Association of Upper Gastrointestinal Surgery of Great Britain and Ireland (AUGIS)/perioperative quality initiative (POQI) consensus statement on prehabilitation in oesophagogastric surgery.

BACKGROUND: Prehabilitation is safe, feasible and may improve a range of outcomes in patients with oesophago-gastric cancer (OGC). Recent studies have suggested the potential of prehabilitation to improve body composition, sarcopenia and physical fitness, reduce surgical complications and improve quality of life. Despite this, prehabilitation services are not offered throughout all OGC centres in the UK. Where prehabilitation is offered, delivery and definitions vary significantly, as do funding sources and access. METHODS: A professional association endorsed series of consensus meetings were conducted using a modified Delphi process developed by the Peri-Operative Quality Initiative (POQI) to identify and refine consensus statements relating to the development and delivery of prehabilitation services for OGC patients. Participants from a variety of disciplines were identified based on a track record of published studies in the field of prehabilitation and/or practice experience encompassing prehabilitation of OGC patients. Approval from the POQI board was obtained and independent supervision provided by POQI. RESULTS: A total of 20 statements were developed and agreed by 26 participants over a preliminary meeting and 2 semi-structured formal POQI meetings. Ten research themes were identified. In the case of one statement, consensus was not reached and the statement was recorded and developed into a research theme. A strong recommendation was made for the majority of the consensus statements (17 of 20). DISCUSSION: Consensus statements encompassing the interventions and outcomes of prehabilitation services in oesophago-gastric cancer surgery have been developed to inform the implementation of programmes.

Identification of neoantigens in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

The AUGIS Survival Predictor: Prediction of Long-Term and Conditional Survival After Esophagectomy Using Random Survival Forests.

OBJECTIVE: The aim of this study was to develop a predictive model for overall survival after esophagectomy using pre/postoperative clinical data and machine learning. SUMMARY BACKGROUND DATA: For patients with esophageal cancer, accurately predicting long-term survival after esophagectomy is challenging. This study investigated survival prediction after esophagectomy using a RandomSurvival Forest (RSF) model derived from routine data from a large, well-curated, national dataset. METHODS: Patients diagnosed with esophageal adenocarcinoma or squamous cell carcinoma between 2012 and 2018 in England and Wales who underwent an esophagectomy were included. Prediction models for overall survival were developed using the RSF method and Cox regression from 41 patient and disease characteristics. Calibration and discrimination (time-dependent area under the curve) were validated internally using bootstrap resampling. RESULTS: The study analyzed 6399 patients, with 2625 deaths during follow-up. Median follow-up was 41 months. Overall survival was 47.1% at 5 years. The final RSF model included 14 variables and had excellent discrimination with a 5-year time-dependent area under the receiver operator curve of 83.9% [95% confidence interval (CI) 82.6%-84.9%], compared to 82.3% (95% CI 81.1%-83.3%) for the Cox model. The most important variables were lymph node involvement, pT stage, circumferential resection margin involvement (tumor at < 1 mm from cut edge) and age. There was a wide range of survival estimates even within TNM staging groups, with quintiles of prediction within Stage 3b ranging from 12.2% to 44.7% survival at 5 years. CONCLUSIONS: An RSF model for long-term survival after esophagectomy exhibited excellent discrimination and well-calibrated predictions. At a patient level, it provides more accuracy than TNM staging alone and could help in the delivery of tailored treatment and follow-up.

Comparison of optimized methodologies for isolating nuclei from esophageal tissue.

Single-nuclei RNA sequencing allows single cell-based analysis in frozen tissue, ameliorating cell recovery biases associated with enzymatic dissociation methods. The authors present two optimized methods for isolating and sequencing nuclei from esophageal tissue using a commercial EZ and citric acid (CA)-based method. Despite high endogenous RNase activity, these protocols produced libraries of expected fragment length (average length EZ: 745 bp; CA: 1232 bp) with comparable complexity (median Transcript/Gene number, EZ: 496/254; CA: 483/256). CA nuclei showed a higher proportion of ribosomal gene reads, potentially reflecting co-isolation of nuclei and adherent ribosomes. The authors identified 11 cell lineages in the combined datasets, with differences in cell type recovery between the two methods, providing utility dependent on experimental needs.

Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Identification of dominant negative human immunodeficiency virus type 1 Vif mutants that interfere with the functional inactivation of APOBEC3G by virus-encoded Vif.

APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1 virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper-deoxyguanine-to-deoxyadenine mutations on positive-strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vif's ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies.

Stably expressed APOBEC3F has negligible antiviral activity.

APOBEC3F (A3F) is a member of the family of cytidine deaminases that is often coexpressed with APOBEC3G (A3G) in cells susceptible to HIV infection. A3F has been shown to have strong antiviral activity in transient-expression studies, and together with A3G, it is considered the most potent cytidine deaminase targeting HIV. Previous analyses suggested that the antiviral properties of A3F can be dissociated from its catalytic deaminase activity. We were able to confirm the deaminase-independent antiviral activity of exogenously expressed A3F; however, we also noted that exogenous expression was associated with very high A3F mRNA and protein levels. In analogy to our previous study of A3G, we produced stable HeLa cell lines constitutively expressing wild-type or deaminase-defective A3F at levels that were more in line with the levels of endogenous A3F in H9 cells. A3F expressed in stable HeLa cells was packaged into Vif-deficient viral particles with an efficiency similar to that of A3G and was properly targeted to the viral nucleoprotein complex. Surprisingly, however, neither wild-type nor deaminase-defective A3F inhibited HIV-1 infectivity. These results imply that the antiviral activity of endogenous A3F is negligible compared to that of A3G.

Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Neoadjuvant therapy followed by surgery is the standard of care for locally advanced esophageal adenocarcinoma (EAC). Unfortunately, response to neoadjuvant chemotherapy (NAC) is poor (20-37%), as is the overall survival benefit at five years (9%). The EAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in EAC, a comparative genomic analysis was performed in 65 patients with extensive clinical and pathological annotation using whole-genome sequencing (WGS). We defined response using Mandard Tumor Regression Grade (TRG), with responders classified as TRG1-2 (n = 27) and non-responders classified as TRG4-5 (n =38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs. 1.70/Mb, p = 0.036) and elevated copy number variation in non-responders (282 vs. 136/patient, p < 0.001). We identified copy number variants unique to each group in our cohort, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of note, NAV3 mutations were exclusively present in the non-responder group with a frequency of 22%. Thus, lower mutation burden, higher chromosomal instability and specific copy number alterations are associated with resistance to NAC.

Outcomes and survival following neoadjuvant chemotherapy versus neoadjuvant chemoradiotherapy for cancer of the esophagus: Inverse propensity score weighted analysis.

BACKGROUND: Esophageal cancer is increasingly common and carries a poor prognosis. The optimal treatment modality for locally advanced cancer is unknown, with current guidance recommending either neoadjuvant chemotherapy (CT) or chemoradiotherapy (CRT) followed by surgery. There is a lack of adequately powered trials comparing CT against CRT. We retrospectively compared CT versus CRT using a propensity score weighting approach. METHODS: Demographic, disease, treatment and outcome data were retrieved from a local database for patients who received neoadjuvant CT or CRT followed by surgery. Inverse probability of treatment weighting (IPTW) was used to balance groups using a propensity score-weighting approach. Groups were assessed for differences in postoperative outcomes and survival. Kaplan-Meier and non-parametric tests were used to compare survival and outcome data as appropriate. RESULTS: Data for 284 patients were retrieved. Following IPTW groups were well matched. No significant differences were seen for postoperative complications (CT 64.9% vs. CRT 63.3%, p = 0.807), including major complications (24.0% vs. 23.6%, p = 0.943) and anastomotic leak (7.8% vs. 5.6%, p = 0.526). Significantly higher rates of clinical regression and complete pathological response were seen following CRT (p = 0.002 for both). Rates of R0 resection were higher with CRT, CT 79.1% vs. CRT 93.1%, p = 0.006. There was no difference between groups for overall or disease-free survival. CONCLUSION: This study suggests that the significant improvements in local tumour response seen after neoadjuvant CRT compared to CT may not translate to different survival outcomes. However, it must be stressed that adequately powered prospective trials are still lacking.

Encapsidation of APOBEC3G into HIV-1 virions involves lipid raft association and does not correlate with APOBEC3G oligomerization.

BACKGROUND: The cellular cytidine deaminase APOBEC3G (A3G), when incorporated into the human immunodeficiency virus type 1 (HIV-1), renders viral particles non-infectious. We previously observed that mutation of a single cysteine residue of A3G (C100S) inhibited A3G packaging. In addition, several recent studies showed that mutation of tryptophan 127 (W127) and tyrosine 124 (Y124) inhibited A3G encapsidation suggesting that the N-terminal CDA constitutes a viral packaging signal in A3G. It was also reported that W127 and Y124 affect A3G oligomerization. RESULTS: Here we studied the mechanistic basis of the packaging defect of A3G W127A and Y124A mutants. Interestingly, cell fractionation studies revealed a strong correlation between encapsidation, lipid raft association, and genomic RNA binding of A3G. Surprisingly, the presence of a C-terminal epitope tag affected lipid raft association and encapsidation of the A3G W127A mutant but had no effect on wt A3G encapsidation, lipid raft association, and interaction with viral genomic RNA. Mutation of Y124 abolished A3G encapsidation irrespective of the presence or absence of an epitope tag. Contrasting a recent report, our co-immunoprecipitation studies failed to reveal a correlation between A3G oligomerization and A3G encapsidation. In fact, our W127A and Y124A mutants both retained the ability to oligomerize. CONCLUSION: Our results confirm that W127 and Y124 residues in A3G are important for encapsidation into HIV-1 virions and our data establish a novel correlation between genomic RNA binding, lipid raft association, and viral packaging of A3G. In contrast, we were unable to confirm a role of W127 and Y124 in A3G oligomerization and we thus failed to confirm a correlation between A3G oligomerization and virus encapsidation.