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Supplementary Figure 2 shows the exome coverage achieved with whole exome sequencing.

Legend for Supplementary Figures, Table and Files

Supplementary Figure 5:Germline TP53 variants in the validation cohort Sanger sequencing-derived electropherograms displaying the germline variant in Exon 4 are shown.

Purpose: Second malignant neoplasms (SMNs) are severe late complications that occur in pediatric cancer survivors exposed to radiotherapy and other genotoxic treatments. To characterize the mutational landscape of treatment-induced sarcomas and to identify candidate SMN-predisposing variants, we analyzed germline and SMN samples from pediatric cancer survivors.Experimental Design: We performed whole-exome sequencing (WES) and RNA sequencing on radiation-induced sarcomas arising from two pediatric cancer survivors. To assess the frequency of germline TP53 variants in SMNs, Sanger sequencing was performed to analyze germline TP53 in 37 pediatric cancer survivors from the Childhood Cancer Survivor Study (CCSS) without any history of a familial cancer predisposition syndrome but known to have developed SMNs.Results: WES revealed TP53 mutations involving p53′s DNA-binding domain in both index cases, one of which was also present in the germline. The germline and somatic TP53-mutant variants were enriched in the transcriptomes for both sarcomas. Analysis of TP53-coding exons in germline specimens from the CCSS survivor cohort identified a G215C variant encoding an R72P amino acid substitution in 6 patients and a synonymous SNP A639G in 4 others, resulting in 10 of 37 evaluable patients (27%) harboring a germline TP53 variant.Conclusions: Currently, germline TP53 is not routinely assessed in patients with pediatric cancer. These data support the concept that identifying germline TP53 variants at the time a primary cancer is diagnosed may identify patients at high risk for SMN development, who could benefit from modified therapeutic strategies and/or intensive posttreatment monitoring. Clin Cancer Res; 23(7); 1852–61. ©2016 AACR.

Supplementary Figure 1: Radiotherapy-induced sarcomas in pediatric cancer survivors Radiation dosimetry, imaging and histology are shown.

Supplementary File 1. SNVs for Patient 1's SMN;Supplementary File 2. SNVs for Patient 2's SMN;Supplementary File 3. List of dinucleotide substitutions;Supplementary File 4. TP53 germline sequencing primers

Supplementary Figure 4 displays Copy number alterations and allele frequencies across all chromosomes for SMNs.

Supplementary Table 1. Treatment exposures and TP53 Variant status in the Validation cohort Tumor, TP53 variant status (G215C, A639G), and treatment exposure details are shown for each validation sample (VS).

Supplementary Figure 3: Somatic variants in radiation-induced SMNs Frequencies of specific types of base substitutions in SNVs (synonymous and non-synonymous SNVs) pooled from all samples are shown.

Radiotherapy-induced second malignant neoplasms (SMNs) are a severe late complication in pediatric cancer survivors. Germline mutations in tumor suppressor genes contribute to SMNs; however, the most relevant germline variants mediating susceptibility are not fully defined. The authors performed matched whole-exome sequencing analyses of germline and tumor DNA from 4 pediatric solid tumor survivors who subsequently developed radiation-associated SMNs. Pathogenic and predicted deleterious germline variants were identified for each patient and validated with Sanger sequencing. These germline variants were compared with germline variants in a cohort of 59 pediatric patients diagnosed with primary sarcomas. Pathway analysis was performed to test for similarities in the germline variant profiles between individuals diagnosed with SMNs or primary sarcomas. One index patient was found to have a pathogenic germline monoallelic mutation in the MUTYH gene, which encodes the base excision repair enzyme adenine DNA glycosylase. This specific germline mutation is associated with a form of familial adenomatous polyposis, a new diagnosis in the patient. Germline-level genetic similarity exists between SMN-developing patients and patients developing primary sarcomas, with relevant genes involved in signal transduction and DNA repair mechanisms. The authors identify a germline MUTYH mutation in a pediatric cancer survivor developing an SMN. Germline mutations involving specific pathways such as base excision repair may identify individuals at risk for developing SMNs. The composition of germline variants in individual patients may enable estimates of patient-specific risk for developing SMNs. The authors anticipate that further analyses of germline genomes and epigenomes will reveal diverse genes and mechanisms influencing cancer risk.

Single nucleotide variants (SNVs) identified in cancer genomes can be de-convolved using non-negative matrix factorization (NMF) into discrete trinucleotide-based mutational signatures indicative of specific cancer-causing processes. The stability of NMF-generated mutational signatures depends upon the numbers of variants available for analysis. In this work, we sought to assess whether data from well-controlled mouse models can compensate for scarce human data for some cancer types. High quality sequencing data from radiotherapy-induced cancers is particularly scarce and the mutational processes defining ionizing radiation (IR)-induced mutagenesis in vivo are poorly defined. Here, we combine sequencing data from mouse models of IR-induced malignancies and human IR-induced malignancies. To determine whether the signatures identified from IR-exposed subjects can be differentiated from other mutagenic signatures, we included data from an ultraviolet radiation (UV)-induced human skin cancer and from a mouse model of urethane-induced cancers. NMF distinguished all three mutagens and in the pooled analysis IR was associated with mutational signatures common to both species. These findings illustrate the utility of pooled analysis of mouse and human sequencing data.

Purpose: Second malignant neoplasms (SMNs) are severe late complications that occur in pediatric cancer survivors exposed to radiotherapy and other genotoxic treatments. To characterize the mutational landscape of treatment-induced sarcomas and to identify candidate SMN-predisposing variants, we analyzed germline and SMN samples from pediatric cancer survivors. Experimental Design: We performed whole-exome sequencing (WES) and RNA sequencing on radiation-induced sarcomas arising from two pediatric cancer survivors. To assess the frequency of germline TP53 variants in SMNs, Sanger sequencing was performed to analyze germline TP53 in 37 pediatric cancer survivors from the Childhood Cancer Survivor Study (CCSS) without any history of a familial cancer predisposition syndrome but known to have developed SMNs. Results: WES revealed TP53 mutations involving p53′s DNA-binding domain in both index cases, one of which was also present in the germline. The germline and somatic TP53-mutant variants were enriched in the transcriptomes for both sarcomas. Analysis of TP53-coding exons in germline specimens from the CCSS survivor cohort identified a G215C variant encoding an R72P amino acid substitution in 6 patients and a synonymous SNP A639G in 4 others, resulting in 10 of 37 evaluable patients (27%) harboring a germline TP53 variant. Conclusions: Currently, germline TP53 is not routinely assessed in patients with pediatric cancer. These data support the concept that identifying germline TP53 variants at the time a primary cancer is diagnosed may identify patients at high risk for SMN development, who could benefit from modified therapeutic strategies and/or intensive posttreatment monitoring. Clin Cancer Res; 23(7); 1852–61. ©2016 AACR.

SummaryIonizing radiation (IR) is a mutagen that promotes tumorigenesis in multiple exposure contexts. One severe consequence of IR is the development of second malignant neoplasms (SMNs), a radiotherapy-associated complication in survivors of cancers, particularly pediatric cancers. SMN genomes are poorly characterized, and the influence of genetic background on genotoxin-induced mutations has not been examined. Using our mouse models of SMNs, we performed whole exome sequencing of neoplasms induced by fractionated IR in wild-type and Nf1 mutant mice. Using non-negative matrix factorization, we identified mutational signatures that did not segregate by genetic background or histology. Copy-number analysis revealed recurrent chromosomal alterations and differences in copy number that were background dependent. Pathway analysis identified enrichment of non-synonymous variants in genes responsible for cell assembly and organization, cell morphology, and cell function and maintenance. In this model system, ionizing radiation and Nf1 heterozygosity each exerted distinct influences on the mutational landscape.

// Bryan J. Thibodeau 1 , Vincent Lavergne 2 , Nayana Dekhne 3 , Pamela Benitez 3 , Mitual Amin 4 , Samreen Ahmed 1 , Jean L. Nakamura 2 , Philip R. Davidson 5 , Alice O. Nakamura 5 , Inga S. Grills 6 , Peter Y. Chen 6 , Jessica Wobb 6 and George D. Wilson 1, 6 1 Beaumont BioBank, Beaumont Health, Royal Oak, MI, USA 2 Department of Radiation Oncology, University of California, San Francisco, CA, USA 3 Department of Surgery, Beaumont Health, Royal Oak, MI, USA 4 Department of Pathology, Beaumont Health, Royal Oak, MI, USA 5 Department of Finance and Statistical Analysis, University of Alberta, Edmonton, Alberta, Canada 6 Department of Radiation Oncology, Beaumont Health, Royal Oak, MI, USA Correspondence to: George D. Wilson, email: george.wilson@beaumont.edu Keywords: angiosarcoma; radiation-associated; breast; mutational signature; next generation sequencing Received: September 15, 2017 Accepted: October 13, 2017 Published: January 19, 2018 ABSTRACT Purpose: Radiation-associated breast angiosarcomas are a rare complication of radiation therapy for breast carcinoma. With relatively little is known about the genetic abnormalities present in these secondary tumors, we examined genomic variation in biospecimens from radiation-associated breast angiosarcomas. Experimental Design: Patients were identified that had a previous breast cancer diagnosis, received radiation therapy, and developed angiosarcoma in the ipsilateral breast as the earlier cancer. Tumor regions were isolated from archival blocks using subsequent laser capture microdissection. Next generation sequencing was performed using a targeted panel of 160 cancer-related genes. Genomic variants were identified for mutation and trinucleotide-based mutational signature analysis. Results: 44 variants in 34 genes were found in more than two thirds of the cases; this included 12 variants identified as potentially deleterious. Of particular note, the BRCA1 DNA damage response pathway was highly enriched with genetic variation. In a comparison to local recurrences, 14 variants in 11 genes were present in both the primary and recurrent lesions including variants in genes associated with the DNA damage response machinery. Furthermore, the mutational signature analysis shows that a previously defined IR signature is present in almost all of the current samples characterized by predominantly C→T substitutions. Conclusions: While radiation-associated breast angiosarcomas are relatively uncommon, their prognosis is very poor. These data demonstrate a mutational pattern associated with genes involved in DNA repair. While important in revealing the biology behind these tumors, it may also suggest new treatment strategies that will prove successful.

Introduction: Ionizing radiation is a known mutagen and can cause cancers in different contexts, for example second malignant neoplasms (SMNs). SMNs are therapy-induced malignancies and severe late complications that develop in cancer survivors, particularly survivors of pediatric cancers who have received radiotherapy. The mutational landscape of ionizing radiation-induced tumorigenesis is not well-characterized on a genome level. Because ionizing radiation produces DNA damage that differs from damage produced by other genotoxins (UV for example), the mutational landscape of ionizing-radiation induced neoplasms may differ from the mutational landscape induced by other mutagens. Defining the mutational landscape of malignancies induced by ionizing radiation may reveal biological mechanisms that specifically contribute to this process. This insight has clinical implications, particularly with regard to understanding the pathogenesis of SMNs. We previously developed mouse models of SMNs by delivering focal, fractionated irradiation to wildtype and Nf1 mutant mice, and established that the Nf1+/- genetic background is sensitized to radiation-induced tumorigenesis. Diverse malignancies recapitulating clinical SMNs arose in irradiated wildtype and Nf1 mutant mice. The goal of this study is to characterize the mutational profile of tumors induced by ionizing radiation that models radiotherapy delivered to patients and is typically responsible for SMNs. Experimental Procedures: Whole exome sequencing was performed on 25 ionizing radiation-induced malignancies generated from our mouse models (sarcomas, carcinomas and hematopoietic malignancies) and a germline control. Malignancies from Nf1 mutant and wildtype mice were sequenced. Indexed paired-end libraries were prepared using the Agilent SureSelectXT Mouse All Exon kit covering all Ensembl genes and miRNAs (50Mb), Sequencing was performed using Illumina HiSeq2000 technology. Alignments were processed and variants were called according to standard practices. Exome sequencing of a 129/Sv-C57BL/6 heterozygote was used as a normal control and only somatic variants were considered for analysis. Each exome was sequenced to a minimum of 5 Gb. Results: In the entire cohort, 7566 somatic mutations were identified, of which 5187 were non-synonymous. Tumors had an average of 200 mutations (range, 31-594). Most nucleotide substitutions were C -> T transitions. In addition to examining somatic variants comprised of nucleotide substitutions, we analyzed immediately flanking sequence context for each somatic variant using an approach developed at the Wellcome Trust Sanger Institute. We applied non-negative matrix factorization methods to exome data and extracted 3 stable and distinctive mutational signatures. These signatures are characterized by unique, context-dependent, patterns of base substitutions, and were present in neoplasms arising from wildtype or Nf1+/- genetic backgrounds. Pathway analysis and functional validation of recurrently mutated genes are underway. Conclusions: Ionizing radiation-induced malignancies display a unique mutational landscape that is shared among different tumor histologies. Three distinct signatures were identified and these are not genotype-dependent (wildtype vs. Nf1 mutant). The enrichment of specific substitutions in each of the signatures implicates discrete mechanisms of DNA repair and operational enzymes. These mechanisms and recurrently mutated genes are being functionally tested in tumor cell lines we established from our mouse models. Ionizing radiation-induced malignancies possess a mutational landscape that is distinguishable from those associated with other common mutagens. Citation Format: Amy L. Sherborne, Philip R. Davidson, Katharine Yu, Alice O. Nakamura, Jean L. Nakamura. Mutational analysis of ionizing radiation-induced neoplasms. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-03.

Objective: Ionizing radiation (IR) is utilized in diagnostic and therapeutic medical applications, but is also a well-recognized mutagen. The in vivo genetic events promoting malignancy formation after IR exposure are not understood in detail. The mutational landscape of IR-induced tumorigenesis may yield insights into how IR-induced genomic injury leads to cancer and the important endogenous mechanisms that safeguard genomic integrity. We previously developed mouse models of IR-induced malignancies initiated by focal, fractionated irradiation similar to clinical radiotherapy. Whole exome sequencing (WES) of these mouse model-derived malignancies revealed stable trinucleotide-based (comprised of a somatic single nucleotide variant and the 5' and 3' flanking bases) mutational signatures that were independent of tumor type or genetic background, suggestive of a unique IR-specific imprint on tumor exomes. On the basis of these findings, we hypothesized that clinically derived IR-induced malignancies (secondary cancers, distinguishable from recurrent primary cancers) from patients also harbor distinct mutational signatures mirroring those identified in our mouse models. Our goal is to determine whether IR-induced malignancies from patients harbor mutational signatures similar to experimentally derived IR-induced malignancies arising in mice. Methods: WES was performed on genomic DNA isolated from two separate clinical IR-induced malignancies, both sarcomas arising in patients who were survivors of pediatric cancers. Radiotherapy exposure was verified by review of volumetric dosimetry. A UV-induced cutaneous squamous cell carcinoma (clinical patient sample) was included as a negative control and also analyzed by WES utilizing the same bioinformatics pipeline. Matched germline and tumor DNA were analyzed. Exome capture, high throughput sequencing (Illumina), read alignment and variant calling were performed using standard industry-accepted approaches. IR-induced tumors from mice and clinical samples were analyzed separately, then pooled into a single analysis to identify mutational signatures common to mice and humans. For all samples, single nucleotide variants (SNVs) were analyzed using non-negative matrix factorization (NMF) to estimate trinucleotide-based mutational signatures. Results: The two IR-induced clinical malignancies harbored 194 somatic SNVs and 361 somatic SNVs. The UV-induced cutaneous carcinoma harbored 2,113 somatic SNVs. NMF analysis of the pooled somatic SNVs from human tumor samples only support the extraction of two signatures, one of which clearly is exclusive to the UV-induced skin cancer, while the other is present almost exclusively in IR-induced malignancies (the number of mutations assigned to the signatures differs between the skin cancer and IR samples with p-value = 8.3x10-179 by a likelihood ratio test). Despite being extracted from a single tumor sample, the UV signature was virtually identical to previously published UV signature. To determine whether IR-associated mutational signatures were stable across species we pooled mouse and human IR-induced tumor SNVs for NMF analysis, which identified common signatures that are not highly correlated with each other, suggesting that they represent separate biological processes. Conclusions: IR-induced tumorigenesis is associated with stable trinucleotide-based mutational signatures that are conserved in human malignancies and mouse malignancies. These signatures are highly distinguishable from that associated with UV. These conserved signatures, developing in multiple irradiated tissue types, may reflect mutational processes associated with in vivo IR exposure and possibly demonstrate non-lethal but pathogenic somatic variants related to DNA repair. This abstract is also being presented as Poster B42. Citation Format: Philip Davidson, Amy Sherborne, Barry Taylor, Alice Nakamura, Jean Nakamura. Ionizing radiation-induced tumorigenesis is associated with exome-wide mutational signatures conserved in mice and humans [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR10.

Purpose: Second malignant neoplasms (SMNs) are therapy-induced malignancies and severe late complications that develop in pediatric cancer survivors. Ionizing radiation (IR) is a known mutagen and can cause SMNs. The mutational landscape of fractionated IR-induced tumorigenesis is not well-characterized on a genome level and but may reveal biological mechanisms that specifically contribute the development of SMNs. To study the influence of clinically relevant radiation delivery and germline mutations in a tumor suppressor gene, we previously developed mouse models of SMNs by delivering focal, fractionated irradiation to wildtype and Nf1 mutant mice. Irradiated mice developed diverse malignancies replicating the sarcomas and carcinomas observed as SMNs in pediatric cancer survivors. The goal of this study is to characterize the mutational profile of tumors induced by ionizing radiation that models clinical radiotherapy and to determine whether germline Nf1 mutations independently influence the mutational landscape. Materials/Methods: Whole exome sequencing was performed on 25 IR-induced malignancies arising in wildtype and Nf1 mutant mice. Indexed paired-end libraries were prepared using the Agilent SureSelectXT Mouse All Exon kit and sequencing was performed using Illumina HiSeq2000 technology (Illumina, San Diego, CA, USA). Alignments and somatic variants were identified using established procedures. Each exome was sequenced to a minimum of 5 Gb. Results: 6,623 somatic mutations were identified, of which 4,633 were non-synonymous. Tumors had an average mutation rate of 265 total SNVs/sample. Most nucleotide substitutions were C -> T or G-> A transitions. We analyzed the immediately flanking sequence context for each somatic variant using non-negative matrix factorization, and extracted 3 stable and distinctive mutational signatures. These signatures are present in multiple types of radiation-induced histologies, are uniquely distinguishable from mutational signatures of other well-recognized mutagens such as UV, and are similarly present in tumors arising in wildtype mice as well as Nf1 mutant mice, suggesting that the IR mutational signature persists in genetic backgrounds either resistant or susceptible to IR-induced tumorigenesis. We compared copy number alterations between tumors from wildtype and Nf1 mutant mice, and found significant differences between genetic backgrounds. Conclusions: IR-induced malignancies possess distinguishable and unique mutational signatures characterized by base substitutions occurring in very specific and unique sequence contexts. This analysis suggests that IR and genetic background influence the mutational landscape of tumors in very specific and discrete ways that are distinguishable from other cancer-promoting processes. SMNs from pediatric cancer survivors may harbor distinctive mutational motifs, and continued studies are needed to examine this. This data also suggest that the mutational landscape may differ between malignancies from different germline mutations or genetic backgrounds in pediatric cancer survivors. From a clinical standpoint, defining distinct mutational mechanisms in SMNs may improve the ability to predict which pediatric cancer survivors are at greatest risk for SMN formation as well as enable the development of strategies to mitigate and manage this risk. Citation Format: Amy Sherborne, Philip Davidson, Katharine Yu, Alice Nakamura, Mamunur Rashid, Jean Nakamura. Mutational analysis of a mouse model of second malignant neoplasms. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A50.

Data from 139 graduates, of the Bachelor of Science program in Occupational Therapy at the University, of Alberta were analyzed, to determine the relationship among pre-admission required courses, grads point average in each of four years, and clinical performance as measured by ratings, on the Report of Clinical Fieldwork from the Canadian, Association of Occupational, Therapy (CAOT Form) and by the Field, work Performance Report from, the American. Occupational Therapy Association (AOTA Form). Relationships between pre-admission interview ratings and both measures of clinical performance were lower, than relationships of the clinical measures with four of the eight academic measures in the case of the CAOT Form, and five of the eight academic measures in the case of the AOTA Form. Results are discussed in terms of admission criteria for occupational therapy programs in general and? the University of Alberta program in particular.