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To evaluate the cost-effectiveness of obtaining a preoperative type and screen (T/S) for common urologic procedures.A decision tree model was constructed to track surgical patients undergoing two preoperative blood ordering strategies as follows: obtaining a preoperative T/S versus not doing so. The model was applied to the National (Nationwide) Inpatient Sample (NIS) data, from January 1, 2006 to September 30, 2015. Cost estimates for the model were created from combined patient-level data with published costs of a T/S, type and crossmatch (T/C), a unit of pRBC, and one unit of emergency-release transfusion (ERT). The primary outcome was the incremental cost per ERT prevented, expressed as an incremental cost-effectiveness ratio (ICER) between the two preoperative blood ordering strategies. A cost-effectiveness analysis determined the ICER of obtaining preoperative T/S to prevent an emergency-release transfusion (ERT), with a willingness-to-pay threshold of $1,500.00.A total of 4,113,144 surgical admissions from 2006 to 2015 were reviewed. The overall transfusion rate was 10.54% (95% CI, 10.17-10.91) for all procedures. The ICER of preoperative T/S was $1500.00 per ERT prevented. One-way sensitivity analysis demonstrated that the risk of transfusion should exceed 4.12% to justify preoperative T/S.Routine preoperative T/S for radical prostatectomy (rate = 3.88%) and penile implants (rate = .91%) does not represent a cost-effective practice for these surgeries. It is important for urologists to review their institution T/S policy to reduce inefficiencies within the preoperative setting.

Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology, especially for highly aggressive cancers with a propensity for metastatic spread. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, a large panel of models is needed to fully elucidate key aspects of disease biology and to recapitulate clinically-relevant phenotypes. We describe the development and characterization of osteosarcoma patient-derived xenografts (PDXs) and a panel of PDX-derived cell lines. Matched patient samples, PDXs, and PDX-derived cell lines were comprehensively evaluated using whole genome sequencing and RNA sequencing. PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication (WGD) in a subset of cell lines. These cell line models were heterogeneous in their metastatic capacity and their tissue tropism as observed in both intravenous and orthotopic models. As proof-of-concept study, we used one of these models to test the preclinical effectiveness of a CDK inhibitor on the growth of metastatic tumors in an orthotopic amputation model. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden in this model.Graphical Abstract

For many pediatric cancer patients, commonly used gene-panel sequencing tests yield few actionable results, partly due to the complex genomic alterations present. We hypothesized that an unbiased approach, combining whole-genome (WGS) and RNA sequencing (RNAseq), could overcome this and lead to a more comprehensive understanding of these diseases. While prior studies have evaluated WGS and RNAseq in pediatric cancers, few focused primarily on metastatic or relapsed disease. We also placed special focus on longitudinal profiling of patients, including with additional deep sequencing, to capture tumor evolution at the primary and metastatic sites, and to quantify the utility of resampling. We assembled a cohort of 191 high-risk pediatric oncology patients, including solid tumors, CNS tumors, and leukemias/lymphomas. We have representation of patients with relapsed/refractory disease (68), metastatic disease at diagnosis (10), rare diagnoses (19), prior cancer history, and estimated overall survival Multiple sampling from the same patient identified drastic spatial and temporal differences in the genomes and transcriptomes of these tumors. Using the Jaccard index as a measure of concordance between samples shows dynamic changes between samples collected at different time points across multiple modalities (range 0-1, 1 is identical); SNVs ranged from 0.01-0.79, SVs 0.01-0.73, major CNAs 0.07-0.99, minor CNAs 0.38-0.99, up expression outliers 0.12-0.56, down expression outliers 0.04-0.54, and fusions 0-1. Potentially biologically significant differences in therapy-induced mutations by platinum agents were also observed, highlighting the impact of therapy on tumor evolution. Clonal architectures were extracted from deep resequencing and show extensive spatial, temporal, and metastatic heterogeneity in these rare and highly aggressive malignancies that is not captured by WGS alone. Identifying clinically relevant evolution remains a challenge in most patients, but our results suggest that resampling of pediatric tumors at relapse or metastasis will be important for the effectiveness of targeted therapies in the future. Citation Format: Henry J. Martell, Avanthi T. Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, María V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, Alejandro Sweet-Cordero. Longitudinal profiling of high-risk pediatric malignancies using a multiomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1509.

Odontogenic sinusitis (ODS) is underrepresented in the literature compared to other forms of rhinosinusitis, specifically in sinusitis guidelines and position statements. ODS publication characteristics could help explain why ODS has received less attention in sinusitis guidelines and position statements. The purpose of this study was to explore trends in the quantity and quality of ODS studies over 3 decades from 1990 to 2019. A systematic review was performed to identify all ODS studies from 1990 to 2019. The following variables from all ODS studies were compared between and across the 3 decades: authors’ specialties, journal specialties, authors’ geographic origins (continents), study topics, study designs, and evidence levels. From 1990 to 2019, there were 254 ODS studies that met inclusion criteria. Numbers of publications increased each decade, with 161 being published from 2010 to 2019. Otolaryngologists and dental authors published over 75% of ODS studies each decade, with 60–75% of ODS articles being published in otolaryngology or dental journals. European and Asian authors published the most ODS studies each decade. Overall, 92–100% of ODS publications per decade were level 4 and 5 evidence, with no significant changes between or across decades. While numbers of ODS publications increased each decade from 1990 to 2019, evidence levels remained low without significant changes over time. Otolaryngologists and dental authors published the majority of ODS studies each decade, with a minority of these studies being multidisciplinary. More ODS studies are needed across all aspects of the condition, and future projects would benefit from improved study designs and multidisciplinary collaboration.

OBJECTIVE Determine the effect of patient demographics and surgical approach on patient outcomes after tracheal resection in the management of thyroid cancer. STUDY DESIGN Systematic review and meta-analysis. METHODS Systematic review of literature was performed using PubMed, Embase, and Cochrane Library to identify patients with thyroid carcinoma who underwent tracheal resection. Pooled estimates for patient demographics, presenting findings, complications, and outcomes are determined using random-effects meta-analyses. RESULTS Ninety-six relevant studies encompassing 1,179 patients met inclusion criteria. Meta-analysis pooled rates of complications: 1.7% (confidence interval [CI] 0.8-2.5; P

Acute invasive fungal sinusitis (AIFS) is a potentially life-threatening diagnosis in immunocompromised patients. Identifying patients who could benefit from evaluation and intervention can be challenging for referring providers and otolaryngologists alike. We aimed to develop and validate an accessible diagnostic tool to estimate the probability of AIFS.Retrospective chart review from 1999 to 2017 identified all patients evaluated for possible AIFS at a tertiary care center. AIFS was diagnosed by pathologic confirmation of fungal tissue angioinvasion. Stepwise selection and univariate logistic regression were used to screen risk factors for a multivariable predictive model. Model performance was assessed using Tukey's goodness-of-fit test and the area under the receiver operator characteristic curve (AUC). Model coefficients were internally validated using bootstrapping with 1000 iterations.A total of 283 patients (244 negative controls, 39 with AIFS) were included. Risk factors in our final diagnostic model included: fever ≥38°C (log-odds ratio [LOR] 1.72; 95% CI, 0.53 to 2.90), unilateral facial swelling, pain, or erythema (LOR 2.84; 95% CI, 1.46 to 4.23), involvement of the orbit or pterygopalatine fossa on imaging (LOR 3.02; 95% CI, 1.78 to 4.26), and mucosal necrosis seen on endoscopy (LOR 5.52; 95% CI, 3.81 to 7.24), with p0.01 for all factors. The model had adequate goodness of fit (p0.05) and discrimination (AUC = 0.96).We present an internally validated diagnostic tool to stratify the risk for AIFS. The estimated risk may help determine which patients can be observed with serial nasal endoscopy, which ones could be biopsied, and which ones would benefit from immediate surgical intervention.

Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.

Objective: To evaluate the cost-effectiveness of open versus endoscopic surgical repair of Zenker’s diverticulum. Methods: In this study, an economic decision tree was utilized to compare the cost-effectiveness of open surgery compared to endoscopic surgery. The primary outcome in this analysis was the incremental cost-effectiveness ratio (ICER) that was calculated based on the economic decision tree. The probability of post-operative esophageal perforation complications, revision rates, and effectiveness of each procedure along with associated costs were extracted to construct the decision tree. Univariate sensitivity analysis was then utilized to determine how changes in esophageal perforation rate affect the cost-effectiveness of each surgical approach. Results: The ICER of open surgery for Zenker’s diverticulum was $67 877, above most acceptable willingness to pay (WTP) thresholds. Additionally, if the probability of esophageal perforation with endoscopic surgery is above 5%, then open surgery becomes a more cost-effective option. Probabilistic sensitivity analysis using Monte Carlo simulations also showed that at the WTP thresholds of $30 000 and $50 000, endoscopic surgery is the most cost-effective method with 83.9% and 67.6% certainty, respectively. Conclusion: Open surgery and endoscopic surgery are 2 treatment strategies for Zenker’s diverticulum that each have their own advantages and disadvantages that can complicate the decision-making process. With no previous cost-effectiveness analysis of open versus endoscopic surgery for Zenker’s diverticulum, our results support the endoscopic approach at most common WTP thresholds. Particularly with the current focus on rising healthcare costs, our results can serve as an important adjunct to medical decision-making for patients undergoing treatment for Zenker’s diverticulum.

Objective This study aims to describe presenting characteristics of patients diagnosed with non-invasive chronic rhinosinusitis (CRS) following liver or kidney transplant and determine factors associated with disease-related complications, selection of endoscopic sinus surgery (ESS), and disease resolution in this population. Study design Retrospective chart review. Setting An academic tertiary care center (Mayo Clinic, Rochester, Minnesota). Subjects and methods Liver and kidney transplant recipients evaluated by Mayo Clinic otolaryngologists for CRS between 1998 and 2018 were identified. Univariate and multivariate logistic regression analyses were used to determine patient factors and treatment modalities associated with developing complications, selection of ESS, and disease resolution. Results Fifty-seven patients met inclusion criteria. No patients developed intraorbital or intracranial complications of their CRS. Multivariate modeling demonstrated that the presence of polyps (P = 0.036) was associated with undergoing ESS within one year of presentation. A higher Lund–Mackay (LM) computed tomography score (P = 0.023) and older age (P = 0.018) were significantly associated with decreased disease resolution. No other factors were significantly associated with the use of endoscopic sinus surgery within one year of otolaryngology presentation or resolution of CRS in this cohort. Conclusion The risk of developing CRS-related intraorbital or intracranial complications in this immunecompromised patient cohort may be lower than originally thought. For liver- and kidney-recipients stable on immunosuppressive medication for many years, prognostic factors for CRS may mirror those for immunocompetent patients.

Odontogenic sinusitis (ODS) is underrepresented in the literature compared to other forms of rhinosinusitis, specifically in sinusitis guidelines and position statements. ODS publication characteristics could help explain why ODS has received less attention in sinusitis guidelines and position statements. The purpose of this study was to explore trends in the quantity and quality of ODS studies over 3 decades from 1990 to 2019.A systematic review was performed to identify all ODS studies from 1990 to 2019. The following variables from all ODS studies were compared between and across the 3 decades: authors' specialties, journal specialties, authors' geographic origins (continents), study topics, study designs, and evidence levels.From 1990 to 2019, there were 254 ODS studies that met inclusion criteria. Numbers of publications increased each decade, with 161 being published from 2010 to 2019. Otolaryngologists and dental authors published over 75% of ODS studies each decade, with 60-75% of ODS articles being published in otolaryngology or dental journals. European and Asian authors published the most ODS studies each decade. Overall, 92-100% of ODS publications per decade were level 4 and 5 evidence, with no significant changes between or across decades.While numbers of ODS publications increased each decade from 1990 to 2019, evidence levels remained low without significant changes over time. Otolaryngologists and dental authors published the majority of ODS studies each decade, with a minority of these studies being multidisciplinary. More ODS studies are needed across all aspects of the condition, and future projects would benefit from improved study designs and multidisciplinary collaboration.

The use of sequencing-based assays for clinical management of pediatric cancer patients has become increasingly common. However, for many pediatric patients, gene panel based sequencing tests yield few actionable results. Given the complex genomic alterations present in many pediatric cancers, especially high-risk solid tumors, we hypothesized that an unbiased approach might reveal more actionable findings and lead to a more comprehensive understanding of these diseases. To accomplish this, we integrated whole-genome sequencing (WGS) with RNAseq in the analysis of a pediatric oncology cohort, with a focus on longitudinal cases to capture potential tumor evolution in metastatic or treated cases. Our cohort consists of 269 high-risk pediatric oncology patients, including patients with relapsed/refractory disease, metastatic disease at diagnosis, prior cancer history, a rare diagnosis, or an estimated overall survival Our results show that the integration of WGS and RNAseq can provide more and higher-quality actionable information than either modality alone, whilst also capturing the majority of actionable variants detected by panel sequencing. RNAseq identified not only druggable fusions and expression outliers, but also many rare and novel fusions. WGS provided fusion validation but highlighted the limitations of WGS alone in identifying fusions resulting from complex SVs. Conversely, WGS was adept at capturing genome-wide patterns of CNAs and loss of heterozygosity that are missed by gene-centric panels. Further RNAseq integration enabled prioritization of expressed SNVs as well as CNAs and SVs that significantly alter gene expression. We also used WGS to extract mutational signatures and tracked their evolution across longitudinal samples. We found potentially biologically significant differences in therapy-induced mutations caused by platinum and alkylating agents. Our unbiased approach has enabled further discovery that advances our understanding of these rare and highly aggressive malignancies. Citation Format: Henry J. Martell, Avanthi Tayi Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Maria V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, E. Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 54.

Objective The study aimed to evaluate the cost-effectiveness of obtaining preoperative type and screens (T/S) for common endonasal skull base procedures, and determine patient and hospital factors associated with receiving blood transfusions. Study Design Retrospective database analysis of the 2006 to 2015 National (nationwide) Inpatient Sample and cost-effectiveness analysis. Main Outcome Measures Multivariate regression analysis was used to identify factors associated with transfusions. A cost-effectiveness analysis was then performed to determine the incremental cost-effectiveness ratio (ICER) of obtaining preoperative T/S to prevent an emergency-release transfusion (ERT), with a willingness-to-pay threshold of $1,500. Results A total of 93,105 cases were identified with an overall transfusion rate of 1.89%. On multivariate modeling, statistically significant factors associated with transfusion included nonelective admission (odds ratio [OR]: 2.32; 95% confidence interval [CI]: 1.78–3.02), anemia (OR: 4.42; 95% CI: 3.35–5.83), coagulopathy (OR: 4.72; 95% CI: 2.94–7.57), diabetes (OR: 1.45; 95% CI: 1.14–1.84), liver disease (OR: 2.37; 95% CI: 1.27–4.43), pulmonary circulation disorders (OR: 3.28; 95% CI: 1.71–6.29), and metastatic cancer (OR: 5.85; 95% CI: 2.63–13.0; p Conclusion Routine preoperative T/S does not represent a cost-effective practice for these surgeries using nationally representative data. A selective T/S policy for high-risk patients may reduce costs.

This cohort study evaluates the feasibility and utility of incorporating comparative gene expression information into the precision medicine framework for difficult-to-treat pediatric and young adult patients with cancer., Key Points Question Is it feasible and useful to compare the tumor RNA sequencing data of a child or young adult with the tumor RNA sequencing data of thousands of other patients (of all ages) in a research setting? Findings Among 144 tumor samples from children and young adults, comparative RNA sequencing analysis, conducted across 4 precision medicine studies in the United States and Canada, was feasible and potentially useful for 99 of 144 pediatric and young adult cancer samples. In contrast, DNA mutation information was potentially useful for only 34 of 74 samples. Meaning This study’s findings suggest that open sharing and combined analysis of tumor RNA sequencing data from pediatric and young adult patients treated on different clinical trials may represent a feasible approach and may produce useful clinical and biological information for individual patients., Importance Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expression information is necessary for a complete understanding of the tumorigenic processes. Objective To evaluate the feasibility and utility of incorporating comparative gene expression information into the precision medicine framework for difficult-to-treat pediatric and young adult patients with cancer. Design, Setting, and Participants This cohort study was conducted as a consortium between the University of California, Santa Cruz (UCSC) Treehouse Childhood Cancer Initiative and clinical genomic trials. RNA sequencing (RNA-Seq) data were obtained from the following 4 clinical sites and analyzed at UCSC: British Columbia Children’s Hospital (n = 31), Lucile Packard Children’s Hospital at Stanford University (n = 80), CHOC Children’s Hospital and Hyundai Cancer Institute (n = 46), and the Pacific Pediatric Neuro-Oncology Consortium (n = 24). The study dates were January 1, 2016, to March 22, 2017. Exposures Participants underwent tumor RNA-Seq profiling as part of 4 separate clinical trials at partner hospitals. The UCSC either downloaded RNA-Seq data from a partner institution for analysis in the cloud or provided a Docker pipeline that performed the same analysis at a partner institution. The UCSC then compared each participant’s tumor RNA-Seq profile with more than 11 000 uniformly analyzed tumor profiles from pediatric and young adult patients with cancer, downloaded from public data repositories. These comparisons were used to identify genes and pathways that are significantly overexpressed in each patient’s tumor. Results of the UCSC analysis were presented to clinical partners. Main Outcomes and Measures Feasibility of a third-party institution (UCSC Treehouse Childhood Cancer Initiative) to obtain tumor RNA-Seq data from patients, conduct comparative analysis, and present analysis results to clinicians; and proportion of patients for whom comparative tumor gene expression analysis provided useful clinical and biological information. Results Among 144 samples from children and young adults (median age at diagnosis, 9 years; range, 0-26 years; 72 of 118 [61.0%] male [26 patients sex unknown]) with a relapsed, refractory, or rare cancer treated on precision medicine protocols, RNA-Seq–derived gene expression was potentially useful for 99 of 144 samples (68.8%) compared with DNA mutation information that was potentially useful for only 34 of 74 samples (45.9%). Conclusions and Relevance This study’s findings suggest that tumor RNA-Seq comparisons may be feasible and highlight the potential clinical utility of incorporating such comparisons into the clinical genomic interpretation framework for difficult-to-treat pediatric and young adult patients with cancer. The study also highlights for the first time to date the potential clinical utility of harmonized publicly available genomic data sets.

Purpose Evaluate trends in mortality due to acute epiglottitis before and after adoption of Haemophilus influenza Type b vaccination (Hib) in pediatric and adult populations. Materials and methods Patients who died from acute epiglottis from 1979 to 2017 identified using National Vital Statistics System. Mortality rates calculated using age-adjusted US census data expressed in rate per 100,000 individuals. Trends analyzed using the National Cancer Institute Joinpoint Regression Program (version 4.7.0; Bethesda, Maryland). Results 1187 epiglottitis-related deaths were identified over thirty-nine years. Total deaths decreased from 65 in 1979 to 15 in 2017. Adult deaths accounted for 63.5% and decreased from 0.015 per 100,000 individuals (24 deaths) in 1979 to 0.006 per 100,000 individuals (14 deaths) in 2017. Best fitting log-liner regression model showed APC of −3.5% (95% CI, −4.2 to −2.7%) from 1979 to 2017. Pediatric and adolescent deaths accounted for 443 (37.3%) deaths, decreasing from 0.064 per 100,000 individuals (41 deaths) in 1979 to 0.001 per 100,000 individuals (1 death) in 2017. APC was −11.1% (95% CI, −13.8% to −8.3%) in 1979 to 1990; 46.5% (95% CI, −16.6% to 157.3%) in 1990 to 1993; −61.6% (95% CI, −88% to 23%) in 1993 to 1996; and 1.1% (95% CI, −2.4% to 4.7%) in 1996 to 2017. Conclusions Mortality from acute epiglottitis decreased after widespread adoption of Hib vaccination in the US. Adults are now more likely than children to die of acute epiglottitis. Further research including multi-institutional cohort studies must be done to elucidate causative factors contributing to remaining cases of mortality.

Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for more than 30 years. Osteosarcoma is characterized by widespread and recurrent somatic copy-number alterations (SCNA) and structural rearrangements. In contrast, few recurrent point mutations in protein-coding genes have been identified, suggesting that genes within SCNAs are key oncogenic drivers in this disease. SCNAs and structural rearrangements are highly heterogeneous across osteosarcoma cases, suggesting the need for a genome-informed approach to targeted therapy. To identify patient-specific candidate drivers, we used a simple heuristic based on degree and rank order of copy-number amplification (identified by whole-genome sequencing) and changes in gene expression as identified by RNA sequencing. Using patient-derived tumor xenografts, we demonstrate that targeting of patient-specific SCNAs leads to significant decrease in tumor burden, providing a road map for genome-informed treatment of osteosarcoma. Significance: Osteosarcoma is treated with a chemotherapy regimen established 30 years ago. Although osteosarcoma is genomically complex, we hypothesized that tumor-specific dependencies could be identified within SCNAs. Using patient-derived tumor xenografts, we found a high degree of response for “genome-matched” therapies, demonstrating the utility of a targeted genome-informed approach. This article is highlighted in the In This Issue feature, p. 1

Clinical use of gene-panel based sequencing has become increasingly common in the management of pediatric cancer patients. For many patients, gene-panel tests have identified clinically actionable findings. However, highly targeted approaches will miss unanticipated (but potentially clinically meaningful) or novel alterations. In diseases with unknown or complex etiologies, including many pediatric high-risk and solid tumors, an unbiased approach may yield more actionable findings. To accomplish this, we examined the feasibility and utility of whole genome sequencing (WGS) and RNA sequencing (RNAseq) in the management of high-risk pediatric oncology patients. Here we describe our experience with an expanded cohort of over 100 high-risk pediatric oncology patients, with a combination of solid tumors, brain tumors, and leukemia/lymphomas represented. The majority of patients were deemed high-risk due to relapsed/refractory disease. An additional group of patients were defined as high-risk at time of initial diagnosis due to metastatic disease, a rare tumor, prior history of another cancer type, an undifferentiated tumor, or less than 50% estimated overall survival. WGS (tumor/germline) and RNAseq were used to characterize available samples and compared to results from panel testing for each patient (performed as part of their clinical evaluation). When possible, multiple samples from an individual patient were collected (i.e. specimens obtained at biopsy, resection, relapse, and/or from metastatic sites). Somatic DNA samples were sequenced to an average depth of at least 60X and germline samples to at least 30X. RNAseq was performed to a depth of at least 20 million paired-end reads for each sample. WGS samples were analyzed for single nucleotide variants (SNVs), structural rearrangements (SV), and copy-number alterations (CNA). RNAseq samples were analyzed to identify known and novel gene-fusions, to measure allele specific expression of SNVs, and to perform gene-expression outlier analysis. Expression of variants (SNV/SV) identified using WGS were confirmed using RNAseq. For gene expression outliers detected using RNAseq, the WGS data was used to predict possible mechanisms for the aberrant expression (such as CNA, gene fusions, or promoter hijacking). This analysis suggests that WGS and RNAseq analysis is feasible in a clinical setting and can reliably identify variants reported on gene panel tests. However, the use of WGS/RNAseq resulted in additional clinically informative findings while also enabling novel research to further advance our understanding of these rare and highly aggressive pediatric malignancies. Citation Format: Marcus R. Breese, Avanthi T. Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, E. Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3665.

Clinical use of gene-panel based tumor sequencing has expanded exponentially over the past few years. While in some cases this molecular testing identifies clinically actionable findings, these highly targeted approaches may miss unanticipated, clinically meaningful or novel alterations. In cancers with poorly understood etiologies, including many pediatric solid or high-risk tumors, an unbiased approach may prove more useful. We sought to explore the feasibility and utility of whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) in comparison to commercially available targeted gene-panel testing in pediatric oncology. Herein we describe our experience with an initial cohort of 58 high-risk pediatric oncology patients (37 solid tumors, 11 brain tumors, and 10 leukemia/lymphomas). The majority of patients (n=40) had relapsed/refractory disease. An additional eighteen patients were defined as high-risk at time of initial diagnosis due to metastatic disease, a rare tumor, prior history of another cancer type, an undifferentiated tumor, or less than 50% survival. A total of 102 samples were obtained from these 58 patients, with 70 samples originating at the primary sites of disease and 32 samples from metastatic sites. Thirty-one samples were chemotherapy/radiation therapy naïve. A combination of WGS and RNA-seq were used to characterize available samples and compared to results from panel testing for that patient (performed as part of their clinical evaluation). Where possible, fresh frozen tissue (FFT) samples were obtained during clinically indicated surgical procedures. When FFT was unavailable, formalin-fixed, paraffin-embedded (FFPE) samples were used. When possible, multiple samples from an individual patient were collected (i.e., specimens obtained at biopsy, resection, relapse, and/or from metastatic sites). Germline DNA was isolated from peripheral blood, with the exception of leukemia patients where saliva was used. Somatic DNA samples were sequenced to an average depth of at least 60X and germline samples to at least 30X. Somatic RNA-seq was performed to a depth of at least 20 million paired-end reads for each sample. In-house as well as published tools and algorithms were used to analyze DNA samples for single-nucleotide variants (SNVs), structural rearrangements, and copy-number alterations. RNA samples were analyzed to identify known and novel gene fusions, to measure allele specific expression of SNVs, and to perform gene-expression outlier analysis. Consistent with previous observations, the mutational burden across pediatric cancers was low. While common mutations were identified, there was a long tail of mutations that occurred at a low frequency. As anticipated, samples obtained post-chemotherapy had a higher mutational burden than treatment-naïve samples. TP53 was the most commonly mutated gene, but we also identified SNVs in other genes commonly mutated in cancer, such as ASXL1, NOTCH2, and RB1. Other novel recurring variants were discovered, further analysis of which is ongoing. Canonical gene fusions were detected in 8/8 patients as well as potentially novel fusions, confirmation of which is also ongoing. In nearly all patients, variants identified by gene panels were also identified through WGS/RNA-seq analysis; however, in 2 instances, variants reported by gene panel testing were reclassified as germline using our tumor/normal WGS analysis. These results indicate that integrated WGS and RNA-seq analysis is feasible in the clinical setting and can reliably identify variants reported on commercially available gene panel testing. However, this approach also resulted in additional clinically relevant findings and allows for novel discovery that will further advance our understanding of these rare and highly aggressive pediatric malignancies. Citation Format: Marcus R. Breese, Avanthi T. Shah, Bogdan Tanasa, Alex G. Lee, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Florette K. Hazard, Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B25.

A promising tool for noninvasive disease monitoring is analysis of circulating tumor DNA (ctDNA). Healthy individuals carry 1-10 ng/ml of cell-free DNA (cfDNA) in the blood; in oncology patients, ctDNA, which is released from tumor cells, comprises a fraction of the cfDNA and carries tumor-specific alterations, such as mutations, translocations, and copy number alterations. Most ctDNA assays lack robust translocation detection capabilities, since they are designed for adult cancers, which are commonly characterized by mutations and copy number alterations. Even assays that are designed specifically for translocation detection have inherent limitations. They are often PCR-based with laborious methods. First, the patient’s primary tumor sample is sequenced to determine the unique sequence across the translocation breakpoint. This is followed by design of patient-specific primers that can only be used for that individual patient. We sought to design an off-the-shelf, broadly applicable ctDNA assay for translocation detection across pediatric Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), and synovial sarcoma (SS). Recent work by our collaborators at Stanford University led to the development of CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), a method capable of ultraspecific and ultrasensitive detection of ctDNA. Utilizing COSMIC and TCGA data, recent sequencing publications defining the landscape of pediatric sarcomas, and our own in-house sequencing data, we designed a pediatric sarcoma CAPP-Seq selector. This selector is comprised of biotinylated oligonucleotides that tile across the introns where translocation breakpoints occur in these pediatric sarcomas. The selector is applied to a sequencing library prepared from patient cfDNA to enrich for the genomic regions of interest via hybrid capture. The resulting enriched library undergoes next-generation sequencing to allow for detection and quantification of circulating tumor DNA. We have isolated cfDNA from pediatric sarcoma patients and found that their cfDNA levels are higher than levels found in adult oncology patients and healthy individuals, likely due to a large fraction of contributing ctDNA. We have applied our selector to pretreatment plasma samples from 5 EWS patients, 2 OS patients, 4 RMS patients, and 1 SS patient. We detected translocations in 10/12 of these samples. Tumor was available for 7/12 of these patients, and we were able to confirm our plasma results by whole-genome sequencing of the tumor, as a validation of our findings. Additionally, we have applied our selector to serial plasma samples collected over the course of treatment and found that ctDNA levels correlate with clinical status. We have detected translocations at allelic frequencies Citation Format: Avanthi Tayi Shah, Tej D. Azad, Jake J. Chabon, Marcus Breese, Bogdan Tanasa, Aviv Spillinger, Stanley G. Leung, Maximilian Diehn, Ash A. Alizadeh, E. Alejandro Sweet-Cordero. Quantitating circulating tumor DNA in translocation-positive sarcoma patients using CAPP-Seq [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B49.

Osteosarcoma (OS) patients who relapse after initial therapy or present with metastatic disease have an extremely poor prognosis. Chemotherapy regimens for these patients have limited efficacy and significant toxicities. Thus, new therapeutic approaches are urgently needed. OS is characterized by numerous copy-number alterations (CNAs) and structural variations (SVs) in cancer-relevant genes. In contrast, recurrent point mutations are not seen. Thus, OS is a C-class (copy number-driven) rather than an M-class (mutation-driven) cancer. However, little is known with regards to whether copy-number alterations can be used to select therapies for aggressive cancers such as OS. The genomic heterogeneity of OS suggests that there may be different oncogenic drivers in subsets of patients. Thus, a systematic effort to identify targetable, patient-specific key driver genes (likely CNAs) is required. We established a clinically annotated patient-derived tumor xenograft (PDTX) bank of 16 OS samples obtained at diagnosis, after surgical resection, and from metastasis, thus representing the full spectrum of disease. Comparison between PDTXs with a corresponding matched primary tumor demonstrated high correlation in copy number (by WGS for 12 samples) and gene expression (by RNAseq for 13 samples), suggesting that PDTXs are faithful preclinical models for OS. To identify recurrent CNAs, we analyzed this WGS dataset together with a public dataset of OS WGS samples. With this combined dataset of 69 samples from 52 patients, we searched for recurrent CNAs across an actionable cancer gene list and identified genes amplified at least 4-fold in at least 2 samples. The two most frequently amplified genes in OS are CCNE1 and MYC. Other frequent alterations were those in the PI3K pathway (PTEN loss and/or AKT amplification), AURKB amplification, CDK4 amplification, and VEGFA amplification. Importantly, all of these CNAs were reflected in at least one PDTX model. We hypothesized that in OS some of these CNAs are key cancer drivers that can be targeted for cancer treatment. To test this hypothesis, we rank-ordered the CNAs in 9 PDTXs by the amplitude of the copy number gain. We used this simple heuristic to identify candidate drivers for individual samples. We then identified 6 drugs that could be used to target specific amplified genes and tested these drugs in corresponding CNA-matched PDTX. In all cases, we saw significant growth inhibition in matched PDTXs whereas the effect was minimal in PDTXs treated with unmatched therapies. These results support the hypothesis that specific genes within CNA serve as oncogenic drivers in OS and thus outline a feasible approach to personalized, genome-informed therapy for this disease. This work could serve as the necessary preclinical proof of principle for development of a targeted therapy basket trial for OS. In parallel to these studies and in order to further define the evolutionary trajectory of OS, we have carried out a comprehensive analysis of both spatial and temporal changes that occur in OS samples from the same patient. This has allowed us to begin defining the role of whole-genome duplication events and chromothripsis as well as loss of heterozygosity in the evolution of OS. We are directing our current efforts towards merging this evolutionary analysis with knowledge of possible targetable events to further identify key vulnerabilities that could be exploited for therapeutic benefit. Citation Format: Leanne Sayles, Marcus Breese, Amanda Koehne, Krystal Straessler, Stanley Leung, Aviv Spillinger, Doug Hawkins, Steven Dubois, Alex Lee, Bogdan Tanasa, Kim Miok, Avanthi Shah, Sheri Spunt, Neyssa Marina, Kim Hazard, Alejandro Sweet-Cordero. Genomic analysis of osteosarcoma reveals opportunities for targeted therapy [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR05.

Several recent studies (including BASIC3, iCAT, INFORM, PEDS-MIONCOSEQ) have used whole exome sequencing (WES) and RNA-seq in order to identify targetable chromosomal alterations in a large variety of pediatric cancers. Few studies have attempted to evaluate whether a more comprehensive approach including WGS and RNA-seq could be used to identify novel events relevant to the pathogenesis of advanced pediatric cancer. We analyzed a total of 59 patients (37 solid tumors, 11 brain tumors, and 10 leukemia/lymphomas) to determine the feasibility of using whole-genome sequencing (WGS) technology in conjunction with RNA-seq in order to identify actionable/druggable alterations in the pediatric cancer genomes. WGS analysis has been performed on 75 samples, that were collected from 45 patients, either at diagnosis or at relapse. For WGS analysis of germline-tumor pairs, after performing the sequence alignment with BWA-MEM to gender-specific hg38 genomes, we have used and verified a set of computational methods: 1) MuTect2 for SNV calling, 2) cn.mops for CNV calling, 3) DELLY and LUMPY for SV calling. Here we present our findings on the sets of SNV, CNV, SV, and gene fusions that we have identified by WGSA and RNA-seq, respectively, with a particular emphasis on the druggable alterations, and on the tumor response in the murine PDX models of the pediatric cancers. Consistent with previous observations, the mutational burden across pediatric cancers was low. While common mutations were identified, there was a long-tail of mutations that occurred at a low frequency. As anticipated, samples obtained post-chemotherapy had a higher mutational burden than treatment naive samples. TP53 was the most commonly mutated gene, but we also identified SNVs in other genes commonly mutated in cancer, such as ASXL1, NOTCH2, and RB1. Other novel recurring variants were discovered, further analysis of which is ongoing. Our results indicate that integrated WGS and RNA-seq analysis is feasible in the clinical setting and can reliably identify variants reported on commercially available gene panel testing. However, this approach also resulted in additional clinically relevant findings and allows for novel discovery that will further advance our understanding of these rare and highly aggressive pediatric malignancies. Citation Format: Bogdan Tanasa, Alex Lee, Marcus Breese, Avanthi Shah, Stan Leung, Heng-Yi Liu, Aviv Spillinger, Kimberly Hazard, Arun Rangaswami, Sheri Spunt, Norm Lacayo, Tabitha Cooney, Eric Alejandro Sweet-Cordero. Whole genome sequence analysis informs precision medicine of pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2075.

Osteosarcoma (OS) patients who relapse after initial therapy or present with metastatic disease have an extremely poor prognosis. Chemotherapy regimens for these patients have limited efficacy and significant toxicities. In clinical practice, treatment is rarely informed by the specific genetic events in individual tumors. It is now well established that OS is characterized by numerous copy-number alterations (CNAs) and structural variations (SVs) in cancer-relevant genes. In contrast, recurrent point mutations are not seen. Thus, OS is a “C-class” (copy number driven) rather than an “M-class” (mutation driven) cancer. However, while a great deal of effort has been directed toward the identification of point mutations in druggable cancer genes, much less effort has gone into determining whether copy number alterations can be used to select therapies for aggressive cancers such as OS. The genomic heterogeneity of OS suggests that there may be different oncogenic drivers in subsets of patients. Thus, a systematic effort to identify targetable, patient-specific key driver genes (likely CNAs) is required. We established a clinically annotated patient-derived tumor xenograft (PDTX) bank of over 20 OS samples obtained at diagnosis, after surgical resection and from metastasis, thus representing the full spectrum of disease. Comparison between PDTXs and matched primary tumor demonstrated high correlation in copy number (by WGS) and gene expression (by RNAseq), suggesting that PDTXs are faithful preclinical models for OS. To identify recurrent CNAs, we analyzed this WGS dataset together with a public dataset of an additional 18 WGS samples. With this combined dataset of 42 samples, we searched for recurrent CNAs across a cancer gene list (1256 genes) generated by combining data from COSMIC and CIVIC. We identified 188 cancer genes amplified at least 4-fold in at least 2 samples. We then used a publicly available resource to identify genes considered “actionable” or “druggable” in cancer. The two most frequently amplified genes in OS are CCNE1 (16/42 cases) and MYC (13/42 cases). Other frequent alterations were those in the PI3K pathway (PTEN loss and/or AKT amplification), AURKB amplification and VEGFA amplification. Importantly, all of these CNAs were reflected in at least one PDTX models. We hypothesized that in OS some of these CNAs are key cancer drivers that can be targeted for cancer treatment. To test this hypothesis, we rank-ordered the CNAs in 9 PDTXs by the amplitude of the copy number gain. We used this simple heuristic to identify candidate drivers for individual samples. We then identified 6 drugs that could be used to target specific amplified genes and tested these drugs in corresponding CNA-matched PDTX. In 9/9 cases we saw significant growth inhibition. These results support the hypothesis that specific genes within CNA serve as oncogenic drivers in OS and thus outline a feasible approach to personalized, genome-informed therapy for this disease. Importantly, the drugs tested were not effective for PDTXs that did not carry the CNA. However, in all cases tested we saw only tumor stasis with a single agent. Therefore, combination therapies will be needed to induce adequate tumor cell kill in OS. Citation Format: Leanne Sayles, Marcus Breese, Amanda Koehne, Leung Stan, Marina Neyssa, Spunt Sheri, Lee Alex, Aviv Spillinger, Steve Dubois, Rafi Avedian, Doug Hawkins, Mohler David, Alejandro Sweet-Cordero. Preclinical evaluation of genome-informed therapy for osteosarcoma using patient-derived xenografts [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B05.

Osteosarcoma (OS) is a highly malignant cancer for which no targeted therapies are currently available. Current treatment modalities are limited to intensive, highly toxic chemotherapy and surgical resection. OS is characterized by wide spread copy number alterations and structural rearrangements. In contrast, no recurrent point mutations in protein-coding genes have been identified, suggesting that copy number alterations (CNAs) are key oncogenic drivers in this disease. However, as copy number alterations are highly heterogeneous, it is likely that each tumor has a distinct set of oncogenic drivers, making a unified treatment approach difficult to define. To identify candidate patient-specific drivers, we used a simple heuristic based on degree of amplification (as assessed by Whole Genome Sequencing) and changes in gene expression (as assessed by RNA sequencing). Using patient-derived tumor xenografts (PDTXs), we assessed if individual OS tumors respond to targeted therapy selected based on this approach. We rank-ordered CNAs in 9 PDTXs by the amplitude of the copy number gain and identified 5 pathways for targeted therapy including CCNE1, MYC, CDK4, PTEN/AKT and AURKB. Next, we prioritized drug choices to those that are in clinical trials and that are readily available and identified 5 drug matches for 9 PDTXs. We used the CDK2 inhibitor Dinaciclib for CCNE1 amplification and observed TGI of 85.5% and 67.8% for 2 different CCNE1 amplified PDTXs tested. CDK9 inhibitor AT7519 was used to treat 2 different MYC amplified PDTXs resulting in TGI of 104% and 83.9%. CDK4 amplified PDTXs were treated with CDK4/6 inhibitor, Palbociclib, resulting in TGI of 82.7%. AKT1 inhibitor MK-2206 was used to treat either AKT1 gains or PTEN loss resulting in TGI of 65.6% and 60.8% respectively. AURKB inhibitor AZD-1152 for AURKB amplified PDTXs in combination with cisplatin resulting in TGI 85.8%. These results support the hypothesis that specific genes within CNA regions serve as oncogenic drivers and that these represent therapeutic opportunities in OS. Our studies provide a roadmap for personalized genome-informed therapy of osteosarcoma, a cancer in which no new therapies have been identified in over 30 years. Citation Format: Leanne C. Sayles, Marcus Breese, Amanda L. Koehne, Stanley Leung, Aviv Spillinger, Alex Lee, Avanthi Shah, Krystal Straessler, Sheri Spunt, Neyssa Marina, Damon Jacobson, Raffi S. Avedian, David G. Mohler, Steven DuBois, Douglas S. Hawkins, E. Alejandro Sweet-Cordero. Tumor-specific copy number alterations uncover therapeutic opportunities in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1948. doi:10.1158/1538-7445.AM2017-1948