Fergus Noble

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 αvβ3 and αvβ5, 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.

Risk assessment using a novel score to predict anastomotic leak and major complications after oesophageal resection

BackgroundOesophagectomy is associated with significant morbidity and mortality. A simple score to define a patients risk of developing major complications would be beneficial.MethodsPatients who underwent upper gastrointestinal resections with an oesophageal anastomosis between 2005 and 2010 were reviewed and formed the development dataset with resections performed in 2011 forming a prospective validation dataset. The association between post-operative C-reactive protein (CRP), white cell count (WCC) and albumin levels with anastomotic leak (AL) or major complication including death using the Clavien–Dindo (CD) classification were analysed by receiver operating characteristic curves. After multivariate analysis, from the development dataset, these factors were combined to create a novel score which was subsequently tested on the validation dataset.ResultsTwo hundred fifty-eight patients were assessed to develop the score. Sixty-three patients (25%) developed a major complication, and there were seven (2.7%) in-patient deaths. Twenty-six (10%) patients were diagnosed with AL at median post-operative day 7 (range: 5–15). CRP (p = 0.002), WCC (p < 0.0001) and albumin (p = 0.001) were predictors of AL. Combining these markers improved prediction of AL (NUn score > 10: sensitivity 95%, specificity 49%, diagnostic accuracy 0.801 (95% confidence interval: 0.692–0.909, p < 0.0001)). The validation dataset confirmed these findings (NUn score > 10: sensitivity 100%, specificity 57%, diagnostic accuracy 0.879 (95% CI 0.763–0.994, p = 0.014)) and a major complication or death (NUn > 10: sensitivity 89%, specificity 63%, diagnostic accuracy 0.856 (95% CI 0.709–1, p = 0.001)).ConclusionsBlood-borne markers of the systemic inflammatory response are predictors of AL and major complications after oesophageal resection. When combined they may categorise a patients risk of developing a serious complication with higher sensitivity and specificity.

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

A prospective comparison of totally minimally invasive versus open Ivor Lewis esophagectomy.

The majority of esophagectomies in Western parts of the world are performed by a transthoracic approach reflecting the prevalence of adenocarcinoma of the lower esophagus or esophagogastric junction. Minimally invasive esophagectomy (MIE) has been reported in a variety of formats, but there are no series that directly compare totally minimally invasive thoracolaparoscopic 2 stage esophagectomy (MIE-2) with open Ivor Lewis (IVL). A prospective single-center cohort study of patients undergoing elective MIE-2 or IVL between January 2005 and November 2010 was performed. Short-term clinicopathologic outcomes were recorded using validated systems. One hundred and six patients (median age 66, range 36-85, 88 M : 18 F) underwent two-stage esophagectomy (53 MIE-2 and 53 IVL). Patient demographics (age, sex, body mass index, American Society of Anesthesiologists grade, tumor characteristics, neoadjuvant chemotherapy, and TNM stage) were comparable between the two groups. Outcomes for MIE-2 and IVL were comparable for anastomotic leak rates (5 [9%] vs. 2 [4%], P= 0.241), resection margin clearance (R0) (43 [81%] vs. 38 [72%], P= 0.253), median lymph node yield (19 vs. 18, P= 0.584), and median length of stay (12 [range 7-91] vs. 12 [range 7-101] days), respectively. Blood loss was significantly less for MIE-2 compared with IVL (median 300 [range 0-1250] mL vs. 400 [range 0-3000] mL, respectively, P= 0.021). MIE-2 in this series of selected patients supports its efficacy, when performed by an experienced minimally invasive surgical team. A well-designed multicenter trial addressing clinical effectiveness is now required.

Refining pathological evaluation of neoadjuvant therapy for adenocarcinoma of the esophagus

AIM To assess tumour regression grade (TRG) and lymph node downstaging to help define patients who benefit from neoadjuvant chemotherapy. METHODS Two hundred and eighteen consecutive patients with adenocarcinoma of the esophagus or gastro-esophageal junction treated with surgery alone or neoadjuvant chemotherapy and surgery between 2005 and 2011 at a single institution were reviewed. Triplet neoadjuvant chemotherapy consisting of platinum, fluoropyrimidine and anthracycline was considered for operable patients (World Health Organization performance status ≤ 2) with clinical stage T2-4 N0-1. Response to neoadjuvant chemotherapy (NAC) was assessed using TRG, as described by Mandard et al. In addition lymph node downstaging was also assessed. Lymph node downstaging was defined by cN1 at diagnosis: assessed radiologically (computed tomography, positron emission tomography, endoscopic ultrasonography), then pathologically recorded as N0 after surgery; ypN0 if NAC given prior to surgery, or pN0 if surgery alone. Patients were followed up for 5 years post surgery. Recurrence was defined radiologically, with or without pathological confirmation. An association was examined between t TRG and lymph node downstaging with disease free survival (DFS) and a comprehensive range of clinicopathological characteristics. RESULTS Two hundred and eighteen patients underwent esophageal resection during the study interval with a mean follow up of 3 years (median follow up: 2.552, 95%CI: 2.022-3.081). There was a 1.8% (n = 4) inpatient mortality rate. One hundred and thirty-six (62.4%) patients received NAC, with 74.3% (n = 101) of patients demonstrating some signs of pathological tumour regression (TRG 1-4) and 5.9% (n = 8) having a complete pathological response. Forty four point one percent (n = 60) had downstaging of their nodal disease (cN1 to ypN0), compared to only 15.9% (n = 13) that underwent surgery alone (pre-operatively overstaged: cN1 to pN0), (P < 0.0001). Response to NAC was associated with significantly increased DFS (mean DFS; TRG 1-2: 5.1 years, 95%CI: 4.6-5.6 vs TRG 3-5: 2.8 years, 95%CI: 2.2-3.3, P < 0.0001). Nodal down-staging conferred a significant DFS advantage for those patients with a poor primary tumour response to NAC (median DFS; TRG 3-5 and nodal down-staging: 5.533 years, 95%CI: 3.558-7.531 vs TRG 3-5 and no nodal down-staging: 1.114 years, 95%CI: 0.961-1.267, P < 0.0001). CONCLUSION Response to NAC in the primary tumour and in the lymph nodes are both independently associated with improved DFS.

A comparison of primary oesophageal squamous epithelial cells with HET-1A in organotypic culture

Background Information. Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5‐year survival of less than 15%. Recent evidence suggests that stromal—epithelial interactions are fundamental in carcinogenesis. The advent of co‐culture techniques permits the investigation of cross‐talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET‐1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro.

The relevance of the Siewert classification in the era of multimodal therapy for adenocarcinoma of the gastro‐oesophageal junction

The Siewert classification has been used to plan treatment for tumours of the gastro‐oesophageal junction since its proposal in the 1980s. The purpose of this study was to assess its continued relevance by evaluating whether there were differences in the biology and clinical characteristics of adenocarcinomas by Siewert type, in a contemporary cohort of patients, in whom the majority had received neoadjuvant chemotherapy.

Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4

Abstract Background Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4’s role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9–15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69–7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65–0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%–79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%–64.0% decrease across different models, P ≤ .04). Conclusions These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.

Systematic review and meta-analysis of immunohistochemical prognostic biomarkers in resected oesophageal adenocarcinoma

Background:Oesophageal adenocarcinoma (OAC) is one of the fastest rising malignancies with continued poor prognosis. Many studies have proposed novel biomarkers but, to date, no immunohistochemical markers of survival after oesophageal resection have entered clinical practice. Here, we systematically review and meta-analyse the published literature, to identify potential biomarkers.Methods:Relevant articles were identified via Ovid medline 1946–2013. For inclusion, studies had to conform to REporting recommendations for tumor MARKer (REMARK) prognostic study criteria. The primary end-point was a pooled hazard ratio (HR) and variance, summarising the effect of marker expression on prognosis.Results:A total of 3059 articles were identified. After exclusion of irrelevant titles and abstracts, 214 articles were reviewed in full. Nine molecules had been examined in more than one study (CD3, CD8, COX-2, EGFR, HER2, Ki67, LgR5, p53 and VEGF) and were meta-analysed. Markers with largest survival effects were COX-2 (HR=2.47, confidence interval (CI)=1.15–3.79), CD3 (HR=0.51, 95% CI=0.32–0.70), CD8 (HR=0.55, CI=0.31–0.80) and EGFR (HR=1.65, 95% CI=1.14–2.16).Discussion:Current methods have not delivered clinically useful molecular prognostic biomarkers in OAC. We have highlighted the paucity of good-quality robust studies in this field. A genome-to-protein approach would be better suited for the development and subsequent validation of biomarkers. Large collaborative projects with standardised methodology will be required to generate clinically useful biomarkers.

Strategies to improve outcomes in esophageal adenocarcinoma

Esophageal adenocarcinoma is one of the fastest rising cancers in Western society. Incidence has increased by 600% within the last 30 years. Rates of diagnosis and death run parallel due to the poor prognosis and a lack of effective treatments. Potentially curative treatments are followed by high rates of disease recurrence. For the majority of patients, who present with advanced disease, we have no effective treatment. We discuss the key areas of progress in this demanding field and offer our views on the direction of future research and treatment.