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The raw material for viral evolution is provided by intra-host mutations occurring during replication, transcription or post-transcription. Replication and transcription of Coronaviridae proceed through the synthesis of negative-sense 'antigenomes' acting as templates for positive-sense genomic and subgenomic RNA. Hence, mutations in the genomes of SARS-CoV-2 and other coronaviruses can occur during (and after) the synthesis of either negative-sense or positive-sense RNA, with potentially distinct patterns and consequences. We explored for the first time the mutational spectrum of SARS-CoV-2 (sub)genomic and anti(sub)genomic RNA. We use a high-quality deep sequencing dataset produced using a quantitative strand-aware sequencing method, controlled for artefacts and sequencing errors, and scrutinized for accurate detection of within-host diversity. The nucleotide differences between negative- and positive-sense strand consensus vary between patients and do not show dependence on age or sex. Similarities and differences in mutational patterns between within-host minor variants on the two RNA strands suggested strand-specific mutations or editing by host deaminases and oxidative damage. We observe generally neutral and slight negative selection on the negative strand, contrasting with purifying selection in ORF1a, ORF1b and S genes of the positive strand of the genome.

BACKGROUND: Immunotherapy has brought substantial benefit for patients with advanced non-small cell lung cancer (NSCLC); however, resistance may occur, of which oligoprogression is most common. There are no standard strategies to overcome acquired resistance, thus exploring potential effective approaches is critical. Our study evaluated the clinical outcome of combing stereotactic body radiotherapy (SBRT) with checkpoint inhibitors (CPIs) in oligoprogressive NSCLC. METHODS: We retrospectively reviewed patients with advanced NSCLC who received SBRT for oligoprogressive lesions after acquired resistance to CPIs in our hospital between January 2015 and January 2021. Acquired resistance was defined as initial complete/partial response (CR/PR) followed by progression/death. Oligo patterns of acquired resistance were defined as progression in ≤2 sites of disease. We evaluated the local control rate (LR), progression-free survival (PFS-PO), overall survival (OS-PO), and safety of combing SBRT after oligoprogression. RESULTS: Among 177 patients reviewed, 24 patients were included. Fifteen (62.5%) were diagnosed with adenocarcinoma, and 20 (83.3%) were with stage IV. Before oligoprogression, immunotherapy was used as first-line treatment in 16 (66.7%) patients, and 4 (16.7%) received monotherapy. After combing SBRT with CPIs, the median PFS-PO and OS-PO were 11 months (95% CI: 8–NA) and 34 months (95% CI: 19-NA). The median LC of 34 oligoprogressed lesions was 43 months (95% CI: 7.7–78.3). The 1- and 2-year LC rates were 100% and 81.8%, respectively. Patients with adenocarcinoma, lung immune prognostic index (LIPI) (≥1), and positive PD-L1 tended to achieve favorable survival benefits. CONCLUSIONS: We observed considerable benefit of local control and survival by combing SBRT in patients with oligoprogression after required resistance to CPIs in NSCLC. The adverse events are well managed. Our results suggest that combing SBRT with CPIs could be a potential strategy to overcome acquired resistance.