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Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

123 abc lane, Townsville, ZZ 00000, USA
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Intra-tumoral heterogeneity in diffuse intrinsic pontine and midline high-grade gliomas
Clinical
DIPG, Childhood (Brain Cancer)
Lay Summary

Background: Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor that affects approximately 150 children in North America each year.1 Though radiation therapy can prolong survival by 2-3 months, adjuvant chemotherapy has not improved outcome. The median survival is less than 12 months and has remained unchanged for the last 30 years.3 Knowledge of the pathogenesis of DIPG has been historically limited by lack of tissue availability, as diagnostic biopsy has not been routinely performed due to safety concerns.2 More recently, however, tissue procurement through autopsy programs3-5 and slowly growing acceptance of the safety of DIPG biopsy6-9 has provided the tissue necessary to gain critical knowledge of the genetic and epigenetic landscape of DIPG and has enabled development of in vitro and in vivo models through which novel therapies may be thoroughly tested.10,11 
 Since 2012, high-throughput sequencing studies have yielded unprecedented insight into the biologic basis of pediatric high-grade glioma (HGG) and DIPG.12-18 Midline, non-brainstem HGGs harbor a genomic profile akin to DIPG (e.g. frequent H3K27M mutation) and carry a similarly dismal prognosis.16,19 Mutations of chromatin remodeling genes H3F3A, HIST1H3B, and HIST1H3C are present in ~80% of thalamic HGGs19 and 70-96% of DIPGs.4-9 More recently discovered ACVR1 mutations are found in ~25% of DIPGs. In addition, mutations in other canonical cancer pathways, including (RTK)-RAS-PI3K, TP53, and RB1, are commonly found in HGG and DIPG.15,20 Unfortunately, our vastly improved understanding of tumor biology has not yet translated into a therapeutic breakthrough for children with HGG and DIPG. One critical factor that will significantly impact guiding appropriate therapy is histologic and molecular intra-tumoral heterogeneity. Although such heterogeneity has been well-documented in adult HGG21-23, there is little understanding of intra-tumoral variation in pediatric HGG and DIPG. Studies of DIPG at autopsy, including our own investigations with correlative post-mortem MRI, have revealed distant tumor metastases to the periventricular and frontal brain regions.5,24 Interestingly, we have observed some histologic variability between primary and distant tumor. Preliminary WES of primary, contiguous, and metastatic sites from 8 patients in our cohort also revealed relevant genomic heterogeneity (Figure 2). Although histone mutations, if present in the primary site, were detected at all disseminated sites, several interesting variations were seen among other clinically relevant mutations. Specifically, in one patient (patient 3) with six regions sequenced (all containing H3.3K27M), we detected regional variations in TP53 mutations. The primary tumour site (primary pons) contained a TP53 p.Arg248Gln alteration (allele frequency, AF = 0.46). Only 1 of 5 contiguous and metastatic sites (leptomeningeal spread) contained this alteration at an AF of 0.10. These 5 sites also harbored a TP53 p.Arg.275His alteration, which was not detected in the primary pons. In this same patient, a PDGFRA p.Glu229Lys alteration and ~8 copy amplification was detected in the right posterior pons, but not the primary pons or any other contiguous and metastatic sites. The leptomeningeal component did exhibit a PDGFRA copy number gain of ~4 copies. We hypothesize that genomic heterogeneity exists within the primary tumor and between primary and distant disease and that better understanding of clonal evolution of DIPG and midline HGG will guide the rational design of molecularly targeted therapies for these lethal brain tumors.  Specific Aim 1: To define spatial intra-tumoral heterogeneity using WES on contiguous, non-contiguous, and distant metastatic sites in DIPG and midline HGG obtained at autopsy  Specific Aim 2: To demonstrate patterns of branched clonal evolution of DIPG and midline HGG using high-depth WES on contiguous, non-contiguous, and distant metastatic sites  Specific Aim 3: To define temporal intra-tumoral heterogeneity using WES on matched biopsy and autopsy tumor specimens from patients with DIPG and midline HGG  
 Methods/Budget: We request $200,000 to perform WES on matched primary and metastatic (n=10) and matched biopsy and autopsy (n=3) specimens from patients with DIPG or midline HGG who underwent surgical biopsy and/or autopsy at Cincinnati Children’s Hospital Medical Center (CCHMC) or The Hospital for Sick Children. A portion of funding will go toward salary support for the study PIs and for bioinformatics analyses. Complete clinical annotation is available for all patients. Clinical Significance: Characterization of spatial and temporal heterogeneity and establishment of clonal evolutionary patterns in DIPG and midline HGG will have significant clinical impacts. First, this work will define the utility of primary tumor biopsy, which may misrepresent targetable genomic lesions across all disease locations, and a potential role for re-biopsy at progression. Additionally, exploration of early somatic events using high-depth sequencing and phylogenetic analyses has potential to define true disease “drivers” common across disease compartments that, if molecularly targeted, may hold more potent therapeutic potential.

Executive Summary

Background: Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor that affects approximately 150 children in North America each year.1 Though radiation therapy can prolong survival by 2-3 months, adjuvant chemotherapy has not improved outcome. The median survival is less than 12 months and has remained unchanged for the last 30 years.3 Knowledge of the pathogenesis of DIPG has been historically limited by lack of tissue availability, as diagnostic biopsy has not been routinely performed due to safety concerns.2 More recently, however, tissue procurement through autopsy programs3-5 and slowly growing acceptance of the safety of DIPG biopsy6-9 has provided the tissue necessary to gain critical knowledge of the genetic and epigenetic landscape of DIPG and has enabled development of in vitro and in vivo models through which novel therapies may be thoroughly tested.10,11 
 Since 2012, high-throughput sequencing studies have yielded unprecedented insight into the biologic basis of pediatric high-grade glioma (HGG) and DIPG.12-18 Midline, non-brainstem HGGs harbor a genomic profile akin to DIPG (e.g. frequent H3K27M mutation) and carry a similarly dismal prognosis.16,19 Mutations of chromatin remodeling genes H3F3A, HIST1H3B, and HIST1H3C are present in ~80% of thalamic HGGs19 and 70-96% of DIPGs.4-9 More recently discovered ACVR1 mutations are found in ~25% of DIPGs. In addition, mutations in other canonical cancer pathways, including (RTK)-RAS-PI3K, TP53, and RB1, are commonly found in HGG and DIPG.15,20 Unfortunately, our vastly improved understanding of tumor biology has not yet translated into a therapeutic breakthrough for children with HGG and DIPG. One critical factor that will significantly impact guiding appropriate therapy is histologic and molecular intra-tumoral heterogeneity. Although such heterogeneity has been well-documented in adult HGG21-23, there is little understanding of intra-tumoral variation in pediatric HGG and DIPG. Studies of DIPG at autopsy, including our own investigations with correlative post-mortem MRI, have revealed distant tumor metastases to the periventricular and frontal brain regions.5,24 Interestingly, we have observed some histologic variability between primary and distant tumor. Preliminary WES of primary, contiguous, and metastatic sites from 8 patients in our cohort also revealed relevant genomic heterogeneity (Figure 2). Although histone mutations, if present in the primary site, were detected at all disseminated sites, several interesting variations were seen among other clinically relevant mutations. Specifically, in one patient (patient 3) with six regions sequenced (all containing H3.3K27M), we detected regional variations in TP53 mutations. The primary tumour site (primary pons) contained a TP53 p.Arg248Gln alteration (allele frequency, AF = 0.46). Only 1 of 5 contiguous and metastatic sites (leptomeningeal spread) contained this alteration at an AF of 0.10. These 5 sites also harbored a TP53 p.Arg.275His alteration, which was not detected in the primary pons. In this same patient, a PDGFRA p.Glu229Lys alteration and ~8 copy amplification was detected in the right posterior pons, but not the primary pons or any other contiguous and metastatic sites. The leptomeningeal component did exhibit a PDGFRA copy number gain of ~4 copies. We hypothesize that genomic heterogeneity exists within the primary tumor and between primary and distant disease and that better understanding of clonal evolution of DIPG and midline HGG will guide the rational design of molecularly targeted therapies for these lethal brain tumors.  Specific Aim 1: To define spatial intra-tumoral heterogeneity using WES on contiguous, non-contiguous, and distant metastatic sites in DIPG and midline HGG obtained at autopsy  Specific Aim 2: To demonstrate patterns of branched clonal evolution of DIPG and midline HGG using high-depth WES on contiguous, non-contiguous, and distant metastatic sites  Specific Aim 3: To define temporal intra-tumoral heterogeneity using WES on matched biopsy and autopsy tumor specimens from patients with DIPG and midline HGG  
 Methods/Budget: We request $200,000 to perform WES on matched primary and metastatic (n=10) and matched biopsy and autopsy (n=3) specimens from patients with DIPG or midline HGG who underwent surgical biopsy and/or autopsy at Cincinnati Children’s Hospital Medical Center (CCHMC) or The Hospital for Sick Children. A portion of funding will go toward salary support for the study PIs and for bioinformatics analyses. Complete clinical annotation is available for all patients. Clinical Significance: Characterization of spatial and temporal heterogeneity and establishment of clonal evolutionary patterns in DIPG and midline HGG will have significant clinical impacts. First, this work will define the utility of primary tumor biopsy, which may misrepresent targetable genomic lesions across all disease locations, and a potential role for re-biopsy at progression. Additionally, exploration of early somatic events using high-depth sequencing and phylogenetic analyses has potential to define true disease “drivers” common across disease compartments that, if molecularly targeted, may hold more potent therapeutic potential.

Description of Research Proposal

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.

Integer fermentum vestibulum lacinia. Duis id aliquam nibh, ut mattis leo. Nulla ac dui at sapien posuere facilisis ut quis ex. Aliquam vestibulum blandit tristique. Integer pretium dui ac nulla accumsan, et finibus velit euismod. Proin placerat, nunc eu sodales facilisis, tellus justo efficitur risus, non blandit diam nulla ac ligula. Aliquam ullamcorper quam leo, porttitor dictum ex tempor ac. Ut efficitur, justo et auctor volutpat, ex ex pulvinar est, sed consequat turpis leo nec ipsum. Nunc tempor, turpis ut ullamcorper tempor, dolor dui varius dui, et congue quam nisi vel nunc.

Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Donec faucibus, turpis sit amet maximus dapibus, sapien nisl bibendum turpis, pharetra commodo tellus libero vel nulla. Sed nec velit viverra, congue sapien et, gravida libero. Proin eget ante eget turpis egestas accumsan. Aliquam arcu nibh, aliquam rhoncus vulputate in, pellentesque at sem. Maecenas cursus tempus nibh id tempus. Mauris dolor sapien, lacinia sit amet condimentum at, dapibus eget lectus. Phasellus vel pellentesque ex. Nunc aliquam in ligula at tristique. In mollis suscipit felis eu finibus. Nullam non dignissim nibh, nec suscipit ex. Suspendisse tincidunt et mauris id finibus. Aliquam vehicula a sem quis venenatis.

Suspendisse leo odio, rutrum et viverra ut, consequat finibus enim. Vivamus dolor nisl, viverra eu egestas vel, blandit in nulla. Curabitur auctor purus non est volutpat bibendum. Proin fringilla magna sed metus maximus, in dictum neque suscipit. Sed ornare ut mi ut sodales. Nulla efficitur urna nunc, non molestie nunc egestas ut. Nunc arcu lorem, semper ut tincidunt ac, eleifend quis elit.

Sed at tortor et tortor tincidunt feugiat id in dui. Vivamus eget justo nisl. Aenean congue laoreet nisl a elementum. Nunc consectetur velit non ligula sollicitudin, quis eleifend urna sollicitudin. Sed tincidunt, nisl quis varius venenatis, dui massa condimentum tellus, sed sollicitudin diam magna mollis augue. Sed venenatis commodo purus id malesuada. Aenean volutpat elit vel gravida consectetur. Vestibulum diam quam, lacinia ac tortor eget, tincidunt dapibus dui. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Proin nisl leo, pretium sed arcu imperdiet, hendrerit sollicitudin sem. Duis non magna at nunc sagittis ullamcorper a id est. Maecenas cursus nisl in faucibus hendrerit.

Sed hendrerit vitae purus et tempor. Aenean vitae varius velit. Nullam aliquet ipsum elit. Mauris vestibulum purus et metus imperdiet, quis gravida eros pretium. Curabitur rutrum nunc vitae tincidunt condimentum. Aenean sit amet augue velit. Nunc tristique quis lorem id pharetra. Pellentesque sollicitudin, eros sed egestas rutrum, nulla nisl sodales elit, ut imperdiet nunc lectus sit amet nulla. Ut malesuada finibus libero. Curabitur mi dolor, sollicitudin quis bibendum quis, dictum sed enim.

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.

Sed egestas arcu in dui euismod, eget faucibus massa iaculis. Etiam efficitur lectus et purus lobortis, ac blandit eros rutrum. Proin bibendum consectetur leo vel gravida. Etiam et ultricies sapien. Nam lacinia tellus erat, id facilisis est consequat et. Morbi quis risus in neque iaculis pharetra ut consectetur libero. Aenean feugiat tempor mi eu posuere.

Budget

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.

Collaborations and Conflicts of Interest

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.