Chromoplexy Is a Frequent Early Clonal Event in EWSR1-Rearranged Round Cell Sarcomas That Can Be Detected Using Clinically Validated Targeted Sequencing Panels.

Chromoplexy is a phenomenon defined by large-scale chromosomal chained rearrangements. A previous study observed chromoplectic events in a subset of Ewing sarcomas (ES), which was linked to an increased relapse rate. Chromoplexy analysis could potentially facilitate patient risk stratification, particularly if it could be detected with clinically applied targeted next-generation sequencing (NGS) panels. Using DELLY, a structural variant (SV) calling algorithm that is part of the MSK-IMPACT pipeline, we characterized the spectrum of SVs in EWSR1-fused round cell sarcomas, including 173 ES and 104 desmoplastic small round cell tumors (DSRCT), to detect chromoplexy and evaluate its association with clinical and genomic features. Chromoplectic events were detected in 31% of the ES cases and 19% of the DSRCT cases. EWSR1 involvement accounted for 76% to 93% of these events, being rearranged with diverse noncanonical gene partners across the genome, involving mainly translocations but also intrachromosomal deletions and inversions. A major breakpoint cluster was located on EWSR1 exons 8-13. In a subset of cases, the SVs disrupted adjacent loci, forming deletion bridges. Longitudinal sequencing and breakpoint allele fraction analysis showed that chromoplexy is an early event that remains detectable throughout disease progression and likely develops simultaneously with the driver fusion. The presence of chromoplexy was validated in an external ES patient cohort with whole exome sequencing. Chromoplexy was significantly more likely to be present in cases that were metastatic at presentation. Together, this study identifies chromoplexy as a frequent genomic alteration in diverse EWSR1-rearranged tumors that can be captured by targeted NGS panels. SIGNIFICANCE: Chromoplexy is detectable using targeted NGS in a substantial portion of EWSR1-rearranged round cell sarcomas as an early and persistent clonal event, expanding the genomic complexity of fusion-associated sarcomas.

Antibiotics are associated with worse outcomes in lung cancer patients treated with chemotherapy and immunotherapy.

Anti-PD(L)-1 inhibition combined with platinum doublet chemotherapy (Chemo-IO) has become the most frequently used standard of care regimen in patients with non-small cell lung cancer (NSCLC). The negative impact of antibiotics on clinical outcomes prior to anti-PD(L)-1 inhibition monotherapy (IO) has been demonstrated in multiple studies, but the impact of antibiotic exposure prior to initiation of Chemo-IO is controversial. We assessed antibiotic exposures at two time windows: within 60 days prior to therapy (-60 d window) and within 60 days prior to therapy and 42 days after therapy (-60 + 42d window) in 2028 patients with advanced NSCLC treated with Chemo-IO and IO monotherapy focusing on objective response rate (ORR: rate of partial response and complete response), progression-free survival (PFS), and overall survival (OS). We also assessed impact of antibiotic exposure in an independent cohort of 53 patients. Univariable and multivariable analyses were conducted along with a meta-analysis from similar studies. For the -60 d window, in the Chemo-IO group (N = 769), 183 (24%) patients received antibiotics. Antibiotic exposure was associated with worse ORR (27% vs 40%, p = 0.001), shorter PFS (3.9 months vs. 5.9 months, hazard ratio [HR] 1.35, 95%CI 1.1,1.6, p = 0.0012), as well as shorter OS (10 months vs. 15 months, HR 1.50, 95%CI 1.2,1.8, p = 0.00014). After adjusting for known prognostic factors in NSCLC, antibiotic exposure was independently associated with worse PFS (HR 1.39, 95%CI 1.35,1.7, p = 0.002) and OS (HR 1.61, 95%CI 1.28,2.03, p < 0.001). Similar results were obtained in the -60 + 42d window, and also in an independent cohort. In a meta-analysis of patients with NSCLC treated with Chemo-IO (N = 4) or IO monotherapy (N = 13 studies) antibiotic exposure before treatment was associated with worse OS among all patients (n = 11,351) (HR 1.93, 95% CI 1.52, 2.45) and Chemo-IO-treated patients (n = 1201) (HR 1.54, 95% CI 1.28, 1.84). Thus, antibiotics exposure prior to Chemo-IO is common and associated with worse outcomes, even after adjusting for other factors. These results highlight the need to implement antibiotic stewardship in routine oncology practice.

Clinical and molecular features of acquired resistance to immunotherapy in non-small cell lung cancer.

Although immunotherapy with PD-(L)1 blockade is routine for lung cancer, little is known about acquired resistance. Among 1,201 patients with non-small cell lung cancer (NSCLC) treated with PD-(L)1 blockade, acquired resistance is common, occurring in >60% of initial responders. Acquired resistance shows differential expression of inflammation and interferon (IFN) signaling. Relapsed tumors can be separated by upregulated or stable expression of IFNγ response genes. Upregulation of IFNγ response genes is associated with putative routes of resistance characterized by signatures of persistent IFN signaling, immune dysfunction, and mutations in antigen presentation genes which can be recapitulated in multiple murine models of acquired resistance to PD-(L)1 blockade after in vitro IFNγ treatment. Acquired resistance to PD-(L)1 blockade in NSCLC is associated with an ongoing, but altered IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance may inform therapeutic strategies to effectively reprogram and reverse acquired resistance.

Intratumoral Escherichia Is Associated With Improved Survival to Single-Agent Immune Checkpoint Inhibition in Patients With Advanced Non-Small-Cell Lung Cancer.

PURPOSE: The impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients with non-small-cell lung cancer (NSCLC) is unknown. Preclinically, intratumoral Escherichia is associated with a proinflammatory tumor microenvironment and decreased metastases. We sought to determine whether intratumoral Escherichia is associated with outcome to ICI in patients with NSCLC. PATIENTS AND METHODS: We examined the intratumoral microbiome in 958 patients with advanced NSCLC treated with ICI by querying unmapped next-generation sequencing reads against a bacterial genome database. Putative environmental contaminants were filtered using no-template controls (n = 2,378). The impact of intratumoral Escherichia detection on overall survival (OS) was assessed using univariable and multivariable analyses. The findings were further validated in an external independent cohort of 772 patients. Escherichia fluorescence in situ hybridization (FISH) and transcriptomic profiling were performed. RESULTS: In the discovery cohort, read mapping to intratumoral Escherichia was associated with significantly longer OS (16 v 11 months; hazard ratio, 0.73 [95% CI, 0.59 to 0.92]; P = .0065) in patients treated with single-agent ICI, but not combination chemoimmunotherapy. The association with OS in the single-agent ICI cohort remained statistically significant in multivariable analysis adjusting for prognostic features including PD-L1 expression (P = .023). Analysis of an external validation cohort confirmed the association with improved OS in univariable and multivariable analyses of patients treated with single-agent ICI, and not in patients treated with chemoimmunotherapy. Escherichia localization within tumor cells was supported by coregistration of FISH staining and serial hematoxylin and eosin sections. Transcriptomic analysis correlated Escherichia-positive samples with expression signatures of immune cell infiltration. CONCLUSION: Read mapping to potential intratumoral Escherichia was associated with survival to single-agent ICI in two independent cohorts of patients with NSCLC.