IntroductionTumor mutational burden (TMB) and APOBEC mutational signatures are potential prognostic markers in patients with advanced urothelial carcinoma (aUC). Their utility in predicting outcomes to specific therapies in aUC warrants additional study.MethodsWe retrospectively reviewed consecutive UC cases assessed with UCSF500, an institutional assay that uses hybrid capture enrichment of target DNA to interrogate 479 common cancer genes. Hypermutated tumors (HM), defined as having TMB ≥10 mutations/Mb, were also assessed for APOBEC mutational signatures, while non-HM (NHM) tumors were not assessed due to low TMB. The logrank test was used to determine if there were differences in overall survival (OS) and progression-free survival (PFS) among patient groups of interest.ResultsAmong 75 aUC patients who had UCSF500 testing, 46 patients were evaluable for TMB, of which 19 patients (41%) had HM tumors and the rest had NHM tumors (27 patients). An additional 29 patients had unknown TMB status. Among 19 HM patients, all 16 patients who were evaluable for analysis had APOBEC signatures. HM patients (N=19) were compared with NHM patients (N=27) and had improved OS from diagnosis (125.3 months vs 35.7 months, p=0.06) but inferior OS for patients treated with chemotherapy (7.0 months vs 13.1 months, p=0.04). Patients with APOBEC (N=16) were compared with remaining 56 patients, comprised of 27 NHM patients and 29 patients with unknown TMB, showing APOBEC patients to have improved OS from diagnosis (125.3 months vs 44.5 months, p=0.05) but inferior OS for patients treated with chemotherapy (7.0 months vs 13.1 months, p=0.05). Neither APOBEC nor HM status were associated with response to immunotherapy.ConclusionsIn a large, single-institution aUC cohort assessed with UCSF500, an institutional NGS panel, HM tumors were common and all such tumors that were evaluated for mutational signature analysis had APOBEC signatures. APOBEC signatures and high TMB were prognostic of improved OS from diagnosis and both analyses also predicted inferior outcomes with chemotherapy treatment.

Patients with non-small cell lung cancer (NSCLC) who have distant metastases have a poor prognosis. To determine which genomic factors of the primary tumor are associated with metastasis, we analyzed data from 759 patients originally diagnosed with stage I–III NSCLC as part of the AACR Project GENIE Biopharma Collaborative consortium. We found that TP53 mutations were significantly associated with the development of new distant metastases. TP53 mutations were also more prevalent in patients with a history of smoking, suggesting that these patients may be at increased risk for distant metastasis. Our results suggest that additional investigation of the optimal management of patients with early-stage NSCLC harboring TP53 mutations at diagnosis is warranted in light of their higher likelihood of developing new distant metastases.

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 <50%. Solid tumors, CNS tumors, and leukemia/lymphomas are all represented. In total, 391 samples were characterized using WGS (tumor ~60X; germline ~30X) and/or RNAseq (tumor, polyA selected, ≥20 million reads). For 85 of these patients, multiple samples were collected at different time points (diagnosis, resection, relapse, etc.) to identify changes in the cancer over time. If panel testing was performed as part of their clinical care, a comparison to the integrated WGS/RNA analysis was made. WGS was used to identify variants (SNVs), structural rearrangements (SVs), mutational signatures, and copy-number alterations (CNAs). RNAseq was used to identify gene expression outliers, gene fusions, and confirm the expression of variants identified using WGS. The combination of WGS and RNAseq was then used to identify and prioritize potentially actionable variants for each patient. 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.

Osteosarcoma (OS) is characterized by widespread somatic copy number alterations (SCNAs) and structural variations (SVs) with few recurrent point mutations. We previously demonstrated proof-of-principle for a genome-informed strategy for treatment of OS based on rank-ordering SCNAs within specific oncogenes identified by WGS and RNAseq. Although we identified several potentially effective therapeutic strategies using this approach, it is likely that effective therapy for OS will require use of combination therapies. To identify new combination therapy approaches for OS, we used a panel of PDX-derived cell lines (PDXC) and performed a combination drug screen. We assessed 5 drug backbones against 15 targeted agents in 8 PDXC and 2 established OS cell lines. When we combined Gemcitabine with agents that target the ATR-CHK1-WEE1 pathway, we observed strong synergy across all PDXC tested. These results were validated in a secondary screen using a combination matrix across 10 PDXC. Use of targeted agents inhibiting ATR, CHK1 or WEE1 in combination with Gemcitabine led to decreased proliferation and a marked increase in apoptosis in vitro. In subcutaneous tumor models, we observed that decreased tumor growth with either ATRi or Gemcitabine alone, whereas tumors shrank when treated with the combination. However, when we treated OS774 in vivo, there was no effect on tumor growth for either single agent alone or when in combination. This effect was dependent on the presence of ATR as a PDXC with no ATR detectable by western blotting showed not effect in vivo. In an orthotopic model in which PDXC are implanted along the tibia, this combination therapy effectively decreased tumor growth. In a lung metastasis model, ATRi and Gemcitabine resulted a durable reduction in metastatic lesions over time. In summary, we have identified a susceptibility to the ATR-CHK1-WEE1 pathway when combined with gemcitabine. These studies suggest that further investigation ATR-CHK1-WEE1 and gemcitabine are warranted to address the unmet need for new therapeutic approaches for relapsed and recurrent OS patients. Citation Format: Leanne C. Sayles, Henry Martell, Amanda Koehne, Kean-Hooi Ang, Chris Wilson, Michelle Arkin, E. Alejandro Sweet-Cordero. ATR-CHK1-WEE1 pathway is a critical dependency in the context of DNA damage and replicative stress in osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B004.