Analysis of the 2017 American Society for Radiation Oncology (ASTRO) Research Portfolio.

PURPOSE: Research in radiation oncology (RO) is imperative to support the discovery of new uses of radiation and improvement of current approaches to radiation delivery and to foster the continued evolution of our field. Therefore, in 2016, the American Society of Radiation Oncology performed an evaluation of research grant funding for RO. METHODS AND MATERIALS: Members of the Society of Chairs of Academic Radiation Oncology Programs (SCAROP) were asked about funded and unfunded grants that were submitted by their departments between the fiscal years 2014 and 2016. Grants were grouped according to broad categories defined by the 2017 American Society of Radiation Oncology Research Agenda. Additionally, active grants in the National Institutes of Health (NIH) Research Portfolio Online Reporting Tools database were collated using RO faculty names. RESULTS: Overall, there were 816 funded (44%) and 1031 unfunded (56%) SCAROP-reported grants. Total grant funding was over $196 million. The US government funded the plurality (42.2%; 345 of 816) of grants compared with nonprofit and industry funders. Investigators from 10 institutions accounted for >75% of funded grants. Of the funded grants, 43.5% were categorized as "genomic influences and targeted therapies." The proportion of funded to unfunded grants was highest within the category of "tumor microenvironment, normal tissue effects, and reducing toxicity" (53.4% funded). "New clinical trial design and big data" had the smallest share of SCAROP grant applications and the lowest percent funded (38.3% of grants). NIH grants to RO researchers in 2014 to 2016 accounted for $85 million in funding. From the 31 responding SCAROP institutions, there was a 28% average success rate for RO proposals submitted to the NIH during this period. CONCLUSIONS: Though RO researchers from responding institutions were relatively successful in obtaining funding, the overall amount awarded remains small. Continued advocacy on behalf of RO is needed, as well as investment to make research careers more attractive areas for emerging faculty.

The role of FREM2 and FRAS1 in the development of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.

Assessing the Training and Research Environment for Genomics, Bioinformatics, and Immunology in Radiation Oncology.

PURPOSE: To assess radiation oncologists' perceptions of training and research opportunities in the fields of genomics, bioinformatics, and immunology. MATERIALS AND METHODS: A 13-item electronic survey was sent to 101 radiation oncology department chairs and administrators. A separate 30-item electronic survey was sent to 132 members of the American Society for Radiation Oncology Science Council as well as to 565 members of the Association of Residents in Radiation Oncology. Survey responses were collected, and results were analyzed using descriptive statistics. RESULTS: Twenty-six department chairs and 91 general respondents submitted responses. Among general respondents, 69% were current trainees and 31% had completed training. The majority of respondents (92%) were affiliated with an academic/university main campus. Approximately half of respondents (43% to 53%) reported no prior formal training in bioinformatics, genomics, or immunology. More than half of department chairs (54% to 58%) and general respondents (57% to 63%) thought that current training opportunities in these areas were absolutely or moderately insufficient. A majority of respondents (53% to 65%) thought that additional training in these areas would provide opportunity for career advancement, and 80% could identify a current or future research project that additional training in these fields would allow them to pursue. More than half of respondents expressed interest in attending a formal training course, and the majority of department chairs (22 of 26 [85%]) reported that they would probably or definitely send trainees or faculty members to a formal training course. CONCLUSION: Among radiation oncologists surveyed, there is a perceived lack of current training opportunities in bioinformatics, genomics, and immunology. A majority of respondents reported an interest in obtaining additional training in these areas and believed that training would provide opportunity for career advancement.

Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.

BACKGROUND: Next-generation sequencing (NGS) data are used for both clinical care and clinical research. DNA sequence variants identified using NGS are often returned to patients/participants as part of clinical or research protocols. The current standard of care is to validate NGS variants using Sanger sequencing, which is costly and time-consuming. METHODS: We performed a large-scale, systematic evaluation of Sanger-based validation of NGS variants using data from the ClinSeq® project. We first used NGS data from 19 genes in 5 participants, comparing them to high-throughput Sanger sequencing results on the same samples, and found no discrepancies among 234 NGS variants. We then compared NGS variants in 5 genes from 684 participants against data from Sanger sequencing. RESULTS: Of over 5800 NGS-derived variants, 19 were not validated by Sanger data. Using newly designed sequencing primers, Sanger sequencing confirmed 17 of the NGS variants, and the remaining 2 variants had low quality scores from exome sequencing. Overall, we measured a validation rate of 99.965% for NGS variants using Sanger sequencing, which was higher than many existing medical tests that do not necessitate orthogonal validation. CONCLUSIONS: A single round of Sanger sequencing is more likely to incorrectly refute a true-positive variant from NGS than to correctly identify a false-positive variant from NGS. Validation of NGS-derived variants using Sanger sequencing has limited utility, and best practice standards should not include routine orthogonal Sanger validation of NGS variants.

FBN1 contributing to familial congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common, life--threatening birth defect. We present a family with recurrent CDH--paraesophageal and central--for whom exome sequencing (ES) revealed a frameshift mutation (c.4969_4970insA, p.Ile1657Asnfs*30) in the fibrillin 1 gene (FBN1) that causes Marfan syndrome. A diagnosis of Marfan syndrome had not been considered previously in this family. However, a review of the literature demonstrated that FBN1 mutations have an unusual pattern of CDH in which paraesophageal hernias are particularly common. Subsequent clinical evaluations revealed evidence for ectopia lentis in affected family members supporting a clinical diagnosis of Marfan syndrome. Since only two other cases of familial CDH have been described in association with FBN1 mutations, we investigated an oligogenic hypothesis by examining ES data for deleterious sequence changes in other CDH-related genes. This search revealed putatively deleterious sequence changes in four other genes that have been shown to cause diaphragm defects in humans and/or mice--FREM1, DES, PAX3 and MET. It is unclear whether these changes, alone or in aggregate, are contributing to the development of CDH in this family. However, their individual contribution is likely to be small compared to that of the frameshift mutation in FBN1. We conclude that ES can be used to identify both major and minor genetic factors that may contribute to CDH. These results also suggest that ES should be considered in the diagnostic evaluation of individuals and families with CDH, particularly when other diagnostic modalities have failed to reveal a molecular etiology.

Identification of critical regions and candidate genes for cardiovascular malformations and cardiomyopathy associated with deletions of chromosome 1p36.

Cardiovascular malformations and cardiomyopathy are among the most common phenotypes caused by deletions of chromosome 1p36 which affect approximately 1 in 5000 newborns. Although these cardiac-related abnormalities are a significant source of morbidity and mortality associated with 1p36 deletions, most of the individual genes that contribute to these conditions have yet to be identified. In this paper, we use a combination of clinical and molecular cytogenetic data to define five critical regions for cardiovascular malformations and two critical regions for cardiomyopathy on chromosome 1p36. Positional candidate genes which may contribute to the development of cardiovascular malformations associated with 1p36 deletions include DVL1, SKI, RERE, PDPN, SPEN, CLCNKA, ECE1, HSPG2, LUZP1, and WASF2. Similarly, haploinsufficiency of PRDM16-a gene which was recently shown to be sufficient to cause the left ventricular noncompaction-SKI, PRKCZ, RERE, UBE4B and MASP2 may contribute to the development of cardiomyopathy. When treating individuals with 1p36 deletions, or providing prognostic information to their families, physicians should take into account that 1p36 deletions which overlie these cardiac critical regions may portend to cardiovascular complications. Since several of these cardiac critical regions contain more than one positional candidate gene-and large terminal and interstitial 1p36 deletions often overlap more than one cardiac critical region-it is likely that haploinsufficiency of two or more genes contributes to the cardiac phenotypes associated with many 1p36 deletions.

Novel frem1-related mouse phenotypes and evidence of genetic interactions with gata4 and slit3.

The FRAS1-related extracellular matrix 1 (FREM1) gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organ systems. In humans, recessive mutations in FREM1 cause eye defects, congenital diaphragmatic hernia, renal anomalies and anorectal malformations including anteriorly placed anus. A similar constellation of findings-microphthalmia, cryptophthalmos, congenital diaphragmatic hernia, renal agenesis and rectal prolapse-have been described in FREM1-deficient mice. In this paper, we identify a homozygous Frem1 missense mutation (c.1687A>T, p.Ile563Phe) in an N-ethyl-N-nitrosourea (ENU)-derived mouse strain, crf11, with microphthalmia, cryptophthalmos, renal agenesis and rectal prolapse. This mutation affects a highly conserved residue in FREM1's third CSPG domain. The p.Ile563Phe change is predicted to be deleterious and to cause decreased FREM1 protein stability. The crf11 allele also fails to complement the previously described eyes2 allele of Frem1 (p.Lys826*) providing further evidence that the crf11 phenotype is due to changes affecting Frem1 function. We then use mice bearing the crf11 and eyes2 alleles to identify lung lobulation defects and decreased anogenital distance in males as novel phenotypes associated with FREM1 deficiency in mice. Due to phenotypic overlaps between FREM1-deficient mice and mice that are deficient for the retinoic acid-responsive transcription factor GATA4 and the extracellular matrix protein SLIT3, we also perform experiments to look for in vivo genetic interactions between the genes that encode these proteins. These experiments reveal that Frem1 interacts genetically with Gata4 in the development of lung lobulation defects and with Slit3 in the development of renal agenesis. These results demonstrate that FREM1-deficient mice faithfully recapitulate many of the phenotypes seen in individuals with FREM1 deficiency and that variations in GATA4 and SLIT3 expression modulate some FREM1-related phenotypes in mice.

Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Congenital diaphragmatic hernia (CDH) is a common life-threatening birth defect. Recessive mutations in the FRAS1-related extracellular matrix 1 (FREM1) gene have been shown to cause bifid nose with or without anorectal and renal anomalies (BNAR) syndrome and Manitoba oculotrichoanal (MOTA) syndrome, but have not been previously implicated in the development of CDH. We have identified a female child with an isolated left-sided posterolateral CDH covered by a membranous sac who had no features suggestive of BNAR or MOTA syndromes. This child carries a maternally-inherited ~86 kb FREM1 deletion that affects the expression of FREM1's full-length transcripts and a paternally-inherited splice site mutation that causes activation of a cryptic splice site, leading to a shift in the reading frame and premature termination of all forms of the FREM1 protein. This suggests that recessive FREM1 mutations can cause isolated CDH in humans. Further evidence for the role of FREM1 in the development of CDH comes from an N-ethyl-N-nitrosourea -derived mouse strain, eyes2, which has a homozygous truncating mutation in Frem1. Frem1(eyes2) mice have eye defects, renal agenesis and develop retrosternal diaphragmatic hernias which are covered by a membranous sac. We confirmed that Frem1 is expressed in the anterior portion of the developing diaphragm and found that Frem1(eyes2) embryos had decreased levels of cell proliferation in their developing diaphragms when compared to wild-type embryos. We conclude that FREM1 plays a critical role in the development of the diaphragm and that FREM1 deficiency can cause CDH in both humans and mice.

Mouse model reveals the role of SOX7 in the development of congenital diaphragmatic hernia associated with recurrent deletions of 8p23.1.

Recurrent microdeletions of 8p23.1 that include GATA4 and SOX7 confer a high risk of both congenital diaphragmatic hernia (CDH) and cardiac defects. Although GATA4-deficient mice have both CDH and cardiac defects, no humans with cardiac defects attributed to GATA4 mutations have been reported to have CDH. We were also unable to identify deleterious GATA4 sequence changes in a CDH cohort. This suggested that haploinsufficiency of another 8p23.1 gene may contribute, along with GATA4, to the development of CDH. To determine if haploinsufficiency of SOX7-another transcription factor encoding gene-contributes to the development of CDH, we generated mice with a deletion of the second exon of Sox7. A portion of these Sox7(Δex2/+) mice developed retrosternal diaphragmatic hernias located in the anterior muscular portion of the diaphragm. Anterior CDH is also seen in Gata4(+/-) mice and has been described in association with 8p23.1 deletions in humans. Immunohistochemistry revealed that SOX7 is expressed in the vascular endothelial cells of the developing diaphragm and may be weakly expressed in some diaphragmatic muscle cells. Sox7(Δex2/Δex2) embryos die prior to diaphragm development with dilated pericardial sacs and failure of yolk sac remodeling suggestive of cardiovascular failure. Similar to our experience screening GATA4, no clearly deleterious SOX7 sequence changes were identified in our CDH cohort. We conclude that haploinsufficiency of Sox7 or Gata4 is sufficient to produce anterior CDH in mice and that haploinsufficiency of SOX7 and GATA4 may each contribute to the development of CDH in individuals with 8p23.1 deletions.

A 1q42 deletion involving DISC1, DISC2, and TSNAX in an autism spectrum disorder.

Individuals with autism spectrum disorders have impairments in social, communicative, and behavior development that are often accompanied by abnormalities in cognitive functioning, learning, attention, and sensory processing. In this report, we describe a 3-year-old male child with an autism spectrum disorder who carries a 2 Mb deletion of chromosome 1q42. Array comparative genome hybridization revealed that this deletion involves at least three genes-DISC1, DISC2, and TSNAX-which have been found to be associated with neuropsychiatric disorders and are likely to play key roles in normal CNS development. Further studies revealed that the deletion was inherited from his unaffected mother. This suggests that other genetic and/or environmental factors, some of which may be sex specific, may modify the phenotypic effects of this deletion. While this case provides evidence for the potential role of DISC1, DISC2, and TSNAX in the development of autism spectrum disorders, it is equally clear that caution must be taken when providing families with prognostic information and genetic counseling regarding such deletions.