A Practical Approach to Interpreting Circulating Tumor DNA in the Management of Gastrointestinal Cancers.

BACKGROUND: There is accumulating evidence supporting the clinical use of circulating tumor DNA (ctDNA) in solid tumors, especially in different types of gastrointestinal cancer. As such, appraisal of the current and potential clinical utility of ctDNA is needed to guide clinicians in decision-making to facilitate its general applicability. CONTENT: In this review, we firstly discuss considerations surrounding specimen collection, processing, storage, and analysis, which affect reporting and interpretation of results. Secondly, we evaluate a selection of studies on colorectal, esophago-gastric, and pancreatic cancer to determine the level of evidence for the use of ctDNA in disease screening, detection of molecular residual disease (MRD) and disease recurrence during surveillance, assessment of therapy response, and guiding targeted therapy. Lastly, we highlight current limitations in the clinical utility of ctDNA and future directions. SUMMARY: Current evidence of ctDNA in gastrointestinal cancer is promising but varies depending on its specific clinical role and cancer type. Larger prospective trials are needed to validate different aspects of ctDNA clinical utility, and standardization of collection protocols, analytical assays, and reporting guidelines should be considered to facilitate its wider applicability.

The prognostic impact of peritoneal tumour DNA in gastrointestinal and gynaecological malignancies: a systematic review.

Peritoneal metastases from various abdominal cancer types are common and carry poor prognosis. The presence of peritoneal disease upstages cancer diagnosis and alters disease trajectory and treatment pathway in many cancer types. Therefore, accurate and timely detection of peritoneal disease is crucial. The current practice of diagnostic laparoscopy and peritoneal lavage cytology (PLC) in detecting peritoneal disease has variable sensitivity. The significant proportion of peritoneal recurrence seen during follow-up in patients where initial PLC was negative indicates the ongoing need for a better diagnostic tool for detecting clinically occult peritoneal disease, especially peritoneal micro-metastases. Advancement in liquid biopsy has allowed the development and use of peritoneal tumour DNA (ptDNA) as a cancer-specific biomarker within the peritoneum, and the presence of ptDNA may be a surrogate marker for early peritoneal metastases. A growing body of literature on ptDNA in different cancer types portends promising results. Here, we conduct a systematic review to evaluate the prognostic impact of ptDNA in various cancer types and discuss its potential future clinical applications, with a focus on gastrointestinal and gynaecological malignancies.

Tumor-Infiltrating Neutrophils after Neoadjuvant Therapy are Associated with Poor Prognosis in Esophageal Cancer.

BACKGROUND: In esophageal cancer (EC), there is a paucity of knowledge regarding the interplay between the tumor immune microenvironment and response to neoadjuvant treatment and, therefore, which factors may influence outcomes. Thus, our goal was to investigate the changes in the immune microenvironment with neoadjuvant treatment in EC by assessing the expression of immune related genes and their association with prognosis. METHODS: We examined the transcriptome of paired pre- and post-neoadjuvant treated EC specimens. Based on these findings, we validated the presence of tumor-infiltrating neutrophils using CD15+ immunohistochemistry in a discovery cohort of patients with residual pathologic disease. We developed a nomogram as a predictor of progression-free survival (PFS) incorporating the variables CD15+ cell count, tumor regression grade, and tumor grade. RESULTS: After neoadjuvant treatment, there was an increase in genes related to myeloid cell differentiation and a poor prognosis associated with high neutrophil (CD15+) counts. Our nomogram incorporating CD15+ cell count was predictive of PFS with a C-index of 0.80 (95% confidence interval [CI] 0.68-0.9) and a concordance probability estimate (CPE) of 0.77 (95% CI 0.69-0.86), which indicates high prognostic ability. The C-index and CPE of the validation cohort were 0.81 (95% CI 0.69-0.91) and 0.78 (95% CI 0.7-0.86), respectively. CONCLUSIONS: Our nomogram incorporating CD15+ cell count can potentially be used to identify patients at high risk of recurrent disease and thus stratify patients who will benefit most from adjuvant treatment.

Use of patient-derived explants as a preclinical model for precision medicine in colorectal cancer: A scoping review.

PURPOSE: Whilst the treatment paradigm for colorectal cancer has evolved significantly over time, there is still a lack of reliable biomarkers of treatment response. Treatment decisions are based on high-risk features such as advanced TNM stage and histology. The role of the tumour microenvironment, which can influence tumour progression and treatment response, has generated considerable interest. Patient-derived explant cultures allow preservation of native tissue architecture and tumour microenvironment. The aim of the scoping review is to evaluate the utility of patient-derived explant cultures as a preclinical model in colorectal cancer. METHODS: A search was conducted using Ovid MEDLINE, EMBASE, Web of Science, and Cochrane databases from start of database records to September 1, 2022. We included all peer-reviewed human studies in English language which used patient-derived explants as a preclinical model in primary colorectal cancer. Eligible studies were grouped into the following categories: assessing model feasibility; exploring tumour microenvironment; assessing ex vivo drug responses; discovering and validating biomarkers. RESULTS: A total of 60 studies were eligible. Fourteen studies demonstrated feasibility of using patient-derived explants as a preclinical model. Ten studies explored the tumour microenvironment. Thirty-eight studies assessed ex vivo drug responses of chemotherapy agents and targeted therapies. Twenty-four studies identified potential biomarkers of treatment response. CONCLUSIONS: Given the preservation of tumour microenvironment and tumour heterogeneity, patient-derived explants has the potential to identify reliable biomarkers, treatment resistance mechanisms, and novel therapeutic agents. Further validation studies are required to characterise, refine and standardise this preclinical model before it can become a part of precision medicine in colorectal cancer.

Potential Clinical Utility of a Targeted Circulating Tumor DNA Assay in Esophageal Adenocarcinoma.

OBJECTIVE: To explore the clinical utility of circulating tumor DNA (ctDNA) in esophageal adenocarcinoma (EAC) by developing a cost-effective and rapid technique utilising targeted amplicon sequencing. SUMMARY OF BACKGROUND DATA: Emerging evidence suggests that levels of ctDNA in the blood can be used to monitor treatment response and in the detection of disease recurrence in various cancer types. Current staging modalities for EAC such as computerised tomography of the chest/abdomen/pelvis (CT) and positron emission tomography (PET) do not reliably detect occult micro-metastatic disease, the presence of which signifies a poor prognosis. After curative-intent treatment, some patients are still at high risk of recurrent disease, and there is no widely accepted optimal surveillance tool for patients with EAC. METHODS: Sixty-two patients with EAC were investigated for the presence of ctDNA using a tumor-informed approach. We designed a custom targeted amplicon sequencing panel of target specific primers covering mutational foci in 9 of the most commonly mutated genes in EAC. Serial blood samples were taken before and after neoadjuvant treatment (NAT), and during surveillance. RESULTS: Somatic mutations were detected in pre-treatment biopsy samples of 55 out of 62 (89%) EAC patients. Mutations in TP53 (80%) were the most common. Out of these 55 patients, 20 (36%) had detectable ctDNA at baseline. The majority (90%) of patients with detectable ctDNA had either locally advanced tumors, nodal involvement or metastatic disease. In patients with locally advanced tumors, disease free survival (DFS) was more accurately stratified using pre-treatment ctDNA status [HR 4.34 (95% CI 0.93-20.21); P = 0.05] compared to nodal status on PET-CT. In an exploratory subgroup analysis, patients who are node negative but ctDNA positive have inferior DFS [HR 11.71 (95% CI 1.16-118.80) P = 0.04]. In blood samples taken before and following NAT, clearance of ctDNA after NAT was associated with a favourable response to treatment. Furthermore, patients who are ctDNA positive during post-treatment surveillance are at high risk of relapse. CONCLUSIONS: Our study shows that ctDNA has potential to provide additional prognostication over conventional staging investigation such as CT and PET. It may also have clinical utility in the assessment of response to NAT and as a biomarker for the surveillance of recurrent disease.

Cyclooxygenases and Prostaglandins in Tumor Immunology and Microenvironment of Gastrointestinal Cancer.

Chronic inflammation is a known risk factor for gastrointestinal cancer. The evidence that nonsteroidal anti-inflammatory drugs suppress the incidence, growth, and metastasis of gastrointestinal cancer supports the concept that a nonsteroidal anti-inflammatory drug target, cyclooxygenase, and its downstream bioactive lipid products may provide one of the links between inflammation and cancer. Preclinical studies have demonstrated that the cyclooxygenase-2-prostaglandin E2 pathway can promote gastrointestinal cancer development. Although the role of this pathway in cancer has been investigated extensively for 2 decades, only recent studies have described its effects on host defenses against transformed epithelial cells. Overcoming tumor-immune evasion remains one of the major challenges in cancer immunotherapy. This review summarizes the impacts of the cyclooxygenase-2-prostaglandin E2 pathway on gastrointestinal cancer development. Our focus was to highlight recent advances in our understanding of how this pathway induces tumor immune evasion.

GRB7 is an oncogenic driver and potential therapeutic target in oesophageal adenocarcinoma.

Efficacious therapeutic approaches are urgently needed to improve outcomes in patients with oesophageal adenocarcinoma (OAC). However, oncogenic drivers amenable to targeted therapy are limited and their functional characterisation is essential. Among few targeted therapies available, anti-human epidermal growth factor receptor 2 (HER2) therapy showed only modest benefit for patients with OAC. Herein, we investigated the potential oncogenic role of growth factor receptor bound protein 7 (GRB7), which is reported to be co-amplified with HER2 (ERBB2) in OAC. GRB7 was highly expressed in 15% of OAC tumours, not all of which could be explained by co-amplification with HER2, and was associated with a trend for poorer overall survival. Knockdown of GRB7 decreased proliferation and clonogenic survival, and induced apoptosis. Reverse phase protein array (RPPA) analyses revealed a role for PI3K, mammalian target of rapamycin (mTOR), MAPK, and receptor tyrosine kinase signalling in the oncogenic action of GRB7. Furthermore, the GRB7 and HER2 high-expressing OAC cell line Eso26 showed reduced cell proliferation upon GRB7 knockdown but was insensitive to HER2 inhibition by trastuzumab. Consistent with this, GRB7 knockdown in vivo with an inducible shRNA significantly inhibited tumour growth in cell line xenografts. HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Taken together, our study provides strong evidence for an oncogenic role for GRB7 in OAC and suggests that targeting GRB7 may be a potential therapeutic strategy for this cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Clinical pathways and outcomes of patients with Barrett's esophagus in tertiary care settings: a prospective longitudinal cohort study in Australia, 2008-2016.

BACKGROUND: Clinical services for Barrett's esophagus have been rising worldwide including Australia, but little is known of the long-term outcomes of such patients. Retrospective studies using data at baseline are prone to both selection and misclassification bias. We investigated the clinical characteristics and outcomes of Barrett's esophagus patients in a prospective cohort. METHODS: We recruited patients diagnosed with Barrett's esophagus in tertiary settings across Australia between 2008 and 2016. We compared baseline and follow-up epidemiological and clinical data between Barrett's patients with and without dysplasia. We calculated age-adjusted incidence rates and estimated minimally and fully adjusted hazard ratios (HR) to identify those clinical factors related to disease progression. RESULTS: The cohort comprised 268 patients with Barrett's esophagus (median follow-up 5 years). At recruitment, 224 (84%) had no dysplasia, 44 (16%) had low-grade or indefinite dysplasia (LGD/IND). The age-adjusted incidence of esophageal adenocarcinoma (EAC) was 0.5% per year in LGD/IND compared with 0.1% per year in those with no dysplasia. Risk of progression to high-grade dysplasia/EAC was associated with prior LGD/IND (fully adjusted HR 6.55, 95% confidence interval [CI] 1.96-21.8) but not long-segment disease (HR 1.03, 95%CI 0.29-3.58). CONCLUSIONS: These prospective data suggest presence of dysplasia is a stronger predictor of progression to cancer than segment length in patients with Barrett's esophagus.

Mutant p53 Mediates Sensitivity to Cancer Treatment Agents in Oesophageal Adenocarcinoma Associated with MicroRNA and SLC7A11 Expression.

TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.

Bridging the molecular divide: alcohol-induced downregulation of PAX9 and tumour development.

The pathogenesis of oro-oesophaeal squamous cell carcinoma is causally linked to the consumption of alcohol. Beyond the carcinogenic effects of ethanol and its metabolites via DNA damage, the precise mechanisms by which alcohol drives tumourigenesis remain to be fully elucidated. A novel contributor now revealed is aberrant differentiation and proliferation mediated by suppression of PAX9, a key regulator of normal squamous maturation in oro-oesophageal tissues. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The prognostic value of TP53 mutations in oesophageal adenocarcinoma: a systematic review and meta-analysis.

OBJECTIVE: To clarify the prognostic role of tumour protein 53 (TP53) mutations in patients with oesophageal adenocarcinoma (OAC) as there is a need for biomarkers that assist in guiding management for patients with OAC. DESIGN: A systematic review was conducted using MEDLINE, Embase, PubMed and Current Contents Connect to identify studies published between January 1990 and February 2015 of oesophageal cancer populations (with OAC diagnoses >50% of cases) that measured tumoural TP53 status and reported hazard ratios (HR), or adequate data for estimation of HR for survival for TP53-defined subgroups. Risk of bias for HR estimates was assessed using prespecified criteria for the appraisal of relevant domains as defined by the Cochrane Prognosis Methods Group including adherence to Grading of Recommendations, Assessment, Development and Evaluation and REporting recommendations for tumor MARKer prognostic studies guidelines, as well as assay method used (direct TP53 mutation assessment vs immunohistochemistry) and adjustment for standard prognostic factors. A pooled HR and 95% CI were calculated using a random-effects model. RESULTS: Sixteen eligible studies (11 with OAC only and 5 mixed histology cohorts) including 888 patients were identified. TP53 mutations were associated with reduced survival (HR 1.48, 95% CI 1.16 to 1.90, I2=33%). A greater prognostic effect was observed in a sensitivity analysis of those studies that reported survival for OAC-only cohorts and were assessed at low risk of bias (HR 2.11, 95% CI 1.35 to 3.31, I2=0%). CONCLUSIONS: Patients with OAC and TP53 gene mutations have reduced overall survival compared with patients without these mutations, and this effect is independent of tumour stage.

Intramuscular Transplantation Improves Engraftment Rates for Esophageal Patient-Derived Tumor Xenografts.

BACKGROUND: Recently, there has been an increase in the availability of targeted molecular therapies for cancer treatment. The application of these approaches to esophageal cancer, however, has been hampered by the relative lack of appropriate models for preclinical testing. Patient-derived tumor xenograft (PDTX) models are gaining popularity for studying many cancers. Unfortunately, it has proven difficult to generate xenografts from esophageal cancer using these models. The purpose of this study was to improve the engraftment efficiency of esophageal PDTXs. METHODS: Fresh pieces of esophageal tumors obtained from endoscopic biopsies or resected specimens were collected from 23 patients. The tumors were then coated in Matrigel and transplanted in immunocompromised mice subcutaneously (n = 6) and/or using a novel implantation technique whereby the tumor is placed in a dorsal intramuscular pocket (n = 18). They are then monitored for engraftment. RESULTS: With the novel intramuscular technique, successful engraftment was achieved for all 18 patient tumors. Among these PDTXs, 13 recapitulated the original patient tumors with respect to degree of differentiation, molecular and genetic profiles, and chemotherapeutic response. Lymphomatous transformation was observed in the other five PDTXs. Successful engraftment was achieved for only one of six patient tumors using the classic subcutaneous approach. DISCUSSION: We achieved a much higher engraftment rate of PDTXs using our novel intramuscular transplant technique than has been reported in other published studies. It is hoped that this advancement will help expedite the development and testing of new therapies for esophageal cancer.

Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvß3 and αvß5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

The Genetics of Barrett's Esophagus: A Familial and Population-Based Perspective.

Barrett's esophagus (BE) is intestinal metaplasia of the lower esophagus and a precursor lesion for esophageal adenocarcinoma (EAC). Both are important health issues as they have rising incidences in the Western world. Improving the management of BE relies on understanding the underlying biology of this disease, but the exact biological mechanisms have been difficult to determine. BE is generally thought to be an acquired condition that develops secondarily to chronic gastroesophageal reflux. However, multiple reports of familial clustering of patients with BE and/or EAC suggest a possible inherited predisposition to BE may be driving this condition, at least in a subset of patients. Identifying the genetic variants that predispose to BE in these families would open up the possibility for blood-based screening tests that could inform decision-making in regard to surveillance strategies, particularly for relatives of patients with BE and/or EAC. Perhaps more importantly, understanding the genetic mechanisms that predispose to BE may provide valuable insights into the biology of this condition and potentially identify novel targets for therapeutic intervention. Here we review the current evidence for a genetic predisposition to BE and discuss the potential implications of these findings.

APR-246 potently inhibits tumour growth and overcomes chemoresistance in preclinical models of oesophageal adenocarcinoma.

OBJECTIVES: p53 is a critical tumour suppressor and is mutated in 70% of oesophageal adenocarcinomas (OACs), resulting in chemoresistance and poor survival. APR-246 is a first-in-class reactivator of mutant p53 and is currently in clinical trials. In this study, we characterised the activity of APR-246 and its effect on p53 signalling in a large panel of cell line xenograft (CLX) and patient-derived xenograft (PDX) models of OAC. DESIGN: In vitro response to APR-246 was assessed using clonogenic survival, cell cycle and apoptosis assays. Ectopic expression, gene knockdown and CRISPR/Cas9-mediated knockout studies of mutant p53 were performed to investigate p53-dependent drug effects. p53 signalling was examined using quantitative RT-PCR and western blot. Synergistic interactions between APR-246 and conventional chemotherapies were evaluated in vitro and in vivo using CLX and PDX models. RESULTS: APR-246 upregulated p53 target genes, inhibited clonogenic survival and induced cell cycle arrest as well as apoptosis in OAC cells harbouring p53 mutations. Sensitivity to APR-246 correlated with cellular levels of mutant p53 protein. Ectopic expression of mutant p53 sensitised p53-null cells to APR-246, while p53 gene knockdown and knockout diminished drug activity. Importantly, APR-246 synergistically enhanced the inhibitory effects of cisplatin and 5-fluorouracil through p53 accumulation. Finally, APR-246 demonstrated potent antitumour activity in CLX and PDX models, and restored chemosensitivity to a cisplatin/5-fluorouracil-resistant xenograft model. CONCLUSIONS: APR-246 has significant antitumour activity in OAC. Given that APR-246 is safe at therapeutic levels our study strongly suggests that APR-246 can be translated into improving the clinical outcomes for OAC patients.

Characterization of a novel tumorigenic esophageal adenocarcinoma cell line: OANC1.

BACKGROUND: Esophageal adenocarcinoma (EAC) has a very high case fatality rate and is one of the fastest rising cancers worldwide. At the same time, research into EAC has been hampered by a relative lack of pre-clinical models, including representative cell lines. AIM: The purpose of this study was to establish and characterize a new EAC cell line. METHODS: Tumor cells were isolated from EAC tissue by enzymatic digestion. Origin of the cell line was confirmed by microsatellite based genotyping. A panel of cancer-related genes was screened for mutations by targeted deep sequencing, Sanger sequencing and high resolution melting.CDKN2A promoter methylation was assessed by methylation specific high resolution melting. HER2 amplification was assessed by fluorescent in situ hybridization. Immunohistochemistry was used to assess expression of markers in xenografts grown in SCID mice. RESULTS: A novel EAC cell line, OANC1, was derived from a Barrett's-associated EAC. Microsatellite-based genotyping of OANC1 and patient DNA confirmed the origin of the cell line. Sequencing of OANC1 DNA identified homozygous TP53 missense (c.856G[A, p.E286K)and SMAD4 nonsense (c.1333C[T, p.R445X) mutations.OANC1 are tumorigenic when injected sub-cutaneously into SCID mice and xenografts were positive for columnar, glandular and intestinal epithelial markers commonly expressed in EAC. Xenografts exhibited strong p53 expression, consistent with a TP53 mutation. Some proteins, including p16, EGFR and b-catenin, had heterogeneous expression patterns across xenograft cross-sections, indicative of tumor heterogeneity. CONCLUSIONS: OANC1 represents a valuable addition to the limited range of pre-clinical models for EAC. This new cell line will be a useful model system for researchers studying both basic and translational aspects of this disease.

Modelling esophageal adenocarcinoma and Barrett's esophagus with patient-derived organoids.

Currently, esophageal adenocarcinoma (EAC) research is hindered by a dearth of adequate models to study this disease. Traditional cell line and genetically engineered mouse models are lacking in biological and physiological significance, whilst the inefficiency of patient-derived xenografts limit their potential applications. This review describes the landscape of EAC research using patient-derived organoids (PDOs). Here, we detail the methods of establishment and optimization of EAC PDO cultures, as well as current and prospective applications of these models. We further highlight a crucial knowledge gap in the mechanisms of EAC transformation from its precursor lesion, Barrett's esophagus (BE). As such, we also describe the culture requirements of BE PDOs and attempts to model tumorigenesis using PDO models.