Tetraspanin 6 is a regulator of carcinogenesis in colorectal cancer.

Early stages of colorectal cancer (CRC) development are characterized by a complex rewiring of transcriptional networks resulting in changes in the expression of multiple genes. Here, we demonstrate that the deletion of a poorly studied tetraspanin protein Tspan6 in Apcmin/+ mice, a well-established model for premalignant CRC, resulted in increased incidence of adenoma formation and tumor size. We demonstrate that the effect of Tspan6 deletion results in the activation of EGF-dependent signaling pathways through increased production of the transmembrane form of TGF-α (tmTGF-α) associated with extracellular vesicles. This pathway is modulated by an adaptor protein syntenin-1, which physically links Tspan6 and tmTGF-α. In support of this, the expression of Tspan6 is frequently decreased or lost in CRC, and this correlates with poor survival. Furthermore, the analysis of samples from the epidermal growth factor receptor (EGFR)-targeting clinical trial (COIN trial) has shown that the expression of Tspan6 in CRC correlated with better patient responses to EGFR-targeted therapy involving Cetuximab. Importantly, Tspan6-positive patients with tumors in the proximal colon (right-sided) and those with KRAS mutations had a better response to Cetuximab than the patients that expressed low Tspan6 levels. These results identify Tspan6 as a regulator of CRC development and a potential predictive marker for EGFR-targeted therapies in CRC beyond RAS pathway mutations.

A highly predictive autoantibody-based biomarker panel for prognosis in early-stage NSCLC with potential therapeutic implications.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. Surgical resection remains the definitive curative treatment for early-stage disease offering an overall 5-year survival rate of 62%. Despite careful case selection, a significant proportion of early-stage cancers relapse aggressively within the first year post-operatively. Identification of these patients is key to accurate prognostication and understanding the biology that drives early relapse might open up potential novel adjuvant therapies. METHODS: We performed an unsupervised interrogation of >1600 serum-based autoantibody biomarkers using an iterative machine-learning algorithm. RESULTS: We identified a 13 biomarker signature that was highly predictive for survivorship in post-operative early-stage lung cancer; this outperforms currently used autoantibody biomarkers in solid cancers. Our results demonstrate significantly poor survivorship in high expressers of this biomarker signature with an overall 5-year survival rate of 7.6%. CONCLUSIONS: We anticipate that the data will lead to the development of an off-the-shelf prognostic panel and further that the oncogenic relevance of the proteins recognised in the panel may be a starting point for a new adjuvant therapy.

The role of B lymphocytes in the immuno-biology of non-small-cell lung cancer.

Tumour-infiltrating immune cells have been widely implicated to play a significant role in carcinogenesis, through both pro- or anti-tumour effects. The multi-faceted effects of lung cancer associated T lymphocytes have been extensively studied, and yet, the role of B lymphocytes remains an area less studied. In this review, we will describe the current understanding of the role of tumour-infiltrating B lymphocytes in NSCLC, discuss their prognostic significance, their functionality within the tumour microenvironment and ultimately how we might harness B-cell biology to develop B-cell therapeutic strategies in cancer.

Docetaxel versus active symptom control for refractory oesophagogastric adenocarcinoma (COUGAR-02): an open-label, phase 3 randomised controlled trial.

BACKGROUND: Second-line chemotherapy for patients with oesophagogastric adenocarcinoma refractory to platinum and fluoropyrimidines has not shown benefits in health-related quality of life (HRQoL). We assessed whether the addition of docetaxel to active symptom control alone can improve survival and HRQoL for patients. METHODS: For this open-labelled, multicentre trial, we recruited patients aged 18 years or older from 30 UK centres. Patients were eligible if they had an advanced, histologically confirmed adenocarcinoma of the oesophagus, oesophagogastric junction, or stomach that had progressed on or within 6 months of treatment with a platinum-fluoropyrimidine combination. Patients could have an Eastern Cooperative Oncology Group performance status of 0-2. We randomly assigned patients using a central, computerised minimisation procedure to receive docetaxel plus active symptom control, or active symptom control alone (1:1; stratified by disease status, disease site, duration of response to previous chemotherapy, and performance status). Docetaxel was given at a dose of 75 mg/m(2) by intravenous infusion every 3 weeks for up to six cycles. The primary endpoint was overall survival, analysed by intention to treat. This is the report of the planned final analysis. This study is an International Standardised Randomised Controlled Trial, number ISRCTN13366390. FINDINGS: Between April 21, 2008, and April 26, 2012, we recruited 168 patients, allocating 84 to each treatment group. After a median follow-up of 12 months [IQR 10-21]) and 161 (96%) deaths (80 in the docetaxel group, 81 in the active symptom control group), median overall survival in the docetaxel group was 5.2 months (95% CI 4.1-5.9) versus 3.6 months (3.3-4.4) in the active symptom control group (hazard ratio 0.67, 95% CI 0.49-0.92; p=0.01). Docetaxel was associated with higher incidence of grade 3-4 neutropenia (12 [15%] patients vs no patients), infection (15 [19%] patients vs two [3%] patients), and febrile neutropenia (six [7%] patients vs no patients). Patients receiving docetaxel reported less pain (p=0.0008) and less nausea and vomiting (p=0.02) and constipation (p=0.02). Global HRQoL was similar between the groups (p=0.53). Disease specific HRQoL measures also showed benefits for docetaxel in reducing dysphagia (p=0.02) and abdominal pain (p=0.01). INTERPRETATION: Our findings suggest that docetaxel can be recommended as an appropriate second-line treatment for patients with oesophagogastric adenocarcinoma that is refractory to treatment with platinum and fluoropyrimidine. FUNDING: Cancer Research UK.

Neoadjuvant immunotherapy for dMMR and pMMR colorectal cancers: therapeutic strategies and putative biomarkers of response.

Approximately 15% of locally advanced colorectal cancers (CRC) have DNA mismatch repair deficiency (dMMR), resulting in high microsatellite instability and a high tumour mutational burden. These cancers are frequently sensitive to therapy with immune-checkpoint inhibitors (ICIs) in the metastatic setting. This sensitivity seems to be even more pronounced in locally advanced disease, and organ preservation has become a realistic aim in ongoing clinical trials involving patients with dMMR rectal cancer. By contrast, metastatic CRCs with proficient DNA mismatch repair (pMMR) are generally resistant to ICIs, although a proportion of locally advanced pMMR tumours seem to have a high degree of sensitivity to ICIs. In this Review, we describe the current and emerging clinical evidence supporting the use of neoadjuvant ICIs in patients with dMMR and pMMR CRC, and the potential advantages (based on a biological rationale) of such an approach. We discuss how neoadjuvant 'window-of-opportunity' trials are being leveraged to progress biomarker discovery and we provide an overview of potential predictive biomarkers of response to ICIs, exploring the challenges faced when evaluating such biomarkers in biopsy-derived samples. Lastly, we describe how these discoveries might be used to drive a rational approach to trialling novel immunotherapeutic strategies in patients with pMMR CRC, with the ultimate aim of disease eradication and the generation of long-term immunosurveillance.

TIGIT-based immunotherapeutics in lung cancer.

In this review, we explore the biology of the TIGIT checkpoint and its potential as a therapeutic target in lung cancer. We briefly review a highly selected set of clinical trials that have reported or are currently recruiting in non-small cell and small cell lung cancer, a disease transformed by the advent of PD-1/PD-L1 checkpoint blockade immunotherapy. We explore the murine data underlying TIGIT blockade and further explore the reliance of effective anti-TIGIT therapy on DNAM-1(CD226)-positive activated effector CD8+ T cells. The synergism with anti-PD-1 therapy is also explored. Future directions in the realm of overcoming resistance to checkpoint blockade and extending the repertoire of other checkpoints are also briefly explored.

Deep immune B and plasma cell repertoire in non-small cell lung cancer.

Introduction: B cells, which have long been thought to be minor players in the development of anti-tumor responses, have been implicated as key players in lung cancer pathogenesis and response to checkpoint blockade in patients with lung cancer. Enrichment of late-stage plasma and memory cells in the tumor microenvironment has been shown in lung cancer, with the plasma cell repertoire existing on a functional spectrum with suppressive phenotypes correlating with outcome. B cell dynamics may be influenced by the inflammatory microenvironment observed in smokers and between LUAD and LUSC. Methods: Here, we show through high-dimensional deep phenotyping using mass cytometry (CyTOF), next generation RNA sequencing and multispectral immunofluorescence imaging (VECTRA Polaris) that key differences exist in the B cell repertoire between tumor and circulation in paired specimens from lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Results: In addition to the current literature, this study provides insight into the in-depth description of the B cell contexture in Non-Small Cell Lung Cancer (NSCLC) with reference to broad clinico-pathological parameters based on our analysis of 56 patients. Our findings reinforce the phenomenon of B-cell trafficking from distant circulatory compartments into the tumour microenvironment (TME). The circulatory repertoire shows a predilection toward plasma and memory phenotypes in LUAD however no major differences exist between LUAD and LUSC at the level of the TME. B cell repertoire, amongst other factors, may be influenced by the inflammatory burden in the TME and circulation, that is, smokers and non-smokers. We have further clearly demonstrated that the plasma cell repertoire exists on a functional spectrum in lung cancer, and that the suppressive regulatory arm of this axis may play a significant role in determining postoperative outcomes as well as following checkpoint blockade. This will require further long-term functional correlation. Conclusion: B and Plasma cell repertoire is very diverse and heterogeneous across different tissue compartments in lung cancer. Smoking status associates with key differences in the immune milieu and the consequent inflammatory microenvironment is likely responsible for the functional and phenotypic spectrum we have seen in the plasma cell and B cell repertoire in this condition.

Are we ready to start studies of Th17 cell manipulation as a therapy for cancer?

From a therapeutic perspective, the bourgeoning literature on Th17 cells should allow us to decide whether to rationally pursue the manipulation of Th17 cells in cancer. The purpose of this review is to attempt a synthesis of a number of contradictory conclusions as to the role that these cells are playing in the process of tumourigenesis in order to provide guidance as to whether our current understanding is sufficient to safely pursue Th17-targeted therapy in cancer at this time. Th17 cells are a highly plastic population and the cytokine drivers for Th17 cell generation and skewing will vary between various cancers and importantly between different sites of tumour involvement in any individual patient. The net impact of the pro-angiogenic IL-17 produced not only by Th17 cells but by other cells particularly macrophages and the anti-tumour effects of Th1/Th17 cells will in turn be determined by the complex interplay of diverse chemokines and cytokines in any tumour microenvironment. Th17 cells that fail to home to tumours may be immunosuppressive. The complexity of IL-17 and Th17 dynamics makes easy prediction of the effects of either enhancing or suppressing Th17 cell differentiation in cancer problematic.

Regulatory B cell repertoire defects predispose lung cancer patients to immune-related toxicity following checkpoint blockade.

Checkpoint blockade with Pembrolizumab, has demonstrated durable clinical responses in advanced non-small cell lung cancer, however, treatment is offset by the development of high-grade immune related adverse events (irAEs) in some patients. Here, we show that in these patients a deficient Breg checkpoint fails to limit self-reactive T cell enhanced activity and auto-antibody formation enabled by PD-1/PD-L1 blockade, leading to severe auto-inflammatory sequelae. Principally a failure of IL-10 producing regulatory B cells as demonstrated through functional ex vivo assays and deep phenotyping mass cytometric analysis, is a major and significant finding in patients who develop high-grade irAEs when undergoing treatment with anti-PD1/PD-L1 checkpoint blockade. There is currently a lack of biomarkers to identify a priori those patients at greatest risk of developing severe auto-inflammatory syndrome. Pre-therapy B cell profiling could provide an important tool to identify lung cancer patients at high risk of developing severe irAEs on checkpoint blockade.

Elevated myeloid-derived suppressor cells in pancreatic, esophageal and gastric cancer are an independent prognostic factor and are associated with significant elevation of the Th2 cytokine interleukin-13.

We undertook a comprehensive analysis of circulating myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) in pancreatic, esophageal and gastric cancer patients and investigated whether MDSCs are an independent prognostic factor for survival. We evaluated a series of plasma cytokines and in particular re-evaluated the Th2 cytokine interleukin-13 (IL-13). Peripheral blood was collected from 131 cancer patients (46 pancreatic, 60 esophageal and 25 gastric) and 54 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC (HLADR(-) Lin1(low/-) CD33(+) CD11b(+)) and Treg (CD4(+) CD25(+) CD127(low/-) FoxP3(+)) percentages. Plasma IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, IFN-γ, TNF-α and VEGF levels were analyzed by the Bio-Plex cytokine assay. Plasma arginase I levels were analyzed by ELISA. MDSCs and Tregs were statistically significantly elevated in pancreatic, esophageal and gastric cancer compared with controls, and MDSC numbers correlated with Treg levels. Increasing MDSC percentage was associated with increased risk of death, and in a multivariate analysis, MDSC level was an independent prognostic factor for survival. A unit increase in MDSC percentage was associated with a 22% increased risk of death (hazard ratio 1.22, 95% confidence interval 1.06-1.41). Arginase I levels were also statistically significantly elevated in upper gastrointestinal cancer patients compared with controls. There was Th2 skewing for cytokine production in all three diseases, and importantly there were significant elevations of the pivotal Th2 cytokine interleukin-13, an increase that correlated with MDSC levels.

Paradox breaker BRAF inhibitors have comparable potency and MAPK pathway reactivation to encorafenib in BRAF mutant colorectal cancer.

The BEACON CRC trial demonstrated a survival advantage over chemotherapy for a combination of targeted agents comprising the potent BRAF inhibitor encorafenib together with cetuximab and binimetinib. Resistance to BRAF inhibition in CRC arises in part through the generation and activation of RAF dimers resulting in MEK-ERK pathway reactivation. Paradox breaker BRAF inhibitors, such as PLX8394, are designed to inhibit RAF dimer formation. We analyzed whether paradox breakers reduce pathway reactivation and so have enhanced potency compared with encorafenib in BRAF mutant CRC. The potency of encorafenib and PLX8394 was greater than vemurafenib and the degree of pathway reactivation somewhat less. However, dose response curves for encorafenib and PLX8394 were similar and there was no significant differences in degree of pathway reactivation. To our knowledge these data represent the first comparative data of encorafenib and paradox breaker inhibitors in BRAF mutant CRC. Whilst these results support further investigation of PLX8394, all three agents tested reactivated the pathway in melanoma cells, a disease in which monotherapy is effective. Strategies focused on restricting RAF dimerization fail to address the impact that specific context of BRAF mutation in CRC has on targeted therapy outcomes.

Characterising the impact of pneumonia on outcome in non-small cell lung cancer: identifying preventative strategies.

BACKGROUND: Infections remain a part of the natural course of cancer, and lung cancer patients often present with some form of respiratory infection that can lead to their ultimate demise. METHODS: Data was gathered concerning all unplanned hospital admissions (UHAs) to our centre from three separate patient cohorts; non-small cell lung cancer (NSCLC) patients (cohort 1), "other cancer" patients (breast, prostate, colon) (cohort 2) and all non-cancer patients (cohort 3). RESULTS: Across the three cohorts, there were 455, 1,190 and 54,158 individual patient UHAs to our centre respectively. Within the NSCLC cohort, 164 UHAs were as a direct result of pneumonia (36.0%), compared to 1.3% and 2.2% in the other two cohorts (P<0.0001). In-hospital mortality and length of hospital stay were significantly higher in the pneumonia sub-group of NSCLC patients only compared with the other two patient cohorts (P<0.0001 and P=0.011 respectively). Within the NSCLC cohort, Patient age, pneumococcal vaccination status, pneumonia admission, smoking status and specific tumour stages were identified as significant independent risk factors for in-hospital mortality. Odds ratios of 0.160 for positive vaccination status and 9.522 for pneumonia admission indicate that for NSCLC patients admitted to hospital with a pneumonia without previous pneumococcal vaccination in the last 5 years, the odds of death were almost 60-fold higher. CONCLUSIONS: Vigilance for infection, early diagnosis with adequate assessment and efforts to identify a culprit organism should be a priority when faced with these patients. Infection prevention strategies should be further explored to address this high mortality risk in NSCLC.

KRAS Mutation and Consensus Molecular Subtypes 2 and 3 Are Independently Associated with Reduced Immune Infiltration and Reactivity in Colorectal Cancer.

Purpose:KRAS mutation is a common canonical mutation in colorectal cancer, found at differing frequencies in all consensus molecular subtypes (CMS). The independent immunobiological impacts of RAS mutation and CMS are unknown. Thus, we explored the immunobiological effects of KRAS mutation across the CMS spectrum.Experimental Design: Expression analysis of immune genes/signatures was performed using The Cancer Genome Atlas (TCGA) RNA-seq and the KFSYSCC microarray datasets. Multivariate analysis included KRAS status, CMS, tumor location, MSI status, and neoantigen load. Protein expression of STAT1, HLA-class II, and CXCL10 was analyzed by digital IHC.Results: The Th1-centric co-ordinate immune response cluster (CIRC) was significantly, albeit modestly, reduced in KRAS-mutant colorectal cancer in both datasets. Cytotoxic T cells, neutrophils, and the IFNγ pathway were suppressed in KRAS-mutant samples. The expressions of STAT1 and CXCL10 were reduced at the mRNA and protein levels. In multivariate analysis, KRAS mutation, CMS2, and CMS3 were independently predictive of reduced CIRC expression. Immune response was heterogeneous across KRAS-mutant colorectal cancer: KRAS-mutant CMS2 samples have the lowest CIRC expression, reduced expression of the IFNγ pathway, STAT1 and CXCL10, and reduced infiltration of cytotoxic cells and neutrophils relative to CMS1 and CMS4 and to KRAS wild-type CMS2 samples in the TCGA. These trends held in the KFSYSCC dataset.Conclusions:KRAS mutation is associated with suppressed Th1/cytotoxic immunity in colorectal cancer, the extent of the effect being modulated by CMS subtype. These results add a novel immunobiological dimension to the biological heterogeneity of colorectal cancer. Clin Cancer Res; 24(1); 224-33. ©2017 AACR.

Genomic loss of heterozygosity and survival in the REAL3 trial.

BACKGROUND: Homologous recombination deficiency (HRD) measured using a genomic signature for loss of heterozygosity (LOH) predicts benefit from rucaparib in ovarian cancer. We hypothesized that some oesophagogastric cancers will have high-LOH which would be prognostic in patients treated with platinum chemotherapy. METHODS: Diagnostic biopsy DNA from patients treated in the REAL3 trial was sequenced using the Foundation Medicine T5 next-generation sequencing (NGS) assay. An algorithm quantified the percentage of interrogable genome with LOH. Multidimensional optimization was performed to identify a cut-off dichotomizing the population into LOH-high and low groups associated with differential survival outcomes. RESULTS: Of 158 available samples, 117 were successfully sequenced; LOH was derived for 74 of these. A cut-off of 21% genomic LOH defined an LOH-high subgroup (n=10, 14% of population) who had median overall survival (OS) of 18.3 months (m) versus 11m for the LOH-low group (HR 0.55 95% CI 0.19-0.97, p= 0.10). Progression free survival (PFS) for LOH-high and LOH-low groups was 10.7m and 7.3m (HR 0.61 (95% CI 0.21 - 1.09, p=0.09). Sensitivity analysis censoring operated patients (n=4), demonstrated OS of 18.3m vs. 10.2m (HR 0.43, 95% CI (0.20-0.92), p=0.02; PFS was 10.5m vs. 7.2m (HR 0.55, (95% CI 0.26-1.17), p=0.09 for LOH-high and LOH-low. CONCLUSION: HRD assessment using an algorithmically derived LOH signature on a standard NGS panel identifies oesophagogastric cancer patients with high LOH who have prolonged survival when treated with platinum chemotherapy. Validation work will determine the signature's predictive value in patients treated with a PARP inhibitor and with platinum chemotherapy.

An immunogenomic stratification of colorectal cancer: Implications for development of targeted immunotherapy.

Although tumor infiltrating lymphocyte (TIL) density is prognostic and predictive in colorectal cancer (CRC), the impact of tumor genetics upon colorectal immunobiology is unclear. Identification of genetic factors that influence the tumor immunophenotype is essential to improve the effectiveness of stratified immunotherapy approaches. We carried out a bioinformatics analysis of CRC data in The Cancer Genome Atlas (TCGA) involving two-dimensional hierarchical clustering to define an immune signature that we used to characterize the immune response across key patient groups. An immune signature termed The Co-ordinate Immune Response Cluster (CIRC) comprising 28 genes was coordinately regulated across the patient population. Four patient groups were delineated on the basis of cluster expression. Group A, which was heavily enriched for patients with microsatellite instability (MSI-H) and POL mutations, exhibited high CIRC expression, including the presence of several inhibitory molecules: CTLA4, PDL1, PDL2, LAG3, and TIM3. In contrast, RAS mutation was enriched in patient groups with lower CIRC expression. This work links the genetics and immunobiology of colorectal tumorigenesis, with implications for the development of stratified immunotherapeutic approaches. Microsatellite instability and POL mutations are linked with high mutational burden and high immune infiltration, but the coordinate expression of inhibitory pathways observed suggests combination checkpoint blockade therapy may be required to improve efficacy. In contrast, RAS mutant tumors predict for a relatively poor immune infiltration and low inhibitory molecule expression. In this setting, checkpoint blockade may be less efficacious, highlighting a requirement for novel strategies in this patient group.

Methylation changes in the TFAP2E promoter region are associated with BRAF mutation and poorer overall & disease free survival in colorectal cancer.

INTRODUCTION: BRAF mutant colorectal cancer carries a poor prognosis which is thought to be related to poor response to conventional chemotherapy. BRAF mutation is associated with the serrated tumour phenotype. We hypothesised that one of the mechanisms by which BRAF mutant colorectal cancer demonstrate poor outcomes with chemotherapy is abnormal gene methylation. METHODS: The Cancer Genome Atlas (TCGA) methylation data was analysed using a linear regression model with BRAF mutation as an independent variable. Expression datasets were also obtained to correlate functional changes. Top differentially methylated probes were taken forward for validation by methylation pyrosequencing. These probes were analysed on a cohort of patients enriched for BRAF mutations taken from the VICTOR and QUASAR2 studies. RESULTS: In an analysis of 91 tumours (9 BRAF mutant, 82 wild type), the Illumina probe cg11835197 was the probe identified as top differentially methylated (p = 2.56×10-7, Bayes Factor (BF) =6.54). This probe covered a region -413bp from the promoter region of TFAP2E. We found a complex pattern of CpG specific methylation of this region which was associated with both overall (p=0.044) and disease free (p=0.046) survival. DISCUSSION: BRAF mutant tumours may attain part of their chemoresistance from abnormal TFAP2E methylation, which has not previously been described.

Periodontal disease in a patient receiving Bevacizumab: a case report.

INTRODUCTION: Bevacizumab is a monoclonal antibody that inhibits the action of vascular endothelial growth factor (VEGF) thereby acting as an angiogenesis inhibitor. As a result, supply of oxygen and nutrients to tissues is impaired and tumour cell growth is reduced. Reported side effects due to bevacizumab are hypertension and increased risk of bleeding. Bowel perforation has also been reported. Periodontal disease in patients on bevacizumab therapy has not been reported before. CASE PRESENTATION: We report a case of a forty-three year old woman who developed periodontitis whilst receiving bevacizumab for lung cancer. The periodontal disease remained stable on discontinuation of the drug. CONCLUSION: Further investigations are needed to determine the mechanism for bevacizumab-induced periodontal disease.

Metastatic rectal adenocarcinoma within haemorrhoids: a case report.

INTRODUCTION: Metastatic tumour involvement of the anal canal is rare. Routine pathological evaluation of haemorrhoidectomy specimens has been suggested to be unhelpful and expensive. Selective rather than routine pathological evaluation of haemorrhoidectomy specimens has been recommended. CASE PRESENTATION: We report the case of a 69-year-old woman with metastatic colorectal carcinoma who presented with metastatic carcinoma within thrombosed haemorrhoids. CONCLUSION: We suggest that in patients with colorectal cancer, careful examination of haemorrhoids on colonoscopy as well as histological examination of suspected haemorrhoidal tissue after surgical resection be performed to evaluate for metastasis.