SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

The Impact of Racial Disparities on Outcome in Patients With Stage IIIC Endometrial Carcinoma: A Pooled Data Analysis.

OBJECTIVE: To report the impact of race on clinical outcomes in patients with stage IIIC endometrial carcinoma. MATERIALS AND METHODS: A retrospective multi-institutional study included 90 black and 568 non-black patients with stage IIIC endometrial carcinoma who received adjuvant chemotherapy and radiation treatments. Overall survival (OS) and recurrence-free survival (RFS) were calculated by the Kaplan-Meier method. Propensity score matching (PSM) was conducted. Statistical analyses were conducted using SPSS version 27. RESULTS: The Median follow-up was 45.3 months. black patients were significantly older, had more nonendometrioid histology, grade 3 tumors, and were more likely to have >1 positive paraaortic lymph nodes compared with non-black patients (all P <0.0001). The 5-year estimated OS and RFS rates were 45% and 47% compared with 77% and 68% for black patients versus non-black patients, respectively ( P <0.001). After PSM, the 2 groups were well-balanced for all prognostic covariates. The estimated hazard ratios of black versus non-black patients were 1.613 ( P value=0.045) for OS and 1.487 ( P value=0.116) for RFS. After PSM, black patients were more likely to receive the "Sandwich" approach and concurrent chemoradiotherapy compared with non-black ( P =0.013) patients. CONCLUSIONS: Black patients have higher rates of nonendometrioid histology, grade 3 tumors, and number of involved paraaortic lymph nodes, worse OS, and RFS, and were more likely to receive the "Sandwich" approach compared with non-black patients. After PSM, black patients had worse OS with a nonsignificant trend in RFS. Access to care, equitable inclusion on randomized trials, and identification of genomic differences are warranted to help mitigate disparities.

Development and Clinical Implementation of an Automated Virtual Integrative Planner for Radiation Therapy of Head and Neck Cancer.

Purpose: Head and neck (HN) radiation (RT) treatment planning is complex and resource intensive. Deviations and inconsistent plan quality significantly affect clinical outcomes. We sought to develop a novel automated virtual integrative (AVI) knowledge-based planning application to reduce planning time, increase consistency, and improve baseline quality. Methods and Materials: An in-house write-enabled script was developed from a library of 668 previously treated HN RT plans. Prospective hazard analysis was performed, and mitigation strategies were implemented before clinical release. The AVI-planner software was retrospectively validated in a cohort of 52 recent HN cases. A physician panel evaluated planning limitations during initial deployment, and feedback was enacted via software refinements. A final second set of plans was generated and evaluated. Kolmogorov-Smirnov test in addition to generalized evaluation metric and weighted experience score were used to compare normal tissue sparing between final AVI planner versus respective clinically treated and historically accepted plans. A t test was used to compare the interactive time, complexity, and monitor units for AVI planner versus manual optimization. Results: Initially, 86% of plans were acceptable to treat, with 10% minor and 4% major revisions or rejection recommended. Variability was noted in plan quality among HN subsites, with high initial quality for oropharynx and oral cavity plans. Plans needing revisions were comprised of sinonasal, nasopharynx, P-16 negative squamous cell carcinoma unknown primary, or cutaneous primary sites. Normal tissue sparing varied within subsites, but AVI planner significantly lowered mean larynx dose (median, 18.5 vs 19.7 Gy; P < .01) compared with clinical plans. AVI planner significantly reduced interactive optimization time (mean, 2 vs 85 minutes; P < .01). Conclusions: AVI planner reliably generated clinically acceptable RT plans for oral cavity, salivary, oropharynx, larynx, and hypopharynx cancers. Physician-driven iterative learning processes resulted in favorable evolution in HN RT plan quality with significant time savings and improved consistency using AVI planner.

Meta-Analysis of Candidate Surrogate End Points in Advanced Prostate Cancer.

Meta-Analysis of Surrogate End Points in Prostate CancerGharzai et al. report on the results of a meta-analysis, which concludes that unlike the case in localized prostate cancer, surrogate end points in advanced prostate cancer may not track overall survival.

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'- O methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'- O MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo , using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive to type I interferon (IFN-I) in vitro . Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'- O methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. Importance: Similar to other coronaviruses, disruption of SARS-CoV-2 NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo , our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1, but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'- O methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Zika Virus Infection Alters Gene Expression and Poly-Adenylation Patterns in Placental Cells.

Flaviviruses are small RNA viruses that are mainly transmitted via arthropod vectors and are found in tropic and sub-tropical regions. Most infections are asymptomatic (90-95%), but symptoms can be as severe as hemorrhagic fever and encephalitis. One recently emerged flavivirus is Zika virus (ZIKV), which was originally isolated from rhesus monkeys in Uganda roughly 70 years ago but has recently spread east, reaching S. America in 2015-2016. This outbreak was associated with the development of Guillain-Barré syndrome in adults and microcephaly in infants born to expectant mothers infected early in pregnancy. ZIKV must traverse the placenta to impact the development of the fetus, but the mechanisms responsible are unknown. While flaviviruses are known to disrupt splicing patterns in host cells, little is known about how flaviviruses such as ZIKV impact the alternative polyadenylation (APA) of host transcripts. This is important as APA is well-established as a mechanism in the regulation of mRNA metabolism and translation. Thus, we sought to characterize transcriptomic changes including APA in human placental (JEG3) cells in response to ZIKV infection using Poly(A)-ClickSeq (PAC-Seq). We used our differential Poly(A)-cluster (DPAC) analysis pipeline to characterize changes in differential gene expression, alternative poly-adenylation (APA) and the use of alternative terminal exons. We identified 98 upregulated genes and 28 downregulated genes. Pathway enrichment analysis indicated that many RNA processing and immune pathways were upregulated in ZIKV-infected JEG3 cells. We also updated DPAC to provide additional metrics of APA including the percentage-distal usage index (PDUI), which revealed that APA was extensive and the 3' UTRs of 229 genes were lengthened while 269 were shortened. We further found that there were 214 upregulated and 59 downregulated poly(A)-clusters (PACs). We extracted the nucleotide sequences surrounding these PACs and found that the canonical signals for poly-adenylation (binding site for poly-A binding protein (PABP) upstream and a GU-rich region down-stream of the PAC) were only enriched in the downregulated PACs. These results indicate that ZIKV infection makes JEG3 cells more permissive to non-canonical poly-adenylation signals.

Imaging response assessment for predicting outcomes after bioselection chemotherapy in larynx cancer: A secondary analysis of two prospective trials.

BACKGROUND AND PURPOSE: Bioselection with induction chemotherapy in larynx cancer is associated with excellent larynx preservation and disease-specific survival but requires visual inspection of the primary tumor. We retrospectively compare clinical and imaging response in bioselected patients to develop predictive models of surgeon-assessed response (SR), laryngectomy-free survival (LFS), and overall survival (OS) in bioselected patients. MATERIALS AND METHODS: In a secondary analysis of patients on two single-institution bioselection trials, model building used a regularized regression model (elastic-net) and applied nested cross-validation. Logistic regression-based model was used to predict SR and Cox proportional hazard-based models were used to predict LFS and OS. RESULTS: In 115 patients with a median age of 57 years, most patients had supraglottic tumors (73.0%) and T3/T4 disease (94.8%). Definitive treatment was chemoradiation in 76.5% and laryngectomy in 23.5%. Change in primary tumor (OR = 5.78, p < 0.001) and N-classification (OR = 1.64, p = 0.003) predicted SR (AUC 0.847). Change in tumor volume (HR = 0.58, p < 0.001) predicted LFS (c-index 0.724). N-classification (HR = 1.48, p = 0.04) and pre-chemotherapy tumor volume (HR = 1.30, p = 0.174) predicted OS (c-index 0.552). CONCLUSIONS: Imaging offers a non-invasive opportunity to evaluate response to induction chemotherapy, complementary to surgeon assessment. Further evaluation of approaches to bioselection that optimize generalizability of this paradigm are needed, and clinical trials utilizing imaging to predict outcomes including LFS are warranted.

Covariation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa.

Adaptation of viruses to their environments occurs through the acquisition of both novel single-nucleotide variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

Does Prophylactic Paraortic Lymph Node Irradiation Improve Outcomes in Women With Stage IIIC1 Endometrial Carcinoma?

PURPOSE: To evaluate the impact of prophylactic paraortic lymph node (PALN) radiation therapy (RT) on clinical outcomes in patients with International Federation of Obstetrics and Gynecology 2018 stage IIIC1 endometrial cancer (EC). METHODS AND MATERIALS: A multi-institutional retrospective study included patients with International Federation of Obstetrics and Gynecology 2018 stage IIIC1 EC lymph node assessment, status postsurgical staging, followed by adjuvant chemotherapy and RT using various sequencing regimens. Overall survival (OS) and recurrence-free survival (RFS) rates were estimated by the Kaplan-Meier method. Univariable and multivariable analysis were performed by Cox proportional hazard models for RFS/OS. In addition, propensity score matching was used to estimate the effect of the radiation field extent on survival outcomes. RESULTS: A total of 378 patients were included, with a median follow-up of 45.8 months. Pelvic RT was delivered to 286 patients, and 92 patients received pelvic and PALN RT. The estimated OS and RFS rates at 5 years for the entire cohort were 80% and 69%, respectively. There was no difference in the 5-year OS (77% vs 87%, P = .47) and RFS rates (67% vs 70%, P = .78) between patients treated with pelvic RT and those treated with pelvic and prophylactic PALN RT, respectively. After propensity score matching, the estimated hazard ratios (HRs) of prophylactic PALN RT versus pelvic RT were 1.50 (95% confidence interval, 0.71-3.19; P = .28) for OS and 1.24 (95% confidence interval, 0.64-2.42; P = .51) for RFS, suggesting that prophylactic PALN RT does not improve survival outcomes. Distant recurrence was the most common site of first recurrence, and the extent of RT field was not associated with the site of first recurrence (P = .79). CONCLUSIONS: Prophylactic PALN RT was not significantly associated with improved survival outcomes in stage IIIC1 EC. Distant metastasis remains the most common site of failure despite routine use of systemic chemotherapy. New therapeutic approaches are necessary to optimize the outcomes for women with stage IIIC1 EC.

Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes.

Tau protein is a known contributor in several neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). It is well-established that tau forms pathological aggregates and fibrils in these diseases. Tau has been observed within the nuclei of neurons, but there is a gap in understanding regarding the mechanism by which tau modulates transcription. We are interested in the P301L mutation of tau, which has been associated with FTD and increased tau aggregation. Our study utilized tau-inducible HEK (iHEK) cells to reveal that WT and P301L tau distinctively alter the transcription and alternative polyadenylation (APA) profiles of numerous nuclear precursors mRNAs, which then translate to form proteins involved in chromatin remodeling and splicing. We isolated total mRNA before and after over-expressing tau and then performed Poly(A)-ClickSeq (PAC-Seq) to characterize mRNA expression and APA profiles. We characterized changes in Gene Ontology (GO) pathways using EnrichR and Gene Set Enrichment Analysis (GSEA). We observed that P301L tau up-regulates genes associated with reactive oxygen species responsiveness as well as genes involved in dendrite, microtubule, and nuclear body/speckle formation. The number of genes regulated by WT tau is greater than the mutant form, which indicates that the P301L mutation causes loss-of-function at the transcriptional level. WT tau up-regulates genes contributing to cytoskeleton-dependent intracellular transport, microglial activation, microtubule and nuclear chromatin organization, formation of nuclear bodies and speckles. Interestingly, both WT and P301L tau commonly down-regulate genes responsible for ubiquitin-proteosome system. In addition, WT tau significantly down-regulates several genes implicated in chromatin remodeling and nucleosome organization. Although there are limitations inherent to the model systems used, this study will improve understanding regarding the nuclear impact of tau at the transcriptional and post-transcriptional level. This study also illustrates the potential impact of P301L tau on the human brain genome during early phases of pathogenesis.

RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3'-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. RBFOX2-mediated APA modulates mRNA levels and/or isoform expression of a collection of genes, including contractile and mitochondrial genes. Depletion of RBFOX2 adversely affects mitochondrial health in myoblasts, correlating with disrupted APA of mitochondrial gene Slc25a4. Mechanistically, RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site, enforcing the use of the proximal polyadenylation site. In sum, our results unveil a role for RBFOX2 in fine-tuning expression of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Contemporary Practice Patterns for Palliative Radiation Therapy of Bone Metastases: Impact of a Quality Improvement Project on Extended Fractionation.

PURPOSE: Radiation therapy effectively palliates bone metastases, although variability exists in practice patterns. National recommendations advocate against using extended fractionation (EF) with courses greater than 10 fractions. We previously reported EF use of 14.8%. We analyzed practice patterns within a statewide quality consortium to assess EF use in a larger patient population after implementation of a quality measure focused on reducing EF. METHODS AND MATERIALS: Patients treated for bone metastases within a statewide radiation oncology quality consortium were prospectively enrolled from March 2018 through October 2020. The EF quality metric was implemented March 1, 2018. Data on patient, physician, and facility characteristics; fractionation schedules; and treatment planning and delivery techniques were collected. Multivariable binary logistic regression was used to assess EF. RESULTS: Twenty-eight facilities enrolled 1445 consecutive patients treated with 1934 plans. The median number of treatment plans per facility was 52 (range, 7-307). Sixty different fractionation schedules were used. EF was delivered in 3.4% of plans. Initially, EF use was lower than expected and remained low over time. Significant predictors for EF use included complicated metastasis (odds ratio [OR], 2.04; 95% confidence interval [CI], 1.04-4.02; P = .04), lack of associated central nervous system or visceral disease (OR, 2.27; 95% CI, 1.2-4.2; P = .01), nonteaching versus teaching facilities (OR, 8.97; 95% CI, 2.1-38.5; P < .01), and treating physicians with more years in practice (OR, 12.82; 95% CI, 3.9-42.4; P < .01). CONCLUSIONS: Within a large, prospective population-based data set, fractionation schedules for palliative radiation therapy of bone metastases remain highly variable. Resource-intensive treatments including EF persist, although EF use was low after implementation of a quality measure. Complicated metastases, lack of central nervous system or visceral disease, and treatment at nonteaching facilities or by physicians with more years in practice significantly predict use of EF. These results support ongoing efforts to more clearly understand and address barriers to high-value radiation approaches in the palliative setting.

Patient Burden with Current Surveillance Paradigm and Factors Associated with Interest in Altered Surveillance for Early Stage HPV-Related Oropharyngeal Cancer.

INTRODUCTION: Optimal surveillance paradigms for survivors of early stage human papillomavirus (HPV)-related oropharyngeal cancer are not well defined. This study aimed to characterize patient interest in and factors associated with an altered surveillance paradigm. MATERIALS AND METHODS: We surveyed patients with Stage I or II HPV-related oropharyngeal cancer treated at a tertiary care institution from 2016 to 2019. Primary outcomes were descriptive assessment of patient knowledge, interest in altered surveillance, burdens of in-person appointments, and priorities for surveillance visits. Ordinal regression was used to identify correlates of interest in altered surveillance. RESULTS: Sixty-seven patients completed surveys from February to April 2020 at a median of 21 months since completing definitive treatment. A majority (61%) of patients were interested in a surveillance approach that decreased in-person clinic visits. Patients who self-identified as medical maximizers, had higher worry of cancer recurrence, or were in long-term relationships were less likely to be interested. Patients reported significant burdens associated with surveillance visits, including driving distance, time off work, and nonmedical costs. Patients were most concerned with discussing cancer recurrence (76%), physical quality of life (70%), mortality (61%), and mental quality of life (52%) with their providers at follow-up visits. CONCLUSION: Patients with early stage HPV-related oropharyngeal cancers are interested in altered surveillance approaches, experience significant burdens related to surveillance visits, and have concerns that are not well addressed with current surveillance approaches, including physical and mental quality of life. Optimized surveillance approaches should incorporate patient priorities and minimize associated burdens. IMPLICATIONS FOR PRACTICE: The number of patients with HPV-related oropharyngeal cancers is increasing, and numerous clinical trials are investigating novel approaches to treating these good-prognosis patients. There has been limited work assessing optimal surveillance paradigms in these patients. Patients experience significant appointment-related burdens and have concerns such as physical and mental quality of life. Additionally, patients with early stage HPV-related oropharyngeal cancers express interest in altered surveillance approaches that decrease in-person clinic visits. Optimization of surveillance paradigms to promote broader survivorship care in clinical practice is needed.

A Multi-Institutional Analysis of Adjuvant Chemotherapy and Radiation Sequence in Women With Stage IIIC Endometrial Cancer.

PURPOSE: Our purpose was to evaluate the effect of sequence and type of adjuvant therapy for patients with stage IIIC endometrial carcinoma (EC) on outcomes. METHODS AND MATERIALS: In a multi-institutional retrospective cohort study, patients with stage IIIC EC who had surgical staging and received both adjuvant chemotherapy and radiation therapy (RT) were included. Adjuvant treatment regimens were classified as adjuvant chemotherapy followed by sequential RT (upfront chemo), which was predominant sequence; RT with concurrent chemotherapy followed by chemotherapy (concurrent); systemic chemotherapy before and after RT (sandwich); adjuvant RT followed by chemotherapy (upfront RT); or chemotherapy concurrent with vaginal cuff brachytherapy alone (chemo-brachy). Overall survival (OS) and recurrence-free survival (RFS) rates were estimated by the Kaplan-Meier method. RESULTS: A total of 686 eligible patients were included with a median follow-up of 45.3 months. The estimated 5-year OS and RFS rates were 74% and 66%, respectively. The sequence and type of adjuvant therapy were not correlated with OS or RFS (adjusted P = .68 and .84, respectively). On multivariate analysis, black race, nonendometrioid histology, grade 3 tumor, stage IIIC2, and presence of adnexal and cervical involvement were associated with worse OS and RFS (all P < .05). Regardless of the sequence of treatment, the most common site of first recurrence was distant metastasis (20.1%). Vaginal only, pelvic only, and paraortic lymph node (PALN) recurrences occurred in 11 (1.6%),15 (2.2 %), and 43 (6.3 %) patients, respectively. Brachytherapy alone was associated with a higher rate of PALN recurrence (15%) compared with external beam radiation therapy (5%) P < .0001. CONCLUSIONS: The sequence and type of combined adjuvant therapy did not affect OS or RFS rates. Brachytherapy alone was associated with a higher rate of PALN recurrence, emphasizing the role of nodal radiation for stage IIIC EC. The vast proportion of recurrences were distant despite systemic chemotherapy, highlighting the need for novel regimens.

Intermediate clinical endpoints for surrogacy in localised prostate cancer: an aggregate meta-analysis.

BACKGROUND: The international Intermediate Clinical Endpoints in Cancer of the Prostate working group has established metastasis-free survival as a surrogate for overall survival in localised prostate cancer based on the findings of 19 predominantly radiotherapy-based trials. We sought to comprehensively assess aggregate trial-level performance of commonly reported intermediate clinical endpoints across all randomised trials in localised prostate cancer. METHODS: For this meta-analysis, we searched PubMed for all trials in localised or biochemically recurrent prostate cancer published between Jan 1, 1970, and Jan 15, 2020. Eligible trials had to be randomised, therapeutic, reporting overall survival and at least one intermediate clinical endpoint, and with a sample size of at least 70 participants. Trials of metastatic disease were excluded. Intermediate clinical endpoints included biochemical failure, local failure, distant metastases, biochemical failure-free survival, progression-free survival, and metastasis-free survival. Candidacy for surrogacy was assessed using the second condition of the meta-analytical approach (ie, correlation of the treatment effect of the intermediate clinical endpoint and overall survival), using R2 weighted by the inverse variance of the log intermediate clinical endpoint hazard ratio. The intermediate clinical endpoint was deemed to be a surrogate for overall survival if R2 was 0·7 or greater. FINDINGS: 75 trials (53 631 patients) were included in our analysis. Median follow-up was 9·1 years (IQR 5·7-10·6). Biochemical failure (R2 0·38 [95% CI 0·11-0·64]), biochemical failure-free survival (R2 0·12 [0·0030-0·33]), biochemical failure and clinical failure (R2 0·28 [0·0045-0·65]), and local failure (R2 0·085 [0·00-0·37]) correlated poorly with overall survival. Progression-free survival (R2 0·46 [95% CI 0·22-0·67]) showed moderate correlation with overall survival, and metastasis-free survival (R2 0·78 [0·59-0·89]) correlated strongly. INTERPRETATION: Intermediate clinical endpoints based on biochemical and local failure did not meet the second condition of the meta-analytical approach and are not surrogate endpoints for overall survival in localised prostate cancer. Our findings validate metastasis-free survival as the only identified surrogate endpoint for overall survival to date. FUNDING: Prostate Cancer Foundation and National Institutes of Health.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene.

Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.

Association of Presalvage Radiotherapy PSA Levels After Prostatectomy With Outcomes of Long-term Antiandrogen Therapy in Men With Prostate Cancer.

Importance: In men with recurrent prostate cancer, addition of long-term antiandrogen therapy to salvage radiotherapy (SRT) was associated with overall survival (OS) in the NRG/RTOG 9601 study. However, hormone therapy has associated morbidity, and there are no validated predictive biomarkers to identify which patients derive most benefit from treatment. Objective: To examine the role of pre-SRT prostate-specific antigen (PSA) levels to personalize hormone therapy use with SRT. Interventions: Men were randomized to SRT plus high-dose nonsteroidal antiandrogen (bicalutamide, 150 mg/d) or placebo for 2 years. Design, Setting, and Participants: In this secondary analysis of the multicenter RTOG 9601 double-blind, placebo-controlled randomized clinical trial conducted from 1998 to 2003 by a multinational cooperative group, men with a positive surgical margin or pathologic T3 disease after radical prostatectomy with pre-SRT PSA of 0.2 to 4.0 ng/mL were included. Analysis was performed between March 4, 2019, and December 20, 2019. Main Outcomes and Measures: The primary outcome was overall survival (OS). Secondary end points included distant metastasis (DM), other-cause mortality (OCM), and grades 3 to 5 cardiac and neurologic toxic effects. Subgroup analyses were performed using the protocol-specified PSA stratification variable (1.5 ng/mL) and additional PSA cut points, including test for interaction. Competing risk analyses were performed for DM and other-cause mortality (OCM). Results: Overall, 760 men with PSA elevation after radical prostatectomy for prostate cancer were included. The median (range) age of particpants was 65 (40-83) years. Antiandrogen assignment was associated with an OS benefit in the PSA stratum greater than 1.5 ng/mL (n = 118) with a 25% 12-year absolute benefit (hazard ratio [HR], 0.45; 95% CI, 0.25-0.81), but not in the PSA of 1.5 ng/mL or less stratum (n = 642) (1% 12-year absolute difference; HR, 0.87; 95% CI, 0.66-1.16). In a subanalysis of men with PSA of 0.61 to 1.5 (n = 253), there was an OS benefit associated with antiandrogen assignment (HR, 0.61; 95% CI, 0.39-0.94). In those receiving early SRT (PSA ≤0.6 ng/mL, n = 389), there was no improvement in OS (HR, 1.16; 95% CI, 0.79-1.70), an increased OCM hazard (subdistribution HR, 1.94; 95% CI, 1.17-3.20; P = .01), and an increased odds of late grades 3 to 5 cardiac and neurologic toxic effects (odds ratio, 3.57; 95% CI, 1.09-15.97; P = .05). Conclusions and Relevance: These results suggest that pre-SRT PSA level may be a prognostic biomarker for outcomes of antiandrogen treatment with SRT. In patients receiving late SRT (PSA >0.6 ng/mL, hormone therapy was associated with improved outcomes. In men receiving early SRT (PSA ≤0.6 ng/mL), long-term antiandrogen treatment was not associated with improved OS. Future randomized clinical trials are needed to determine hormonal therapy benefit in this population. Trial Registration: ClinicalTrials.gov Identifier: NCT00002874.