Adrenergic and mesenchymal signatures are identifiable in cell-free DNA and correlate with metastatic disease burden in children with neuroblastoma.

BACKGROUND: Cell-free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES-specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. METHODS: We previously performed an integrative analysis to identify ADRN and MES-specific genes (n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. RESULTS: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < .001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. CONCLUSIONS: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.

Diffuse Pediatric-type High-grade Glioma Arising in an Ovarian Mature Cystic Teratoma.

Immature neuroectodermal tissue can be found in the ovary as part of an immature teratoma or as part of a teratoma with malignant neuroectodermal transformation. Such lesions may closely resemble central nervous system tumors, but their biologic similarity is unclear. We describe an 18-yr-old female who presented with abdominal pain caused by an ovarian mass with widespread metastases. Histology showed a primitive, high-grade tumor arising in the background of a mature teratoma. The tumor was SOX10 positive, with focal expression of GFAP, S100, NSE, and synaptophysin. Molecular analysis demonstrated co-amplification of PDGFRA and KIT , alterations common in high-grade gliomas. By whole-genome methylation profiling, it clustered into the "diffuse pediatric-type high-grade glioma, RTK1 subtype, subclass c" group. Despite progressing through 2 lines of chemotherapy with widespread metastatic disease, she achieved an excellent response to chemotherapy directed toward aggressive germ cell tumors. This case emphasizes the importance of immunohistochemical, genomic, and epigenetic analyses to accurately classify these exceedingly rare tumors and determine the optimal therapy.

Histotripsy induces apoptosis and reduces hypoxia in a neuroblastoma xenograft model.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood, and high-risk disease is resistant to intensive treatment. Histotripsy is a focused ultrasound therapy under development for tissue ablation via bubble activity. The goal of this study was to assess outcomes of histotripsy ablation in a xenograft model of high-risk NB. METHODS: Female NCr nude mice received NGP-luciferase cells intrarenally. Under ultrasound image guidance, histotripsy pulses were applied over a distance of 4-6 mm within the tumors. Bioluminescence indicative of tumor viability was quantified before, immediately after, and 24 h after histotripsy exposure. Tumors were immunostained to assess apoptosis (TUNEL), endothelium (endomucin), pericytes (αSMA), hypoxia (pimonidazole), vascular endothelial growth factor A (VEGFA), and platelet-derived growth factor-B (PDGF-B). The apoptotic cytokine TNFα and its downstream effector cleaved caspase-3 (c-casp-3) were assessed with SDS-PAGE. RESULTS: Histotripsy induced a 50% reduction in bioluminescence compared to untreated controls, with an absence of nuclei in the treatment core surrounded by a dense rim of TUNEL-positive cells. Tumor regions not targeted by histotripsy also showed an increase in TUNEL staining density. Increased apoptosis in histotripsy samples was consistent with increases in TNFα and c-casp-3 relative to controls. Treated tumors exhibited a decrease in hypoxia, VEGF, PDGF-B, and pericyte coverage of vasculature compared to control samples. Further, increases in vasodilation were found in histotripsy-treated specimens. CONCLUSIONS: In addition to ablative effects, histotripsy was found to drive tumor apoptosis through intrinsic pathways, altering blood vessel architecture, and reducing hypoxia.

Efficacy of post-induction therapy for high-risk neuroblastoma patients with end-induction residual disease.

BACKGROUND: High-risk neuroblastoma patients with end-induction residual disease commonly receive post-induction therapy in an effort to increase survival by improving the response before autologous stem cell transplantation (ASCT). The authors conducted a multicenter, retrospective study to investigate the efficacy of this approach. METHODS: Patients diagnosed between 2008 and 2018 without progressive disease with a partial response or worse at end-induction were stratified according to the post-induction treatment: 1) no additional therapy before ASCT (cohort 1), 2) post-induction "bridge" therapy before ASCT (cohort 2), and 3) post-induction therapy without ASCT (cohort 3). χ2 tests were used to compare patient characteristics. Three-year event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and survival curves were compared by log-rank test. RESULTS: The study cohort consisted of 201 patients: cohort 1 (n = 123), cohort 2 (n = 51), and cohort 3 (n = 27). Although the end-induction response was better for cohort 1 than cohorts 2 and 3, the outcomes for cohorts 1 and 2 were not significantly different (P = .77 for EFS and P = .85 for OS). Inferior outcomes were observed for cohort 3 (P < .001 for EFS and P = .06 for OS). Among patients with end-induction stable metastatic disease, 3-year EFS was significantly improved for cohort 2 versus cohort 1 (P = .04). Cohort 3 patients with a complete response at metastatic sites after post-induction therapy had significantly better 3-year EFS than those with residual metastatic disease (P = .01). CONCLUSIONS: Prospective studies to confirm the benefits of bridge treatment and the prognostic significance of metastatic response observed in this study are warranted.

Progress toward liquid biopsies in pediatric solid tumors.

Pediatric solid tumors have long been known to shed tumor cells, DNA, RNA, and proteins into the blood. Recent technological advances have allowed for improved capture and analysis of these typically scant circulating materials. Efforts are ongoing to develop "liquid biopsy" assays as minimally invasive tools to address diagnostic, prognostic, and disease monitoring needs in childhood cancer care. Applying these highly sensitive technologies to serial liquid biopsies is expected to advance understanding of tumor biology, heterogeneity, and evolution over the course of therapy, thus opening new avenues for personalized therapy. In this review, we outline the latest technologies available for liquid biopsies and describe the methods, pitfalls, and benefits of the assays that are being developed for children with extracranial solid tumors. We discuss what has been learned in several of the most common pediatric solid tumors including neuroblastoma, sarcoma, Wilms tumor, and hepatoblastoma and highlight promising future directions for the field.

Association between end-induction response according to the revised International Neuroblastoma Response Criteria (INRC) and outcome in high-risk neuroblastoma patients.

BACKGROUND: The 1993 International Neuroblastoma Response Criteria (INRC) were revised in 2017 to include modern functional imaging studies and methods for quantifying disease in bone marrow. We hypothesized the 2017 INRC would enable more precise assessment of response to treatment and provide superior prognostic information compared with the 1993 criteria. METHODS: High-risk (HR) neuroblastoma patients from two institutions in Chicago diagnosed between 2006 and 2016 were identified. Patients were assessed post induction chemotherapy via the 1993 and 2017 INRC and classified as responder (≥ mixed response [MXR] or ≥ minor response [MR], respectively) or nonresponder (< MXR or < MR). Event-free survival (EFS) and overall survival (OS) for responders versus nonresponders were determined from end induction and stratified by Cox regression. Patients with progressive disease at end induction were eliminated from the EFS analyses but included in the OS analysis. RESULTS: The 1993 criteria classified 52 of the 60 HR patients as responders, whereas 54 responders were identified using the 2017 criteria (Spearman correlation r = 0.82, P < 0.001). No statistically significant difference in EFS was observed for responders versus nonresponders using either criteria (P = 0.48 and P = 0.08). However, superior OS was observed for responders (P = 0.01) using either criteria. Both criteria were sensitive in identifying responders among those with good outcomes. The specificity to identify nonresponders among those with poor outcomes was poor. CONCLUSIONS: In HR neuroblastoma, end-induction response defined by the 1993 or 2017 INRC is associated with survival. Larger cohorts are needed to determine if the 2017 INRC provides more precise prognostication.

Segmental Chromosomal Aberrations in Localized Neuroblastoma Can be Detected in Formalin-Fixed Paraffin-Embedded Tissue Samples and Are Associated With Recurrence.

BACKGROUND: Array comparative genomic hybridization (CGH) analyses of frozen tumors have shown strong associations between the pattern of chromosomal aberrations and outcome in patients with advanced-stage neuroblastoma. New platforms for analyzing chromosomal aberrations using formalin-fixed paraffin-embedded (FFPE) tissue have recently been developed. We sought to determine whether chromosomal microarray analysis (CMA) using FFPE tumors is feasible and if segmental chromosomal aberrations were prognostic of recurrence in localized neuroblastoma. METHODS: Patients with MYCN nonamplified International Neuroblastoma Staging System stage 1 and 2 disease who recurred were identified. CMA was performed with diagnostic FFPE samples using OncoScan™ FFPE Express 2.0. The prognostic significance of chromosomal pattern was validated in 105 patients with available CGH results. RESULTS: In 26 evaluable patients, 11 recurred locally, nine had metastatic relapse, and six remained progression free >3 years from diagnosis. No chromosomal aberrations were identified in four tumors. Numerical chromosomal aberrations (NCAs) without segmental chromosomal aberration (SCA) were identified in 11 patients: six progressed locally, two had metastatic progression and 3 remained progression-free. Eleven patients had SCAs: four progressed locally, six developed metastatic progression and one remained progression-free. Five or more SCAs were only detected in tumors from patients who developed metastases (P = 0.0004). In the validation cohort, SCAs were associated with inferior event-free survival (EFS) compared to NCA (5-year EFS 68% ± 8.3% vs. 91% ± 3.6%, respectively; P = 0.0083). CONCLUSIONS: It is feasible to evaluate chromosomal aberrations using FFPE neuroblastoma tissue. SCA is associated with inferior EFS in localized neuroblastoma patients, and multiple SCAs may be predictive of metastatic relapse.

Second malignancies in patients with neuroblastoma: the effects of risk-based therapy.

BACKGROUND: To investigate the incidence of second malignant neoplasms (SMN) for patients with neuroblastoma, we analyzed patients from the SEER database according to three treatment eras (Era 1: 1973-1989, Era 2: 1990-1996, and Era 3: 1997-2006) corresponding to the introduction of multi-agent chemotherapy, risk-based treatment, and stem cell transplant. PROCEDURES: The SEER database was mined for all patients with neuroblastoma or ganglioneuroblastoma. Cumulative incidence of SMN was calculated with death as a competing risk. A poisson regression model was used to estimate incidence rate ratios and 95% confidence intervals to compare the rates of SMN between patients in different Eras. RESULTS: The analytic cohort included 2,801 patients. Thirty-four patients developed a SMN, accounting for 1.2% of all patients. Of the patients who developed a SMN, 47.1% received radiation for their primary neuroblastoma. Fourteen of the SMN were carcinomas, and 10 were hematologic malignancies, with six cases of acute myelogenous leukemia. There was no difference in the incidence of SMN in Era 1 compared to Era 3 (P = 0.48). The cumulative incidence of SMN at 30 years for high-risk patients was 10.44% (95% CI 3.98-20.52%) compared to 3.57% (95% CI 1.87-6.12%) for non-high-risk patients (P < 0.001). CONCLUSIONS: This study showed no increase in the incidence of SMNs for children treated in the most recent treatment era as compared to earlier Eras. However, as the risk for developing SMN does not plateau, the number of SMNs will likely continue to rise in the cohort of patients treated after 1996. Comprehensive follow-up care for these survivors will be important.

5-Hydroxymethylcytosine Profiling of Cell-Free DNA Identifies Bivalent Genes That Are Prognostic of Survival in High-Risk Neuroblastoma.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high-risk neuroblastoma. METHODS: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found increased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. CONCLUSION: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.

Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Clinical features and outcomes in patients with secondary Ewing sarcoma.

BACKGROUND: Ewing sarcoma (EWS) is rarely diagnosed as a second malignancy. We sought to describe a cohort of patients with secondary EWS and investigate if patient characteristics and survival differ between patients with secondary and primary EWS. PROCEDURE: Patients with EWS or peripheral primitive neuroectodermal tumor (PNET) reported to the Surveillance, Epidemiology, and End Results Program (SEER) database from 1973 to 2008 were evaluated based on primary or secondary tumor sequence. Overall survival was estimated by Kaplan-Meier methods and evaluated using the log-rank test. Competing risk analysis was used to describe risk of death due to malignancy rather than other causes. RESULTS: Fifty-eight cases of secondary EWS were reported, accounting for 2.1% of all EWS cases. The median latency from primary malignancy to secondary EWS was 64 months (range 1-282 months). 12.1% of patients with secondary EWS received radiation to the site of secondary tumor during therapy for their primary malignancy. Patients with secondary EWS were more likely to have axial tumors (77.4% vs. 62.5%; P = 0.03) and smaller tumors (75.0% vs. 48.2% <8 cm; P = 0.001). Five-year overall survival from diagnosis was inferior for patients with secondary compared to primary EWS (34.3% vs. 52.2%; P = 0.002). However, patients with secondary tumors were less likely than those with primary EWS to die from their malignancy [hazard ratio 0.44; 95% confidence interval (CI) 0.23-0.85]. CONCLUSIONS: Secondary EWS accounts for a minority of cases of EWS. Tumor size and site and patient survival differ among patients with primary and secondary EWS.

Perception of transition readiness and preferences for use of technology in transition programs: teens' ideas for the future.

BACKGROUND: Efforts to facilitate transition of care to adult providers for adolescents with chronic disease are not uniformly successful and many patients encounter challenges. The goal of this study was to assess transition readiness and preferences for tools to aid in the transition process with an emphasis on technology and social media. METHODS: We surveyed and performed focus groups on patients aged 13-21 years from a pediatric university-based rheumatology and general pediatric practice. Demographics and transition readiness were assessed using a questionnaire. Transition readiness was assessed by examining patient knowledge and independence with care. Focus groups were conducted to elicit perspectives about desirable features of a transition program and useful tools. RESULTS: Thirty-five patients completed surveys; and 20 patients and 13 of their parents participated in a focus group. The median patient age was 17 years and 74.3% were female. A Likert scale (0-10, 10=most) was used to evaluate concern over changing to an adult medical provider, (mean=6.4, SD=2.6), preparedness for disease self-management (mean=6.0, SD=2.8), and perceived importance of self-managing their condition (mean=7.1, SD=3.1). Themes that emerged from focus groups included a desire for support groups with other teens, a preference for using text messaging for communication and a desire for an online health management program. CONCLUSIONS: Teens with chronic disease are able to identify health maintenance tasks and strategies that will aid in developing independence with healthcare management. These findings support the idea that developing engaging applications and support groups will assist teens in the transitioning.

Breaking Up Isn't Hard to Do: Isolating Cell-free DNA Fragments in Osteosarcoma.

The novel use of blood-based biospecimens from a retrospective cohort of 50 patients with osteosarcoma was recently studied. The potential clinical utility of sorting cell-free DNA by fragment size was defined, with shorter tumor-specific DNA enrichment providing prognostic value and allowing for streamlined molecular profiling of circulating tumor material. See related article by Udomruk et al., p. 2085.

5-hydroxymethylcytosine profiling of cell-free DNA identifies bivalent genes that are prognostic of survival in high-risk neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumor in childhood and epigenetic dysregulation is a key driver of this embryonal disease. In cell-free DNA from neuroblastoma patients with high-risk disease, we found increased 5-hydroxymethylcytosine (5-hmC) deposition on Polycomb Repressive Complex 2 (PRC2) target genes, a finding previously described in the context of bivalent genes. As bivalent genes, defined as genes bearing both activating (H3K4me3) and repressive (H3K27me3) chromatin modifications, have been shown to play an important role in development and cancer, we investigated the potential role of bivalent genes in maintaining a de-differentiated state in neuroblastoma and their potential use as a biomarker. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogenous neuroblastoma cell lines. Through gene set variance analysis, we developed a clinically implementable bivalent signature. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in high-risk neuroblastoma patients. Thus, low expression of bivalent genes is a biomarker of ultra-high-risk disease and may represent a therapeutic opportunity in neuroblastoma.

Methyltransferase Inhibition Enables Tgfß Driven Induction of CDKN2A and B in Cancer Cells.

CDKN2A/B deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three CDKN2A/B transcripts are independently expressed to varying degrees, but one, ARF, is uniformly silenced. Although TGFß induces certain CDKN2A/B transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. CDKN2A/B induction by TGFß correlated with de novo appearance of three H3K27Ac peaks within a 20 kb cis element ∼150 kb proximal to CDKN2A/B. Deleting that segment prevented their induction by TGFß but not a basal increase driven by methyltransferase inhibition alone. Expression of two CDKN2A/B transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb cis elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFß-dependent activation of a remote enhancer to reverse CDKN2A/B silencing. Though focused on CDKN2A/B here, such crosstalk may apply to other TGFß-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.

Adrenergic and mesenchymal signatures are identifiable in cell-free DNA and correlate with metastatic disease burden in children with neuroblastoma.

Background: Cell free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. Methods: We previously performed an integrative analysis to identify ADRN and MES specific genes (n=373 and n=159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. Results: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < 0.001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. Conclusions: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.

T-cell inflammation is prognostic of survival in patients with high-risk neuroblastoma enriched for an adrenergic signature.

Purpose: T-cell inflammation (TCI) has been shown to be a prognostic marker in neuroblastoma, a tumor comprised of cells that can exist in two epigenetic states, adrenergic (ADRN) and mesenchymal (MES). We hypothesized that elucidating unique and overlapping aspects of these biologic features could serve as novel biomarkers. Patients and Methods: We detected lineage-specific, single-stranded super-enhancers defining ADRN and MES specific genes. Publicly available neuroblastoma RNA-seq data from GSE49711 (Cohort 1) and TARGET (Cohort 2) were assigned MES, ADRN, and TCI scores. Tumors were characterized as MES (top 33%) or ADRN (bottom 33%), and TCI (top 67% TCI score) or non-inflamed (bottom 33% TCI score). Overall survival (OS) was assessed using the Kaplan-Meier method, and differences were assessed by the log-rank test. Results: We identified 159 MES genes and 373 ADRN genes. TCI scores were correlated with MES scores (R=0.56, p<0.001 and R=0.38, p<0.001) and anticorrelated with MYCN -amplification (R=-0.29, p<0.001 and -0.18, p=0.03) in both cohorts. Among Cohort 1 patients with high-risk, ADRN tumors (n=59), those with TCI tumors (n=22) had superior OS to those with non-inflammed tumors (n=37) (p=0.01), though this comparison did not reach significance in Cohort 2. TCI status was not associated with survival in patients with high-risk MES tumors in either cohort. Conclusions: High inflammation scores were correlated with improved survival in some high-risk patients with, ADRN but not MES neuroblastoma. These findings have implications for approaches to treating high-risk neuroblastoma.

Persistence of Racial and Ethnic Disparities in Risk and Survival for Patients with Neuroblastoma over Two Decades.

BACKGROUND: Racial/ethnic survival disparities in neuroblastoma were first reported more than a decade ago. We sought to investigate if these disparities have persisted with current era therapy. METHODS: Two patient cohorts were identified in the International Neuroblastoma Risk Group Data Commons (INRGdc) (Cohort 1: diagnosed 2001-2009, n=4359; Cohort 2: diagnosed 2010-2019, n=4891). Chi-squared tests were used to assess the relationship between race/ethnicity and clinical and biologic features. Survival was estimated by the Kaplan-Meier method. Cox proportional hazards regression analyses were performed to investigate the association between racial/ethnic groups and prognostic markers. RESULTS: Significantly higher 5-year event-free survival (EFS) and overall survival (OS) were observed for Cohort 2 compared to Cohort 1 (P<0.001 and P<0.001, respectively). Compared to White patients, Black patients in both cohorts had a higher proportion of high-risk disease (Cohort 1: P<0.001; Cohort 2: P<0.001) and worse EFS (Cohort 1: P<0.001; Cohort 2 P<0.001) and OS (Cohort 1: P<0.001; Cohort 2: P<0.001). In Cohort 1, Native Americans also had a higher proportion of high-risk disease (P=0.03) and inferior EFS/OS. No significant survival disparities were observed for low- or intermediate-risk patients in either cohort or high-risk patients in Cohort 1. Hispanic patients with high-risk disease in Cohort 2 had significantly inferior OS (P=0.047). Significantly worse OS, but not EFS, (P=0.006 and P=0.02, respectively) was also observed among Black and Hispanic patients assigned to receive post-Consolidation dinutuximab on clinical trials (n=885). CONCLUSION: Racial/ethnic survival disparities have persisted over time and were observed among high-risk patients assigned to receive post-Consolidation dinutuximab.

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

PURPOSE: Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS: In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS: In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS (P = .04). The most frequently altered genes in the primary cohort were TP53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort (P = .015) and the validation cohort (P = .012). CONCLUSION: The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Clinical features and outcomes in patients with Ewing sarcoma and regional lymph node involvement.

BACKGROUND: A minority of patients with Ewing sarcoma present with regional lymph node involvement. We investigated if patient characteristics and outcomes differ between patients with Ewing sarcoma with and without regional node involvement. PROCEDURE: Patients <40 years of age with Ewing sarcoma or peripheral primitive neuroectodermal tumor (PNET) reported to the SEER database from 1973 to 2008 were evaluated based on the presence (n = 91) or absence (n = 1,361) of regional node involvement. Patient characteristics were analyzed using Fisher exact tests. Overall survival was estimated by Kaplan-Meier methods and evaluated using log-rank tests and Cox models. RESULTS: Patients with regional node involvement were more likely to have extraskeletal primary tumors (65.9% vs. 31.2%; P < 0.001) and axial tumors (71.1% vs. 59.6%; P = 0.03) compared to patients without regional node involvement. The incidence of regional node involvement was 12.4% for patients with extraskeletal primary tumors compared to 3.2% for patients with skeletal tumors. Five-year overall survival from diagnosis was inferior for patients with regional node involvement compared to those without regional node involvement (45.9% vs. 60.3%; P < 0.001). On multivariate analysis, regional node involvement was predictive of inferior overall survival independent of age, metastatic status, tumor site, and soft tissue origin (hazard ratio 1.59; 95% CI 1.16-2.19). CONCLUSIONS: Patients with extraskeletal Ewing sarcoma should undergo evaluation for regional node involvement. If validated, our findings indicate that regional node involvement may be an independent adverse prognostic factor in Ewing sarcoma, and potentially useful in risk-stratifying patients with otherwise localized disease.