Diffuse intrinsic pontine glioma (DIPG) is a malignant pediatric brain tumor with a very poor outlook, with an expected survival of one year. Lack of access to biological tissue sample continues to be the largest barrier to advancing treatment. In 2016, the World Health Organization (WHO) reclassified DIPG tumors as diffuse midline gliomas with H3K27M mutation, which is a substitution mutation of histone 3 (H3) lysine at site 27 for methionine (K27M). This signaled the growing relevance of histone biology in DIPG, as well as its related epigenetic changes. Although not in DIPG yet, such changes have been detected in the blood from circulating tumor DNA (ctDNA) of patients with other cancers. We hypothesize that establishing a blood test of circulating nucleosomes in the plasma of DIPG patients for H3K27M mutation and associated epigenetic-alterations in histone biology will 1) describe a diagnostic, disease-specific molecular profile for DIPG, and 2) be able to monitor corresponding epigenetic changes with targeted H3K27M therapy.
This proposal is designed to answer the question if we can effectively establish detection methods of DIPG in pediatric patients based on the histone profile. This would be beneficial for many reasons. The first is that a blood test is non-invasive, and compared to surgical intervention to the pediatric brainstem, carries an almost negligible risk to the patient. The second is that a blood test can be repeated. This allows to look at the disease at multiple time points. Currently, surgery is not repeated, and as such, clinicians do not have an idea if the DIPG tumor is progressing or responding to treatment or not. Finally, a blood test will give us insights into how we can develop more effective therapies, because it will not only give us biologic measures about diagnosis, but it will give us a thorough, more rapid modality to assess for response. This means if a trial drug is not showing any evidence that it is affecting the ctDNA, then clinicians can try an alternative drug quicker.
We will aim to do this by testing the blood samples of DIPG patients and animal models for particular markers that reflect the histone biology of DIPG – this will include the characteristic H3K27M mutation and its associated epigenetic changes. There are multiple ways to do this, and we will focus primarily on using different molecular analytic techniques. These have been shown in the past to be sufficient in other solid tumors. Once we are able to show what markers are of the most importance, we will be able to inform clinicians about these findings in the hope that we can expand our concept of an epigenetic blood test for DIPG.
An epigenetic blood test for DIPG has yet to be proposed, and we are excited to propose this idea in the hope that what makes the epigenome stand out in DIPG histone biology will similarly make this proposal stand out as one with great merit and promise to transform DIPG practice for the better.
Introduction
Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain tumor for which curative treatment remains elusive, and optimal management strategies other than radiation therapy are ineffective. This brain tumor constitutes one of the leading causes of cancer-related morbidity and mortality in children, with average survival at 12-14 months despite best standard of care currently available. As such, there is a growing and desperate need to develop novel diagnostic and therapeutic platforms to treat this deadly disease.
Recent advances in our understanding of this disease have uncovered the majority of DIPG tumors have a histone H3 gene mutation that most commonly results in a H3K27M substitution (histone 3 lysine for methionine at site 27 (H3K27M)). In the latest World Health Organization (WHO) Classification of Central Nervous System (CNS) Tumors in 2016, DIPG was subsequently reclassified as a diffuse midline glioma with H3K27M mutation. This signaled the growing relevance of histone proteins in DIPG tumors, as well as their epigenetic changes, primarily the global loss of trimethylation at other wild type H3K27 sites.
Hypotheses
Our first specific aim is to uncover the molecular profile of H3K trimethylation and H3S phosphorylation in circulating nucleosomes isolated from patients plasma with H3K27M tumors and confirm detection of the H3K27M mutation in patient plasma. We hypothesize that the unique epigenetic landscape in H3K27M tumors can be detected in circulating nucleosomes from DIPG patient plasma allowing for the potential of a liquid biopsy.
Our second specific aim is to validate that the unique DIPG histone profile of circulating nucleosomes can be used for a liquid biopsy and monitoring of therapeutic responses in PDX and GEMM mouse models. We hypothesize circulating nucleosomes in plasma in H3K27M xenograft mouse models will not only detect the H3K27M mutation but will be altered by drug treatment and may guide therapeutic response to targeted treatment.
Goals
Liquid biopsy of biofluids has emerged as an exciting clinical prospect that has the potential to reshape our clinical practice and biological understanding of DIPG. This is because not only can it be more universal than surgical biopsy, but it also possesses the ability to stratify potential DIPG cases based on more objective, quantifiable molecular criteria. Furthermore, liquid biopsy presents a less invasive approach, and it has the potential to reveal serial epigenetic data about this disease that we have yet to obtain otherwise, similar to the genetic histone discoveries tissue biopsy afforded scientists years ago. Although the biomarkers of interest and the degree to which they are sensitive and reliable to a clinical diagnosis of DIPG remains under current investigation, all signs indicate that research is progressing in the appropriate direction. Given the significant progress made to date in its rationale and development, it is a realizable hope that liquid biopsy will be able to shed light on this disease very soon, which is our overarching goal.
Background
DIPG is a malignant pediatric brain tumor that constitutes one of the leading causes of cancer-related morbidity and mortality in children.14,15 Average survival is 14 months despite best standard of care currently available.1-3 In the latest World Health Organization (WHO) Classification of Central Nervous System (CNS) Tumors in 2016, DIPG was reclassified as a diffuse midline glioma with H3K27M mutation.15 Biologically, DIPG now is defined by a H3K27M mutation that can occur in several H3 genes, however, most commonly it occurs in the H3F3A and HIST1H3B/C genes, which encode for H3 variants H3.3 and H3.1 respectively, with subsequent H3 epigenetic reprogramming.16 The most common epigenetic changes in the presence of the H3K27M mutation are H3 lysine trimethylation and serine phosphorylation aberrations at numerous sites.17,18 In H3K27M tumors there is global loss of H3K27 trimethylation, which is likely the main driver for tumorigenesis.5,6,19-23 Other significant changes that are more variable include increased trimethylation at H3K4/9, and changes to phosphorylation at H3S10/28.5-9 Increased trimethylation at H3K4/9 activates aberrant transcription, decreased trimethylation at H3K27 alleviates repressed transcription, and alterations in H3S10/28 further modulate these important H3 lysine residues to promote tumorigenesis in H3K27M tumors.18,24-26 These important epigenetic changes create a unique biological signature for DIPG tumors that can be identified as its histone profile.
The propensity for H3K27M tumors to occur in functionally eloquent and anatomically intricate areas historically precluded surgical intervention, and currently, radiographic identification has remained the only feasible universal tool for diagnosis and monitoring.27,28 Recently, biopsy has been shown to be generally safe in a large study cohort,28 however, even in our hands at Mayo Clinic, significant complications can occur including hemorrhage within the brainstem leading to neurological deficit. This highlights the need for a tool or biomarker that can provide molecular data in a non-invasive fashion—one that could be used for potential diagnosis and follow treatment effects. One possible solution is molecular tumor biomarkers obtained from body biofluids.11,29-31 A liquid biopsy is an attractive goal in DIPG care, for it circumvents the risk of major surgery, and is still able to obtain biologic information about the tumor. To this end, cell free tumor DNA in cerebrospinal fluid (CSF) from H3K27M tumors has already been detected in a small cohort of DIPG patients.32,33 However, it is not known if the tumor needs to make contact with a CSF surface to shed this nuclear cargo, which is not global in DIPG patients and furthermore, still requires an invasive procedure.
Another potential biofluid that can be biopsied is the blood plasma and its circulating (plasma) nucleosomes: strands of DNA wrapped around a histone core.7,22,34,35 In solid cancers of the colon and pancreas, epigenetic alterations, such as trimethylation at H3K9 and H4K20, can be reliably detected from plasma of patients with a high degree of sensitivity to the primary tumor suggesting this nuclear data is tumor-derived and indeed circulating.11-13,36,37 Plasma histone profiling is both an exciting and novel avenue of research in DIPG given its unique and significant histone biology. If we are able to demonstrate that genetic information about DIPG can be obtained by means of blood plasma sampling, it will provide clinicians and researchers a more applicable, readily accessible modality to evaluate and monitor the tumor than all the options currently available.
DIPG is a malignant pediatric brain tumor that continues to have a dismal prognosis. Molecularly, we are limited by lack of access to tissue samples. Therapeutically, we are limited by the lack of tools and biomarkers to monitor disease progression and response. This proposal aims to expand upon our promising preliminary data to discover a non-invasive liquid biopsy for diagnostic purposes, and identify a unique histone profile that may validate response to novel therapies.
Clinical significance
The potential for a liquid biopsy in the field of DIPG is significant, especially if the emerging characteristic epigenetic histone profile of these tumors can be unraveled in the bloodstream. Not only will this be a less-invasive procedure which is crucial in the care of pediatric patients, it is also repeatable and potentially could detect epigenetic changes with therapy over time. It is currently unclear if epigenetic changes can predict superior or inferior outcomes, simply because to date there has been no modality to assess epigenetic changes in DIPG patients at more than one time point (tissue biopsy is performed only once) – it is hypothesized that restoration of trimethylation at H3K27 could be one such change that could indicate a favorable prognosis or response.
If novel therapies can be rapidly evaluated for sensitivity based on epigenetic response of these circulating nucleosomes, then clinicians can be better equipped to determine in shorter amounts of time if a certain therapy should be pursued, rather than rely solely on radiographic responses which can take months before any potential change is evident. Additionally, it is envisaged that if analysis of circulating nucleosomes can be optimized, it will facilitate the development of real-time diagnosis and monitoring, which could prove vital in this era of personalized medicine. From a surgical perspective, real-time diagnostics could revolutionize the way these tumors are approached, and open the door for more effective testing of novel therapies intraoperatively which currently cannot be justified at the such speed with the diagnostic tools currently available, i.e. histopathology.
Design and methods
To evaluate H3 lysine trimethylation at H3K4/9/27/36, phosphorylation at H3S10/28, and H3K27M status from blood plasma and surgical tissue of DIPG patients: Prospective DIPG patient samples will be collected during surgical biopsy done on-site at Mayo Clinic by neurosurgeon Dr. David Daniels (IRB #12-003458). Other tumor and plasma samples will be obtained from the tumor biobanks at Mayo Clinic, PCH, and the national DIPG Registry. Histone H3K4/9/27/36 trimethylation and H3S10/28 phosphorylation levels will then be assessed by ELISA using the Cell-Death Detection ELISA plus kit (Roche Diagnostics, US) to first standardize nucleosome content, and then with respective EpiQuik Histone Quantification Kits (Epigentek, US).11 In addition, H3K27M mutation status from the blood plasma sample will be evaluated using PCR and Sanger sequencing.32,33 All these analyses will be conducted in triplicate. Surgical biopsy specimens will be sent to our neuro-pathology core and tissue in excess of diagnosis will banked according to our IRB (#12-003458). The amount for tissue for testing will be largely determined by what is feasible and safe to the patient. Control tissue will be obtained from biobanks with autopsy-acquired normal brain tissue. Trimethylation and phosphorylation levels and H3K27M status of tissue samples will then be assessed using appropriate staining antibodies.
To evaluate the therapeutic response and changes to histone trimethylation and phosphorylation in PDX and GEMM mouse models during a therapeutic course of alisertib, WP1066 or GSKJ4: We will use two orthotopic H3K27M PDX mouse models (Peds8 and Peds17), and one wild-type (DJD14), all which were established in-house. We will use 20 mice total for each cell line, where 10 will receive one of the three drugs, commencing when tumor volume reaches 100mm3. The other n=10 will be used as therapy-free tumor-bearing controls. Tumor growth in the implanted mice will be monitored using bioluminescence imaging regularly (all cell lines express luciferase). Blood plasma will be sampled from whole blood samples collected by periorbital venipuncture with appropriate anesthesia every three days from therapy initiation.43 Once mice reach a moribund state, they will be euthanized, brains removed and tissue processed. Evaluation of H3 lysine trimethylation at H3K4/9/27/36, phosphorylation at H3S10/28, and H3K27M status will be performed in a similar fashion to that of 1A. These studies will be extended to an H3K27M GEMM model Dr. Oren Becher created, that uses the RCAS/Tv-a system we have at Mayo Clinic.
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Pellentesque nibh erat, egestas sit amet sagittis malesuada, rhoncus at neque. Mauris maximus commodo tortor, non egestas magna finibus vitae. Sed hendrerit nulla vel venenatis tempor. Suspendisse tristique tincidunt libero et placerat. Nam tincidunt condimentum lorem, vel pharetra est iaculis sed. Nullam ac tincidunt orci. Quisque pharetra ut sem sit amet aliquam. Phasellus risus libero, varius in condimentum vel, commodo id ipsum. Aliquam in metus cursus, mattis diam ut, aliquam magna. Suspendisse facilisis dui et orci varius, suscipit facilisis augue dapibus. In eget nibh ipsum. Suspendisse eget pharetra est, quis condimentum felis. Fusce scelerisque congue libero, sed aliquam mi elementum a. Etiam scelerisque ante non auctor porta. Nam eu nunc id ex finibus dictum. Praesent dui ex, dictum ac massa eget, rutrum gravida nisi.
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Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.
Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.
Morbi orci urna, ornare non pretium eget, pulvinar eget magna. Ut consectetur efficitur varius. Fusce ac aliquet mauris, at mattis ligula. Quisque est libero, interdum id orci et, ornare luctus diam. Proin commodo lectus id accumsan blandit. Nulla eu turpis interdum, luctus ante ac, imperdiet tellus. In semper enim eu tristique aliquam.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.
Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.