Germline Genetic Testing and Survival Outcomes Among Children With Rhabdomyosarcoma: A Report From the Children's Oncology Group.

Importance: Determining the impact of germline cancer-predisposition variants (CPVs) on outcomes could inform novel approaches to testing and treating children with rhabdomyosarcoma. Objective: To assess whether CPVs are associated with outcome among children with rhabdomyosarcoma. Design, Setting, and Participants: In this cohort study, data were obtained for individuals, aged 0.01-23.23 years, newly diagnosed with rhabdomyosarcoma who were treated across 171 Children's Oncology Group sites from March 15, 1999, to December 8, 2017. Data analysis was performed from June 16, 2021, to May 15, 2023. Exposure: The presence of a CPV in 24 rhabdomyosarcoma-associated cancer-predisposition genes (CPGs) or an expanded set of 63 autosomal-dominant CPGs. Main Outcomes and Measures: Overall survival (OS) and event-free survival (EFS) were the main outcomes, using the Kaplan-Meier estimator to assess survival probabilities and the Cox proportional hazards regression model to adjust for clinical covariates. Analyses were stratified by tumor histology and the fusion status of PAX3 or PAX7 to the FOXO1 gene. Results: In this study of 580 individuals with rhabdomyosarcoma, the median patient age was 5.9 years (range, 0.01-23.23 years), and the male-to-female ratio was 1.5 to 1 (351 [60.5%] male). For patients with CPVs in rhabdomyosarcoma-associated CPGs, EFS was 48.4% compared with 57.8% for patients without a CPV (P = .10), and OS was 53.7% compared with 65.3% for patients without a CPV (P = .06). After adjustment, patients with CPVs had significantly worse OS (adjusted hazard ratio [AHR], 2.49 [95% CI, 1.39-4.45]; P = .002), and the outcomes were not better among patients with embryonal histology (EFS: AHR, 2.25 [95% CI, 1.25-4.06]; P = .007]; OS: AHR, 2.83 [95% CI, 1.47-5.43]; P = .002]). These associations were not due to the development of a second malignant neoplasm, and importantly, patients with fusion-negative rhabdomyosarcoma who harbored a CPV had similarly inferior outcomes as patients with fusion-positive rhabdomyosarcoma without CPVs (EFS: AHR, 1.35 [95% CI, 0.71-2.59]; P = .37; OS: AHR, 1.71 [95% CI, 0.84-3.47]; P = .14). There were no significant differences in outcome by CPV status of the 63 CPG set. Conclusions and Relevance: This cohort study identified a group of patients with embryonal rhabdomyosarcoma who had a particularly poor outcome. Other important clinical findings included that individuals with TP53 had poor outcomes independent of second malignant neoplasms and that patients with fusion-negative rhabdomyosarcoma who harbored a CPV had outcomes comparable to patients with fusion-positive rhabdomyosarcoma. These findings suggest that germline CPV testing may aid in clinical prognosis and should be considered in prospective risk-based clinical trials.

Polymorphisms in the Nonhomologous End-joining DNA Repair Pathway are Associated with HPV Integration in Cervical Dysplasia.

Previous evidence indicates that human papillomavirus (HPV) integration status may be associated with cervical cancer development and progression. However, host genetic variation within genes that may play important roles in the viral integration process is understudied. The aim of this study was to examine the association between HPV16 and HPV18 viral integration status and SNPs in nonhomologous-end-joining (NHEJ) DNA repair pathway genes on cervical dysplasia. Women enrolled in two large trials of optical technologies for cervical cancer detection and positive for HPV16 or HPV18 were selected for HPV integration analysis and genotyping. Associations between SNPs and cytology (normal, low-grade, or high-grade lesions) were evaluated. Among women with cervical dysplasia, polytomous logistic regression models were used to evaluate the effect of each SNP on viral integration status. Of the 710 women evaluated [149 high-grade squamous intraepithelial lesion (HSIL), 251; low-grade squamous intraepithelial lesion (LSIL, 310 normal)], 395 (55.6%) were positive for HPV16 and 192 (27%) were positive for HPV18. Tag-SNPs in 13 DNA repair genes, including RAD50, WRN, and XRCC4, were significantly associated with cervical dysplasia. HPV16 integration status was differential across cervical cytology, but overall, most participants had a mix of both episomal and integrated HPV16. Four tag-SNPs in the XRCC4 gene were found to be significantly associated with HPV16 integration status. Our findings indicate that host genetic variation in NHEJ DNA repair pathway genes, specifically XRCC4, are significantly associated with HPV integration, and that these genes may play an important role in determining cervical cancer development and progression. PREVENTION RELEVANCE: HPV integration in premalignant lesions and is thought to be an important driver of carcinogenesis. However, it is unclear what factors promote integration. The use of targeted genotyping among women presenting with cervical dysplasia has the potential to be an effective tool in assessing the likelihood of progression to cancer.

Germline genetic variants and pediatric rhabdomyosarcoma outcomes: a report from the Children's Oncology Group.

BACKGROUND: Relative to other pediatric cancers, survival for rhabdomyosarcoma (RMS) has not improved in recent decades, suggesting the need to enhance risk stratification. Therefore, we conducted a genome-wide association study for event-free survival (EFS) and overall survival (OS) to identify genetic variants associated with outcomes in individuals with RMS. METHODS: The study included 920 individuals with newly diagnosed RMS who were enrolled in Children's Oncology Group protocols. To assess the association of each single nucleotide polymorphism (SNP) with EFS and OS, we estimated hazard ratios (HRs) and 95% confidence intervals (CIs) using multivariable Cox proportional hazards models, adjusted for clinical covariates. All statistical tests were two sided. We also performed stratified analyses by histological subtype (alveolar and embryonal RMS) and carried out sensitivity analyses of statistically significant SNPs by PAX3/7-FOXO1 fusion status and genetic ancestry group. RESULTS: We identified that rs17321084 was associated with worse EFS (HR = 2.01, 95% CI = 1.59 to 2.53, P = 5.39 × 10-9) and rs10094840 was associated with worse OS (HR = 1.84, 95% CI = 1.48 to 2.27, P = 2.13 × 10-8). Using publicly available data, we found that rs17321084 lies in a binding region for transcription factors GATA2 and GATA3, and rs10094840 is associated with SPAG1 and RNF19A expression. We also identified that CTNNA3 rs2135732 (HR = 3.75, 95% CI = 2.34 to 5.99, P = 3.54 × 10-8) and MED31 rs74504320 (HR = 3.21, 95% CI = 2.12 to 4.86, P = 3.60 × 10-8) were associated with worse OS among individuals with alveolar RMS. CONCLUSIONS: We demonstrated that common germline variants are associated with EFS and OS among individuals with RMS. Additional replication and investigation of these SNP effects may further support their consideration in risk stratification protocols.

Germline Cancer Predisposition Variants in Pediatric Rhabdomyosarcoma: A Report From the Children's Oncology Group.

BACKGROUND: Several cancer-susceptibility syndromes are reported to underlie pediatric rhabdomyosarcoma (RMS); however, to our knowledge there have been no systematic efforts to characterize the heterogeneous genetic etiologies of this often-fatal malignancy. METHODS: We performed exome-sequencing on germline DNA from 615 patients with newly diagnosed RMS consented through the Children's Oncology Group. We compared the prevalence of cancer predisposition variants in 63 autosomal-dominant cancer predisposition genes in these patients with population controls (n = 9963). All statistical tests were 2-sided. RESULTS: We identified germline cancer predisposition variants in 45 RMS patients (7.3%; all FOXO1 fusion negative) across 15 autosomal dominant genes, which was statistically significantly enriched compared with controls (1.4%, P = 1.3 × 10-22). Specifically, 73.3% of the predisposition variants were found in predisposition syndrome genes previously associated with pediatric RMS risk, such as Li-Fraumeni syndrome (TP53) and neurofibromatosis type I (NF1). Notably, 5 patients had well-described oncogenic missense variants in HRAS (p.G12V and p.G12S) associated with Costello syndrome. Also, genetic etiology differed with histology, as germline variants were more frequent in embryonal vs alveolar RMS patients (10.0% vs 3.0%, P = .02). Although patients with a cancer predisposition variant tended to be younger at diagnosis (P = 9.9 × 10-4), 40.0% of germline variants were identified in those older than 3 years of age, which is in contrast to current genetic testing recommendations based on early age at diagnosis. CONCLUSIONS: These findings demonstrate that genetic risk of RMS results from germline predisposition variants associated with a wide spectrum of cancer susceptibility syndromes. Germline genetic testing for children with RMS should be informed by RMS subtypes and not be limited to only young patients.

A prospective study of a simple algorithm to individually dose high-dose methotrexate for children with leukemia at risk for methotrexate toxicities.

PURPOSE: High-dose methotrexate (HDMTX) is critical to the successful treatment of pediatric acute lymphoblastic leukemia (ALL) but can cause significant toxicities. This study prospectively evaluated the effectiveness of a fixed algorithm which requires no real-time pharmacokinetic modeling and no previous patient exposure to HDMTX, to individualize HDMTX dosing for at-risk patients with the aim of avoiding methotrexate-related toxicities. METHODS: We developed a simple algorithm to individualize HDMTX infusions with 0-2 rate adjustments based on methotrexate levels during the infusion. This was a prospective, open-label, study; eligible patients were identified and referred by their oncologist. RESULTS: Fifty-four evaluable cycles of HDMTX (5 g/m2 over 24 h) were administered to 22 patients. Blood samples were obtained in 21 patients to examine single nucleotide polymorphisms (SNPs) related to methotrexate disposition. Twelve (54.5%) subjects had a history of previous HDMTX toxicities including seven (31.8%) who previously required glucarpidase rescue and seven (31.8%) with an entry glomerular filtration rate < 80 ml/min/1.73 m2. 107/110 (97.2%) of methotrexate levels were drawn properly and 100% of algorithm dosing instructions were performed correctly at the bedside. Thirty-five (64.8%) of all cycles and 24 of 33 (72.7%) cycles that required a dose-adjustment had an end 24-h methotrexate level (Cpss) within our goal range of 65 ± 15 µM with only 3 (5.6%) resulting in Cpss higher than goal. Grade 3/4 toxicities were rare; no patients developed > Grade 1 acute kidney injury. CONCLUSION: This algorithm is a simple, safe and effective method for individualizing HDMTX in pediatric patients with ALL. CLINICALTRIALS. GOV REGISTRY: NCT02076997.

Membrane protected apoptotic trophoblast microparticles contain nucleic acids: relevance to preeclampsia.

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA(+) MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies.