able to choose drugs that have the best chance of success against those particular cells. It’s a tremendous first step toward developing personalized treatments that will someday allow us to select the most promising drugs to use in children with DIPG.

Lab Update: Fall 2015

As I write this, it’s hard to believe it’s been nearly four years since I received word that the FDA had approved my clinical trial testing the safety of using convection-enhanced delivery (CED) to deliver drugs directly to the site of a DIPG tumor. It seems like the blink of an eye, and in scientific research terms it is. But I know that those four years also represent nearly 800 children who lost their lives to this dreadful disease while we’ve been searching for a cure.Dr. Mark M. Souweidane

Keeping those children front and center—in my own mind, in the minds of our lab researchers, and most of all in the minds of our families—is a key driver that keeps this project going, and that creates the momentum we need to keep reaching new milestones.

I reached one of those milestones just today, when I operated on the last patient in the trial to receive dose level six. Nearly two dozen children have been treated, without a single significant adverse effect. We are very close to establishing what I have believed for four years: that CED is safe to use in children, and that it can deliver drugs to a tumor in far greater concentrations than can be achieved using traditional chemotherapy.

Now comes the final stage of this trial: dose level seven. After that we hope to expand the trial to more institutions nationwide—treating more children, with a wider range of drugs and dose levels, using single and combination drug therapies tailored specifically to individual tumors.

The cell lines we have growing right now in our lab are providing us with a wealth of information every day about DIPG (see next page for more on these cell lines). We could not being doing this work if it were not for the collaboration among our peers worldwide, the participation of families who are dedicated to finding new treatments, and the generous donors who keep supporting us. I could not be more proud of where we are today, or more excited about where we are headed.


Mark Souweidane Signature

Other Happenings at the CBTP

Things are hopping in the lab these days, with new investigations going on and with summer help and new hires giving us the capacity to get more work done. We’ve recently welcomed lab technician and manager Melanie Schweitzer; instructor Ude Macchani, PhD; medical student Shaikha Al-Qahtani; research assistant Rachel Yanowitch; summer clinical interns Samantha O’Brien and Nicole Michelson; and summer marketing intern Michael Sapunor.

Praj and Fellow
Dr. Prajwal Rajappa and lab manager Melanie Schweitzer
Sixth-year resident Babacar Cisse, MD/PhD, oversees fellow Raymond Xu preparing samples of ependymoma cells for study
Sixth-year resident Babacar Cisse, MD/PhD, oversees fellow Raymond Xu preparing samples of ependymoma cells for study
Dr. Greenfield and our summer interns Michael. Samantha, and Nicole
Dr. Greenfield and our summer interns Michael. Samantha, and Nicole

New Medical Student Fellow to Investigate DIPG Drug Resistance

Medical student Linda Wu is only 25, but she has a long-standing interest in oncology and neuroscience. Wu is a graduate of Cornell University in Ithaca, where she majored in biology with a concentration in neuroscience. She was then accepted into Weill Cornell Medical College, where she has completed three of her four years of study. Between her first and second years she worked on a pediatric neurology research project, and this summer she joins the CBTP lab full-time for a year to work on Dr. Souweidane’s DIPG research.

Linda YueIt might sound strange for a medical student to be taking a year off from her studies instead of spending that year completing her medical degree, but it’s not at all surprising for Wu. Her passion for oncology research made this an easy choice once she heard about the new pediatric neurosurgery Medical Student Fellowship, made possible by a grant from the Rudin Foundation. After her application was accepted by Dr. Souweidane, Wu immediately dug into the project started by Ranjodh Singh (who returns to his medical studies this fall after his own year in the CBTP lab).

Singh spent the past year testing combinations of drugs to fight DIPG; Wu will continue that work by focusing on how tumors might develop resistance to those drugs. Her research will involve monitoring changes along the molecular pathway and investigating which inhibitors would work to defeat the tumor’s resistance.

Wu will return to medical school in the fall of 2016 to complete her fourth year and earn her medical degree.

Lab Update: Summer 2015

One of the interesting things about scientific research is that you don’t always know what you’ll find when you start looking at something, and you can’t predict what seemingly minor discovery will turn out to be a stepping stone to a much larger one. That’s what makes it so important to gather data, analyze it, and turn it over dozens of times, looking for clues you may not recognize at first or new questions you hadn’t previously thought to ask.

Drs. Greenfield and Souweidane
Drs. Greenfield and Souweidane

We’re proud to be contributing to the advancement of neuroscience at a faster pace these days, as the additional resources made possible by your donations allow us to gather and analyze more and more data, with some surprising results. Our team recently published three papers on new findings that add to the knowledge base on pediatric brain tumors:

The September issue of Neurosurgery will report on a study directed by Dr. Greenfield. This new study looked at 97 children—the single largest cohort of children ever studied—with a malignant brain tumor called glioblastoma. While beginning a more detailed genomic analysis, they first investigated more mundane parts of their clinical course such as surgery and radiation. The data confirmed in children what was already known for adults, which is that total resection leads to longer survival times than partial. That may be intuitive, but studying the data produced another, more puzzling result: When tumors can be totally removed, the improvement in outcomes is significantly greater for girls than for boys. Why? We don’t know… yet. This gives the CBTP an important clue as we begin analyzing specific parts of these tumors’ chromosomes and genomes, looking for potential clues as to where the gender bias might arise and if this could offer new targets to investigate as therapeutic options.

The July issue of the Journal of Neurosurgery Pediatrics reports on a case of a young man whose MRI had shown a tumor in the region of the midbrain called the tectum. An endoscopic biopsy, however, revealed additional lesions on the floor of the third ventricle that turned out to be low-grade gliomas. This surprising find not only points out limitations of MRI (which is an excellent imaging tool), but also suggests the tumors spread through the cerebrospinal fluid. Analyzing these samples genetically also offers a fascinating glimpse into a tumor that looks benign, but that spreads like a malignant tumor! All these clues are part of the outside-the-box thinking that our genomic tools and resources allow us to investigate. Dr. Souweidane is the senior author of this paper.

The June issue of the Journal of Neurosurgery includes a study that looked at radionecrosis, a devastating complication of external beam radiation that causes cell death months or even years after treatment, making radiation therapy particularly risky for young children. This new study, co-authored by Dr. Kramer of Memorial Sloan-Kettering and Dr. Souweidane, looked at the incidence of radionecrosis in young patients treated with intraventricular compartmental radio-immunotherapy (cRIT) instead of or in addition to external beam radiation. The study found none of the neurological damage caused by radiation necrosis in those treated with cRIT. This new finding makes radioimmunotherapy a potentially safer weapon against pediatric brain tumors than external beam therapy. It’s part of our commitment to finding not only more effective but also gentler therapies for our children.

Each finding we can publish not only helps our own team as we strive to develop new therapies for rare and inoperable brain tumors, it also goes forth into the scientific community at large, where it just might mean a safer treatment for a child today, or a new piece of the puzzle that will fit into another piece a year from now. We’re proud to be contributing, and we thank our donors for making it possible.


Mark Souweidane SignatureJeffrey Greenfield Signature

New Research on Ependymoma Made Possible Thanks to a St. Baldrick’s Summer Fellowship Grant and Family Donations

Medical student Scott Connors will continue his investigations into recurrent pediatric ependymoma in the CBTP lab this summer, funded by a fellowship grant from the St. Baldrick’s Foundation. St. Baldrick’s, a longtime supporter of the Children’s Brain Tumor Project, awarded the grant for research into the genomics and epigenomics of ependymoma. Much like DIPG and GC, ependymomas—which arise from ependymal cells that line the ventricles of the brain—are rare brain tumors that have been poorly studied to date compared with other pediatric cancers. Thanks to an outpouring of support from the family and friends of Campbell Hoyt, the CBTP has been able to expand seminal studies specific to understanding why recurrent ependymoma is so difficult to cure. The St. Baldrick’s grant will allow us to redouble our efforts on that research and lay the groundwork for future investigations and potential clinical trials.

Alex’s Lemonade Stand Awards Grant to CBTP to Investigate Drug Distribution in CED

Melinda WangThe Alex’s Lemonade Stand Foundation has awarded a $5,000 grant to Melinda Wang, a medical student at Weill Cornell Medical College, to conduct research into the distribution of the therapeutic agent delivered during convection- enhanced delivery (CED). Dr. Souweidane’s clinical trial for DIPG has been testing the safety of using CED to get tumor-fighting drugs directly to the site of a brainstem tumor. A special contrast agent can trace large molecules as the drugs are infused, but there has not been a reliable way to measure the concentration of the smallest molecules. Wang, a first-year medical student, will modify two small-molecule kinase inhibitors, dasatinib and everolimus, to allow them to be monitored by positron emission tomography (PET) and near-infrared fluorescence (NIRF). This will provide, for the first time, accurate real-time images of how small-molecule drugs perform when infused during a CED procedure.

Lab Updates: April 2015

This issue’s lab update is more like a chorus of “On the Road Again,” as we have both been traveling so much this spring. As reluctant as we always are to leave our work here, that travel has been invaluable in the sharing of information and the enhancing of our worldwide collaborations.

Dr. Mark Souweidane and Dr. Jeffrey GreenfieldThe DIPG Workshop in Barcelona in February brought together experts from all across the United States and Europe, from cities including Amsterdam, Helsinki, and London—not to mention institutions such as the NIH, Duke, Dana Farber, Northwestern, and Weill Cornell. The workshop is made possible through the efforts of the Alicia Pueyo Fund, a family foundation dedicated to finding treatments for brainstem gliomas and encouraging collaboration among researchers worldwide. We are honored to participate alongside other international leaders who are striving to defeat DIPG, and grateful to the dedicated families who are at the forefront of the effort, always refusing to take no for an answer.

March meant Paris, for the first-ever International Gliomatosis Cerebri Conference, organized and driven by families affected by GC. That groundbreaking meeting created a framework for important collaborations as we go forward. (Read more about both conferences on page 2.)

This month, we look forward to the annual meeting of the DIPG Collaborative in Chicago. The 2015 Symposium promises to be filled with new research and development about this tumor, which is one of the prime targets of our research efforts here at the CBTP.


Jeffrey Greenfield Signature Mark Souweidane Signature

3 Years of Progress: What Your Donations Have Done

The end of the year is usually a time for reflection, for looking back over the accomplishments and challenges of the year behind us. We have a lot to be proud of this year, but we are even more excited about our plans for 2015, when all the projects we were able to initiate this year move to their next stage. Here is an overview of our many current initiatives, none of which would have been possible with the support of our generous donors.


Mediated Oncogenesis Dr. Greenfield is working in collaboration with Dr. David Allis at The Rockefeller University as well as with researchers at Memorial Sloan-Kettering on this project investigating how histone H3.3 mutations affect the development of pediatric gliomas. New insights into how pediatric gliomas form may help researchers identify the next generation of treatment options. \

Prolonged CED Under the direction of Dr. Zhiping Zhou, Dr. Souweidane’s lab is investigating the feasibility of delivering a weeklong infusion of DIPG-fighting drugs into a tumor instead of a single dose. The procedure has been tested in rats and may hold promise for a future clinical trial.

HDAC Inhibitors Dr. Souweidane’s lab is con-ducting toxicity testing on targeted compounds (HDAC inhibitors) in search of a new weapon against DIPG. The tests will provide safety data needed to progress to clinical trials, where the compounds may be used in children with DIPG.

Multi-drug Therapy for DIPG Before researchers can test a combination of drugs against a tumor, they must identify (or create) an animal model on which to try it. Our researchers are now working on establishing genetically engineered DIPG rodent models to be used for validating multi-drug therapy using CED of targeted compounds.

Sequencing and Bioinformatics Elizabeth Minter was the first patient whose GC tumor was sequenced in the search for genetic clues to how the tumor forms and spreads. Many more such tests are needed to establish patterns and commonalities that allow researchers to identify the best approaches to treatment, and we are now sequencing every tumor we treat. With the addition of the first Ty Louis Campbell Fellow, Sheng Li, in September 2014, we greatly expanded our ability to process all the data generated by each sequencing. By utilizing single-cell gene sequencing technology, we are studying genomic abnormalities and heterogeneity in various types of gliomas. We are exploring the crucial genes and signaling in transformed cells, which make gliomas resistant and cause them to recur after primary therapies. With advanced cell sorting and RNA sequencing technologies, our researchers are dissecting the glioma microenvironment, including macrophages and endothelial cells at the single-cell level, to reveal their diversified roles during glioma progression.


Under the supervision of Dr. Yujie Huang, we are bio-banking tumor tissue and blood samples from pediatric brain tumor patients for study by our precision medicine team. In collaboration with other institutions in the Northeast, we are creating a valuable way for researchers to share data and conclusions, hastening our ability to find answers.


Dr. Souweidane is in the third year of his Phase I trial testing the safety of convection-enhanced delivery (CED) and radioimmunotherapy, which delivers a therapeutic agent directly to a DIPG tumor. With 17 patients treated so far, the trial shows promising results, with no adverse effects reported in any patient. Participants in the trial are now getting the sixth dose level of the seven approved for this trial. As patients are treated, Dr. Souweidane’s team is using MRI-based research to monitor the distribution and clearance rates for drugs delivered to the brain stem; they are using that data to drive their prolonged CED laboratory investigations. Dr. Greenfield is now enrolling patients in a clinical trial testing the safety and effectiveness of intra-arterial (IA) chemotherapy in children with malignant brain tumors. This approach seeks to avoid the side effects of intravenous chemotherapy and improve the concentration of drugs reaching the tumor itself by delivering them directly into the tumor’s blood supply, bypassing the blood-brain barrier. This “super-selective IA cerebral infusion” has been shown to be safe in adults; we hope this trial opens up a new treatment option for children as well.

The year ended with a gratifying new beginning: Dr. Greenfield and his team moved out of their borrowed space into a dedicated laboratory on the sixth floor of the Whitney Pavilion. Thanks to our generous donors, the new lab has some important new equipment and a few new faces, as medical students and research assistants lend a hand.
The year ended with a gratifying new beginning: Dr. Greenfield and his team moved out of their borrowed space into a dedicated laboratory on the sixth floor of the Whitney Pavilion. Thanks to our generous donors, the new lab has some important new equipment and a few new faces, as medical students and research assistants lend a hand.

Lab Update: Winter 2014

November marked the third anniversary of Elizabeth’s Hope, the effort that quickly grew into the Children’s Brain Tumor Project. We had many supporters before then, of course, and we will be forever grateful to the all-important early believers who helped us get started, including the Cristian Rivera Foundation, The Cure Starts Now, the Lyla Nsouli Foundation, St. Baldrick’s, Ian’s Friends, the Dana Foundation, the Beez Foundation, and the Matthew Larson Foundation.

Dr. Mark Souweidane and Dr. Jeffrey GreenfieldThe launch of Elizabeth’s Hope created a new momentum, and 2011 became the tipping point for us. Since then the Children’s Brain Tumor Project has brought together more than 20 families in a drive to fund our research, raising a combined $2.5 million from friends and family members who know all too well just how important this work is.

Thanks to our donors, today we are not only conducting important clinical trials, but we are also able to do the lab work necessary to begin new trials in the near future. Keeping that pipeline open is key to maintaining momentum. (See next page for more on that.)

Our deepest thanks to those who believed in us a decade ago, and to those whose ongoing efforts allow us to continue our work. The next three to five years will almost surely produce significant results—and with your continued support, we will find the answers we need.


Mark Souweidane SignatureJeffrey Greenfield Signature