Total neoadjuvant therapy in oesophageal and gastro-oesophageal junctional adenocarcinoma.

Adenocarcinoma of the oesophagus and gastro-oesophageal junction represent a large burden of cancer death in the Western World with an increasing incidence. In the past two decades, the overall survival of patients on a potentially curative treatment pathway has more than doubled due to the addition of perioperative oncological therapies to surgery. However, patients often fail to respond to oncological treatment or struggle to complete their treatment after surgery. In this review, we discuss the current evidence for total neoadjuvant therapy and options for assessment of treatment response.

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.

Multicenter, Prospective Cohort Study of Oesophageal Injuries and Related Clinical Outcomes (MUSOIC study).

OBJECTIVE: To identify prognostic factors associated with 90-day mortality in patients with oesophageal perforation (OP), and characterize the specific timeline from presentation to intervention, and its relation to mortality. BACKGROUND: OP is a rare gastro-intestinal surgical emergency with a high mortality rate. However, there is no updated evidence on its outcomes in the context of centralized esophago-gastric services; updated consensus guidelines; and novel non-surgical treatment strategies. METHODS: A multi-center, prospective cohort study involving eight high-volume esophago-gastric centers (January 2016 to December 2020) was undertaken. The primary outcome measure was 90-day mortality. Secondary measures included length of hospital and ICU stay, and complications requiring re-intervention or re-admission. Mortality model training was performed using random forest, support-vector machines, and logistic regression with and without elastic net regularisation. Chronological analysis was performed by examining each patient's journey timepoint with reference to symptom onset. RESULTS: The mortality rate for 369 patients included was 18.9%. Patients treated conservatively, endoscopically, surgically, or combined approaches had mortality rates of 24.1%, 23.7%, 8.7%, and 18.2%, respectively. The predictive variables for mortality were Charlson comorbidity index, haemoglobin count, leucocyte count, creatinine levels, cause of perforation, presence of cancer, hospital transfer, CT findings, whether a contrast swallow was performed, and intervention type. Stepwise interval model showed that time to diagnosis was the most significant contributor to mortality. CONCLUSIONS: Non-surgical strategies have better outcomes and may be preferred in selected cohorts to manage perforations. Outcomes can be significantly improved through better risk-stratification based on afore-mentioned modifiable risk factors.

Results of the ARROW survey of anti-reflux practice in the United Kingdom.

Gastro-esophageal reflux disease (GERD) is a common, significant health burden. United Kingdom guidance states that surgery should be considered for patients with a diagnosis of GERD not suitable for long-term acid suppression. There is no consensus on many aspects of patient pathways and optimal surgical technique, and an absence of information on how patients are currently selected for surgery. Further detail on the delivery of anti-reflux surgery (ARS) is required. A United Kingdom-wide survey was designed to gather surgeon opinion regarding pre-, peri- and post-operative practice of ARS. Responses were received from 155 surgeons at 57 institutions. Most agreed that endoscopy (99%), 24-hour pH monitoring (83%) and esophageal manometry (83%) were essential investigations prior to surgery. Of 57 units, 30 (53%) had access to a multidisciplinary team to discuss cases; case-loads were higher in those units (median 50 vs. 30, P < 0.024). The most popular form of fundoplication was a Nissen posterior 360° (75% of surgeons), followed by a posterior 270° Toupet (48%). Only seven surgeons stated they had no upper limit of body mass index prior to surgery. A total of 46% of respondents maintain a database of their practice and less than a fifth routinely record quality of life scores before (19%) or after (14%) surgery. While there are areas of consensus, a lack of evidence to support workup, intervention and outcome evaluation is reflected in the variability of practice. ARS patients are not receiving the same level of evidence-based care as other patient groups.

Impact of postoperative chemotherapy on survival for oesophagogastric adenocarcinoma after preoperative chemotherapy and surgery.

BACKGROUND: Perioperative chemotherapy is widely used in the treatment of oesophagogastric adenocarcinoma (OGAC) with a substantial survival benefit over surgery alone. However, the postoperative part of these regimens is given in less than half of patients, reflecting uncertainty among clinicians about its benefit and poor postoperative patient fitness. This study estimated the effect of postoperative chemotherapy after surgery for OGAC using a large population-based data set. METHODS: Patients with adenocarcinoma of the oesophagus, gastro-oesophageal junction or stomach diagnosed between 2012 and 2018, who underwent preoperative chemotherapy followed by surgery, were identified from a national-level audit in England and Wales. Postoperative therapy was defined as the receipt of systemic chemotherapy within 90 days of surgery. The effectiveness of postoperative chemotherapy compared with observation was estimated using inverse propensity treatment weighting. RESULTS: Postoperative chemotherapy was given to 1593 of 4139 patients (38.5 per cent) included in the study. Almost all patients received platinum-based triplet regimens (4004 patients, 96.7 per cent), with FLOT used in 3.3 per cent. Patients who received postoperative chemotherapy were younger, with a lower ASA grade, and were less likely to have surgical complications, with similar tumour characteristics. After weighting, the median survival time after postoperative chemotherapy was 62.7 months compared with 50.4 months without chemotherapy (hazard ratio 0.84, 95 per cent c.i. 0.77 to 0.94; P = 0.001). CONCLUSION: This study has shown that postoperative chemotherapy improves overall survival in patients with OGAC treated with preoperative chemotherapy and surgery.

Association of Upper Gastrointestinal Surgery of Great Britain and Ireland (AUGIS)/Perioperative Quality Initiative (POQI) consensus statement on intraoperative and postoperative interventions to reduce pulmonary complications after oesophagectomy.

BACKGROUND: Pulmonary complications are the most common morbidity after oesophagectomy, contributing to mortality and prolonged postoperative recovery, and have a negative impact on health-related quality of life. A variety of single or bundled interventions in the perioperative setting have been developed to reduce the incidence of pulmonary complications. Significant variation in practice exists across the UK. The aim of this modified Delphi consensus was to deliver clear evidence-based consensus recommendations regarding intraoperative and postoperative care that may reduce pulmonary complications after oesophagectomy. METHODS: With input from a multidisciplinary group of 23 experts in the perioperative management of patients undergoing surgery for oesophageal cancer, a modified Delphi method was employed. Following an initial systematic review of relevant literature, a range of anaesthetic, surgical, and postoperative care interventions were identified. These were then discussed during a two-part virtual conference. Recommendation statements were drafted, refined, and agreed by all attendees. The level of evidence supporting each statement was considered. RESULTS: Consensus was reached on 12 statements on topics including operative approach, pyloric drainage strategies, intraoperative fluid and ventilation strategies, perioperative analgesia, postoperative feeding plans, and physiotherapy interventions. Seven additional questions concerning the perioperative management of patients undergoing oesophagectomy were highlighted to guide future research. CONCLUSION: Clear consensus recommendations regarding intraoperative and postoperative interventions that may reduce pulmonary complications after oesophagectomy are presented.

ELEVATE - evaluating Temozolomide and Nivolumab in patients with advanced unresectable previously treated oesophagogastric adenocarcinoma with MGMT methylation: study protocol for a single arm phase II trial.

BACKGROUND: For patients with oesophagogastric adenocarcinoma, surgery is the only curative option and despite the use of multimodality therapy, which combines it with chemotherapy and/or radiotherapy, more than 50% of patients will relapse and die. Many UK patients present with advanced disease which is already inoperable or metastatic at diagnosis. For these patients, standard care chemotherapy only offers them survival of less than a year. Nivolumab, a checkpoint blockade inhibitor, has been found to work in some advanced cancers. It is proposed, for those where immunotherapy hasn't worked, that these immunologically evasive tumours need to be sensitized to immunotherapy drugs to allow them to act. METHODS: ELEVATE is a single arm phase II trial testing the overall response to nivolumab following temozolomide treatment in patients with advanced unresectable previously treated adenocarcinoma which is O6-methylguanine-DNA-methyltransferase (MGMT) methylated. 18 patients are being recruited from UK secondary care sites. To be eligible, participants must have been treated with at least 3 months of platinum and fluoropyrimidine chemotherapy. Participants will receive 50 mg/m2 temozolomide continuously for 3 months. If their disease progresses during the 3 months, they will stop temozolomide and start nivolumab at a dose of 240mg every 2 weeks. If there is no progression after 3 months the participant will continue taking temozolomide in combination with nivolumab. All treatment will stop once the participant progresses on nivolumab. The primary endpoint is the best overall response to nivolumab, using both Response Evaluation Criteria in Solid Tumours version 1.1 and immunotherapy modified Response Evaluation Criteria in Solid Tumours. Secondary endpoints include progression-free survival, overall survival, and quality of life. DISCUSSION: ELEVATE will provide evidence for whether giving nivolumab after temozolomide in patients with previously treated advanced oesophagogastric adenocarcinoma is safe and biologically effective prior to future randomised trials. TRIAL REGISTRATIONS: EudraCT Number: 2020-004771-41 (issued 01 October 2020); ISCRTN11398887 (registered 14 July 2021).

Evaluation of postoperative surveillance strategies for esophago-gastric cancers in the UK and Ireland.

Esophago-gastric malignancies are associated with a high recurrence rate; yet there is a lack of evidence to inform guidelines for the standardization and structure of postoperative surveillance after curatively intended treatment. This study aimed to capture the variation in postoperative surveillance strategies across the UK and Ireland, and enquire the opinions and beliefs around surveillance from practicing clinicians. A web-based survey consisting of 40 questions was sent to surgeons or allied health professionals performing or involved in surgical care for esophago-gastric cancers at high-volume centers in the UK. Respondents from each center completed the survey on what best represented their center. The first section of the survey evaluated the timing and components of follow-ups, and their variation between centers. The second section evaluated respondents perspective on how surveillance can be structured. Thirty-five respondents from 27 centers consisting 28 consultants, 6 senior trainees and 1 specialist nurse had completed the questionnaire; 45.7% of responders arranged clinical follow-up at 2-4 weeks. Twenty responders had a specific postoperative surveillance protocol for their patients. Of these, 31.4% had a standardized protocol for all patients, while 25.7% tailored it to patient needs. Patient preference, comorbidities and chance of recurrence were considered as major factors for necessitating more intense surveillance than currently practiced. There is a significant variation in how patients are monitored after surgery between centers in the UK. Randomized controlled trials are necessary to link surveillance strategies to both survival outcomes and quality of life of patients and to evaluate the prognostic value of different postoperative surveillance strategies.

A modified Delphi process to establish future research priorities in malignant oesophagogastric surgery.

BACKGROUND: With rapid advancement in the genomics of oesophagogastric (OG) cancer and raised expectations in clinical outcomes from patients and clinicians alike there is a clear need to determine the current research priorities in OG cancer surgery. The aim of our study was to use a modified Delphi process to determine the research priorities among OG cancer surgeons in the United Kingdom. METHODS: Delphi methodology may be utilised to develop consensus opinion amongst a group of experts. Members of the Association of Upper Gastrointestinal Surgeons of Great Britain and Ireland were invited to submit individual research questions via an online survey (phase I). Two rounds of prioritisation by multidisciplinary expert healthcare professionals (phase II and III) were completed to determine a final list of high priority research questions. All questions submitted and subsequently ranked were analysed on an anonymised basis. RESULTS: In total, 427 questions were submitted in phase I and 75 with an OG cancer focus were taken forward for prioritisation in phase II. Phase III produced a final list of 12 high priority questions with an emphasis on tailored or personalised treatment strategies in OG cancer surgery. CONCLUSION: A modified Delphi process produced a list of 12 high priority research questions in OG cancer surgery. Future studies and awards from funding bodies should reflect this consensus list of prioritised questions in the interest of improving patient care and encouraging collaborative research across multiple centres.

Whole Genome Methylation Analysis of Nondysplastic Barrett Esophagus that Progresses to Invasive Cancer.

OBJECTIVE: To investigate differences in methylation between patients with nondysplastic Barrett esophagus who progress to invasive adenocarcinoma and those who do not. BACKGROUND: Identifying patients with nondysplastic Barrett esophagus who progress to invasive adenocarcinoma remains a challenge. Previous studies have demonstrated the potential utility of epigenetic markers for identifying this group. METHODS: A whole genome methylation interrogation using the Illumina HumanMethylation 450 array of patients with nondysplastic Barrett esophagus who either develop adenocarcinoma or remain static, with validation of findings by bisulfite pyrosequencing. RESULTS: In all, 12 patients with "progressive" versus 12 with "nonprogressive" nondysplastic Barrett esophagus were analyzed via methylation array. Forty-four methylation markers were identified that may be able to discriminate between nondysplastic Barrett esophagus that either progress to adenocarcinoma or remain static. Hypomethylation of the recently identified tumor suppressor OR3A4 (probe cg09890332) validated in a separate cohort of samples (median methylation in progressors 67.8% vs 96.7% in nonprogressors; P = 0.0001, z = 3.85, Wilcoxon rank-sum test) and was associated with the progression to adenocarcinoma. There were no differences in copy number between the 2 groups, but a global trend towards hypomethylation in the progressor group was observed. CONCLUSION: Hypomethylation of OR3A4 has the ability to risk stratify the patient with nondysplastic Barrett esophagus and may form the basis of a future surveillance program.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Androgen Receptor and Androgen-Responsive Gene FKBP5 Are Independent Prognostic Indicators for Esophageal Adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma is a male-dominant disease, but the role of androgens is unclear. AIMS: To examine the expression and clinical correlates of the androgen receptor (AR) and the androgen-responsive gene FK506-binding protein 5 (FKBP5) in esophageal adenocarcinoma. METHODS: Expression of AR and FKBP5 was determined by immunohistochemistry. The effect of the AR ligand 5α-dihydrotestosterone (DHT) on the expression of a panel of androgen-responsive genes was measured in AR-positive and AR-negative esophageal adenocarcinoma cell lines. Correlations in expression between androgen-responsive genes were analyzed in an independent cohort of esophageal adenocarcinoma tissues. RESULTS: There was AR staining in 75 of 77 cases (97 %), and FKBP5 staining in 49 (64 %), all of which had nuclear AR. Nuclear AR with FKBP5 expression was associated with decreased median survival (451 vs. 2800 days) and was an independent prognostic indicator (HR 2.894, 95 % CI 1.396­6.002, p = 0.0043) in multivariable Cox proportional hazards models. DHT induced a significant increase in expression of the androgen-responsive genes FKBP5, HMOX1, FBXO32, VEGFA, WNT5A, and KLK3 only in AR-positive cells in vitro. Significant correlations in expression were observed between these androgen-responsive genes in an independent cohort of esophageal adenocarcinoma tissues. CONCLUSION: Nuclear AR and expression of FKBP5 is associated with decreased survival in esophageal adenocarcinoma.

Pathologic Lymph Node Regression After Neoadjuvant Chemotherapy Predicts Recurrence and Survival in Esophageal Adenocarcinoma: A Multicenter Study in the United Kingdom.

PURPOSE: There is limited evidence regarding the prognostic effects of pathologic lymph node (LN) regression after neoadjuvant chemotherapy for esophageal adenocarcinoma, and a definition of LN response is lacking. This study aimed to evaluate how LN regression influences survival after surgery for esophageal adenocarcinoma. METHODS: Multicenter cohort study of patients with esophageal adenocarcinoma treated with neoadjuvant chemotherapy followed by surgical resection at five high-volume centers in the United Kingdom. LNs retrieved at esophagectomy were examined for chemotherapy response and given a LN regression score (LNRS)-LNRS 1, complete response; 2, <10% residual tumor; 3, 10%-50% residual tumor; 4, >50% residual tumor; and 5, no response. Survival analysis was performed using Cox regression adjusting for confounders including primary tumor regression. The discriminatory ability of different LN response classifications to predict survival was evaluated using Akaike information criterion and Harrell C-index. RESULTS: In total, 17,930 LNs from 763 patients were examined. LN response classified as complete LN response (LNRS 1 ≥1 LN, no residual tumor in any LN; n = 62, 8.1%), partial LN response (LNRS 1-3 ≥1 LN, residual tumor ≥1 LN; n = 155, 20.3%), poor/no LN response (LNRS 4-5; n = 303, 39.7%), or LN negative (no tumor/regression; n = 243, 31.8%) demonstrated superior discriminatory ability. Mortality was reduced in patients with complete LN response (hazard ratio [HR], 0.35; 95% CI, 0.22 to 0.56), partial LN response (HR, 0.72; 95% CI, 0.57 to 0.93) or negative LNs (HR, 0.32; 95% CI, 0.25 to 0.42) compared with those with poor/no LN response. Primary tumor regression and LN regression were discordant in 165 patients (21.9%). CONCLUSION: Pathologic LN regression after neoadjuvant chemotherapy was a strong prognostic factor and provides important information beyond pathologic TNM staging and primary tumor regression grading. LN regression should be included as standard in the pathologic reporting of esophagectomy specimens.

Nasogastric tube drainage and pyloric intervention after oesophageal resection: UK practice variation and effect on outcomes.

BACKGROUND: Over 1500 patients with oesophageal cancer undergo a resection in the UK each year. At surgery, patients commonly have a nasogastric tube (NGT) placed and may undergo a pyloric intervention. There is conflicting evidence on the use of both NGTs and pyloric interventions during oesophageal resections. We performed a national survey of oesophageal centres and assessed practice variation. MATERIAL AND METHODS: An electronic survey was distributed to all resection centres in England, Wales and Scotland. Variations in practice regarding NGTs and pyloric intervention were assessed, and compared to nationally reported centre volumes and length-of-stay data. RESULTS: Most centres (31/39, 79%) responded to the survey. All centres reported routine NGT use. The majority of centres (19/31, 61%) did not perform pyloric interventions. When used, surgical pyloroplasty was the most frequent strategy (8/31, 26%). Routine post-operative radiological assessment was utilised in 9/31 (29%) of centres. Criteria for NGT removal and dietary progression was highly variable, with every centre reporting different protocols. There were no significant differences in practice between high and low volume centres. There were also no trends seen when comparing centres above vs at-or-below the median length-of-stay. The majority (68%) of centres were willing to take part in a trial assessing NGT use and pyloric interventions. CONCLUSIONS: Pyloric intervention use varies widely, with no clear link to outcomes. NGT use remains standard practice despite evidence for safe omission. Surgeons require and recognise the need for a trial to assess requirement for NGTs and pyloric intervention after oesophageal resection.

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.

The Use of miRNAs in Predicting Response to Neoadjuvant Therapy in Oesophageal Cancer.

Oesophageal cancer (OC) is the ninth most common cancer worldwide. Patients receive neoadjuvant therapy (NAT) as standard of care, but less than 20% of patients with oesophageal adenocarcinoma (OAC) or a third of oesophageal squamous cell carcinoma (OSCC) patients, obtain a clinically meaningful response. Developing a method of determining a patient's response to NAT before treatment will allow rational treatment decisions to be made, thus improving patient outcome and quality of life. (1) Background: To determine the use and accuracy of microRNAs as biomarkers of response to NAT in patients with OAC or OSCC. (2) Methods: MEDLINE, EMBASE, Web of Science and the Cochrane library were searched to identify studies investigating microRNAs in treatment naïve biopsies to predict response to NAT in OC patients. (3) Results: A panel of 20 microRNAs were identified as predictors of good or poor response to NAT, from 15 studies. Specifically, miR-99b, miR-451 and miR-505 showed the strongest ability to predict response in OAC patients along with miR-193b in OSCC patients. (4) Conclusions: MicroRNAs are valuable biomarkers of response to NAT in OC. Research is needed to understand the effects different types of chemotherapy and chemoradiotherapy have on the predictive value of microRNAs; studies also require greater standardization in how response is defined.

The first international Delphi consensus statement on Laparoscopic Gastrointestinal surgery.

BACKGROUND: Laparoscopic surgery has almost replaced open surgery in many areas of Gastro-Intestinal (GI) surgery. There is currently no published expert consensus statement on the principles of laparoscopic GI surgery. This may have affected the training of new surgeons. This exercise aimed to achieve an expert consensus on important principles of laparoscopic GI surgery. METHODS: A committee of 38 international experts in laparoscopic GI surgery proposed and voted on 149 statements in two rounds following a strict modified Delphi protocol. RESULTS: A consensus was achieved on 133 statements after two rounds of voting. All experts agreed on tailoring the first port site to the patient, whereas 84.2% advised avoiding the umbilical area for pneumoperitoneum in patients who had a prior midline laparotomy. Moreover, 86.8% agreed on closing all 15 mm ports irrespective of the patient's body mass index. There was a 100% consensus on using cartridges of appropriate height for stapling, checking the doughnuts after using circular staplers, and keeping the vibrating blade of the ultrasonic energy device in view and away from vascular structures. An 84.2% advised avoiding drain insertion through a ≥10 mm port site as it increases the risk of port-site hernia. There was 94.7% consensus on adding laparoscopic retrieval bags to the operating count and ensuring any surgical specimen left inside for later removal is added to the operating count. CONCLUSION: Thirty-eight experts achieved a consensus on 133 statements concerning various aspects of laparoscopic GI Surgery. Increased awareness of these could facilitate training and improve patient outcomes.

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing.

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.

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.