Search

Strong immune responses in primary colorectal cancer correspond with better patient survival following surgery compared with tumors with predominantly stromal microenvironments. However, biomarkers to identify patients with colorectal cancer liver metastases (CRLM) with good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary colorectal cancer and CRLM and to interrogate the underlying functional biology that drives disease progression. Samples from patients undergoing synchronous resection of primary colorectal cancer and CRLM were evaluated in detail through histological assessment, panel genomic and bulk transcriptomic assessment, IHC, and GeoMx spatial transcriptomics (ST) analysis. High immune infiltration of metastases was associated with improved cancer-specific survival. Bulk transcriptomic analysis was confounded by stromal content, but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for type II IFN signaling and MHC-class II antigen presentation. In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T cells and neutrophils with enrichment of Notch and TGFβ signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcomes in patients undergoing synchronous resection of CRLM, suggesting that it has potential as a prognostic biomarker. Furthermore, ST analysis has revealed significant intratumoral and interlesional heterogeneity and identified the underlying transcriptomic programs driving each phenotype. Significance: Spatial transcriptomics uncovers heterogeneity between patients, between matched lesions in the same patient, and within individual lesions and identifies drivers of metastatic progression in colorectal cancer with reactive and suppressed immune microenvironments.

All supplementary figures and supplementary tables 1 and 2 from the manuscript

Gene Set Enrichment Analysis results obtained by interrogating ranked list of differentially expressed KM high vs KM low genes against the REACTOME curated gene set database using ClusterProfiler package

Supplementary details regarding methods and materials

SpatialDecon derived immune cell counts aggregated by Region, KM grade and KRAS status with pairwise comparison between groups. Mann-Whitney test used to determine statistical significance between groups

Strong immune responses in primary colorectal cancer correspond with better patient survival following surgery compared with tumors with predominantly stromal microenvironments. However, biomarkers to identify patients with colorectal cancer liver metastases (CRLM) with good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary colorectal cancer and CRLM and to interrogate the underlying functional biology that drives disease progression. Samples from patients undergoing synchronous resection of primary colorectal cancer and CRLM were evaluated in detail through histological assessment, panel genomic and bulk transcriptomic assessment, IHC, and GeoMx spatial transcriptomics (ST) analysis. High immune infiltration of metastases was associated with improved cancer-specific survival. Bulk transcriptomic analysis was confounded by stromal content, but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for type II IFN signaling and MHC-class II antigen presentation. In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T cells and neutrophils with enrichment of Notch and TGFβ signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcomes in patients undergoing synchronous resection of CRLM, suggesting that it has potential as a prognostic biomarker. Furthermore, ST analysis has revealed significant intratumoral and interlesional heterogeneity and identified the underlying transcriptomic programs driving each phenotype.Significance:Spatial transcriptomics uncovers heterogeneity between patients, between matched lesions in the same patient, and within individual lesions and identifies drivers of metastatic progression in colorectal cancer with reactive and suppressed immune microenvironments.

BackgroundPatients demonstrating strong immune responses to primary colorectal cancer (CRC) have a survival benefit following surgery, while those with predominantly stromal microenvironments do poorly. Biomarkers to identify patients with colorectal cancer liver metastases (CRLM) who have good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary CRC and CRLM, and to interrogate the underlying functional biology that drives disease progression.MethodsPatients undergoing synchronous resection of primary CRC and CRLM underwent detailed histological assessment, panel genomic and bulk transcriptomic assessment, immunohistochemistry (IHC) and GeoMx Spatial Transcriptomics (ST) analysis. Integration with genomic features, pathway enrichment analysis and immune deconvolution were performed.ResultsHigh-immune metastases were associated with improved cancer specific survival (HR, 0.36, P=0.01). Bulk transcriptomic analysis was confounded by stromal content but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for Type II Interferon signalling (NES=-2.05 P.AdjP.AdjP.Adj=0.022) and TGF-β (NES=2.2 P.Adj=0.02) signalling pathways at the metastatic tumor centre.ConclusionsHistological assessment stratifies outcome in patients undergoing synchronous resection of CRLM. ST analysis reveals significant intra-tumoral and inter-lesional heterogeneity with underlying transcriptomic programmes identified in driving each phenotype.TRANSLATIONAL RELEVANCEThe current study demonstrates that accurate histological assessment of immune cell infiltration and stromal content can define survival in patients following resection of oligometastatic liver disease when presenting synchronously with primary colorectal cancer. A spatial transcriptomic approach has demonstrated heterogeneity between patients, between matched lesions in the same patient and within individual lesions. Patients with high immune infiltrates at the invasive margin demonstrated lymphocytic infiltration and associated upregulated adaptive immune pathways in long term survivors. In specimens with low immune infiltrate at the tumor edge a significant reduction in survival was observed, this was determined by upregulated immunosuppressive pathways and a predominance of innate immune cells surrounding metastases. Spatial transcriptomics can be used to examine drivers of metastatic progression in CRC and identifies patients with reactive and suppressed immune microenvironments. Application across a larger cohort will build the cartography of CRLM, while in future, studies may assess application of this technology to pre and post treatment biopsy samples with the aim of predicting individual therapeutic responses. The current study has highlighted discrepancies between bulk and ST derived data whilst demonstrating accuracy of deconvoluted transcriptome to determine immune profiling. Now that ST strategies are becoming more achievable at scale, this has implications for the interpretation of the bulk transcriptomic signatures both of primary and metastatic CRC.

The tumour microenvironment is an important factor for colorectal cancer prognosis, affecting the patient's immune response. Immune checkpoints, which regulate the immune functions of lymphocytes, may provide prognostic power. This study aimed to investigate the prognostic value of the immune checkpoints TIM‐3, LAG‐3 and PD‐1 in patients with stage I–III colorectal cancer. Immunohistochemistry was employed to detect TIM‐3, LAG‐3, PD‐1 and PD‐L1 in 773 patients with stage I–III colorectal cancer. Immune checkpoint protein expression was assessed in tumour cells using the weighted histoscore, and in immune cells within the stroma using point counting. Scores were analysed for associations with survival and clinical factors. High tumoural LAG‐3 (hazard ratio [HR] 1.45 95% confidence interval [CI] 1.00–2.09, p = 0.049) and PD‐1 (HR 1.34 95% CI 1.00–1.78, p = 0.047) associated with poor survival, whereas high TIM‐3 (HR 0.60 95% CI 0.42–0.84, p = 0.003), LAG‐3 (HR 0.58 95% CI 0.40–0.87, p = 0.006) and PD‐1 (HR 0.65 95% CI 0.49–0.86, p = 0.002) on immune cells within the stroma associated with improved survival, while PD‐L1 in the tumour (p = 0.487) or the immune cells within the stroma (p = 0.298) was not associated with survival. Furthermore, immune cell LAG‐3 was independently associated with survival (p = 0.017). Checkpoint expression scores on stromal immune cells were combined into a Combined Immune Checkpoint Stromal Score (CICSS), where CICSS 3 denoted all high, CICSS 2 denoted any two high, and CICSS 1 denoted other combinations. CICSS 3 was associated with improved patient survival (HR 0.57 95% CI 0.42–0.78, p = 0.001). The results suggest that individual and combined high expression of TIM‐3, LAG‐3, and PD‐1 on stromal immune cells are associated with better colorectal cancer prognosis, suggesting there is added value to investigating multiple immune checkpoints simultaneously.

Histological ‘phenotypic subtypes’ that classify patients into four groups (immune, canonical, latent and stromal) have previously been demonstrated to stratify survival in a stage I–III colorectal cancer (CRC) pilot cohort. However, clinical utility has not yet been validated. Therefore, this study assessed prognostic value of these subtypes in additional patient cohorts along with associations with risk of recurrence and response to chemotherapy. Two independent stage I–III CRC patient cohorts (internal and external cohort) were utilised to investigate phenotypic subtypes. The primary endpoint was disease‐free survival (DFS) and the secondary endpoint was recurrence risk (RR). Stage II–III patients, from the SCOT adjuvant chemotherapy trial, were utilised to further validate prognostic value and for exploratory analysis assessing associations with adjuvant chemotherapy. In an 893‐patient internal cohort, phenotypic subtype independently associated with DFS (p = 0.025) and this was attenuated in stage III patients (p = 0.020). Phenotypic subtype also independently associated with RR (p

To understand the relationship between key non-canonical NF-κB kinase IKK-alpha(α), tumour mutational profile and survival in primary colorectal cancer.Immunohistochemical expression of IKKα was assessed in a cohort of 1030 patients who had undergone surgery for colorectal cancer using immunohistochemistry. Mutational tumour profile was examined using a customised gene panel. Immunofluorescence was used to identify the cellular location of punctate IKKα expression.Two patterns of IKKα expression were observed; firstly, in the tumour cell cytoplasm and secondly as discrete 'punctate' areas in a juxtanuclear position. Although cytoplasmic expression of IKKα was not associated with survival, high 'punctate' IKKα expression was associated with significantly reduced cancer-specific survival on multivariate analysis. High punctate expression of IKKα was associated with mutations in KRAS and PDGFRA. Dual immunofluorescence suggested punctate IKKα expression was co-located with the Golgi apparatus.These results suggest the spatial expression of IKKα is a potential biomarker in colorectal cancer. This is associated with a differential mutational profile highlighting possible distinct signalling roles for IKKα in the context of colorectal cancer as well as potential implications for future treatment strategies using IKKα inhibitors.

581 Background: Tumour budding is an independent prognostic factor in colorectal cancer and has recently been defined by the International Consensus Conference on Tumour Budding. The aim was to use the ITBCC budding evaluation method to examine relationships between tumour budding, tumour factors, tumour microenvironment, gene expression profiles and survival in patients with primary operable CRC. Methods: Hematoxylin and Eosin (H&E) stained slides of 953 CRC patients, diagnosed between 1997 and 2007 were evaluated for tumour budding according to the ITBCC-criteria. The tumour microenvironment was evaluated using tumour stroma percentage (TSP) and Klintrup–Makinen (KM) grade to assess the tumour inflammatory cell infiltrate. Differential gene expression was assessed using TempO-Seq gene expression profiling (BioSpyder Technologies Inc., CA, USA) using the Surrogate+Tox targeted panel (2,733 genes selected for biological diversity, maximal information content, and widespread pathway coverage). Results: High budding (n = 269/ 28%) was significantly associated with TNM stage (P < 0.001), venous invasion (P < 0.001), weak KM grade (P < 0.001), high TSP (P < 0.001) and reduced cancer specific survival (CSS) (HR = 5.04; 95% confidence interval [CI], 3.50-9.51; P < 0.001) and was independent of venous invasion, KM grade, and Ki67 proliferation index. RNA expression analysis was employed using TempO-Seq to determine differential gene expression between tumours with (n = 8) and without budding (n = 18). Three genes were identified as significantly differentially expressed: S100A2 (S100 calcium binding protein A2) was upregulated by 2.9 fold (padj < 0.00001); REG1A (regenerating family member 1 alpha) was downregulated by 4.7 fold (padj < 0.01) and LCN2 (lipocalin 2) was downregulated by 2.2 fold (padj < 0.01). Conclusions: Tumour budding stratifies patients’ survival in primary operable colorectal cancer and associates with differing gene expression profiles and factors of the tumour. Therefore, the ITBCC budding evaluation method should be used to assess tumour budding as supplement the TNM staging system and can help to further subdivide colorectal cancer into new prognostic groups.

Background Despite advances in therapies to treat breast cancer, over 100,000 patients die in the UK of this disease per year, highlighting the need to develop effect predictive and prognostic markers for patients with primary operable ductal breast cancer. Therefore, the aim of the present study was to examine the relationship between membranous, cytoplasmic and nuclear expression of focal adhesion kinase (phosphorylated at Y 397, Y 861 and Y 925), molecular subtypes, tumour microenvironment and survival in patients with primary operable ductal breast cancer. Methods Four hundred and seventy-four patients presenting between 1995 and 1998 with primary operable ductal breast cancer were included in this study. Using tissue microarrays expression of membranous, cytoplasmic and nuclear tumour cell phosphorylation of FAK at Y397, Y861 and Y925 was assessed, and associations with clinicopathological characteristics, tumour microenvironment and cancer-specific survival (CSS) were examined. Results No significant association was observed for ph-FAK Y861 with survival at all sites. However, high expression of membranous ph-FAK Y397 was associated with increased tumour grade (P Conclusion The results of the present study show that membranous and nuclear ph-FAK Y397 and cytoplasmic ph-FAK Y925 were associated with prognosis in patients with primary operable ductal breast cancer. In addition, high expression of nuclear ph-FAK Y397 was an independent prognostic factor in patients with primary operable ductal breast cancer and could be incorporated into clinical practice.

Breast cancer is a heterogeneous disease, which over time acquires various adaptive changes leading to more aggressive biological characteristics and development of treatment resistance. Several mechanisms of resistance have been established; however, due to the complexity of oestrogen receptor (ER) signalling and its crosstalk with other signalling networks, various areas still need to be investigated. This article focusses on the role of nuclear factor kappa B (NF-KB) as a key link between inflammation and cancer and addresses its emerging role as a key player in endocrine therapy resistance. Understanding the precise mechanism of NF-KB-driven endocrine therapy resistance provides a possible opportunity for therapeutic intervention.

Background: As new systemic therapies emerge for the treatment of breast cancer, new prognostic markers are required to help stratify patients into higher and lower risk groups to aid treatment decision making. Features of the tumour microenvironment, such as tumour necrosis, tumour-stroma percentage (TSP), and tumour budding have been shown to have prognostic value in some cancers. However, their role in breast cancer is unclear. Methods: Patients who underwent surgery for primary operable breast cancer in 2 centres between 1995-2007 and who had paraffin-embedded tissue blocks available were identified. Clinicopathological details and survival data were obtained from patient records. Haematoxylin & Eosin-stained slides were visually assessed within a set visual field for TSP (50% tumour stroma), tumour necrosis (25% necrosis) and tumour budding (20 buds). A combined score of tumour necrosis and tumour budding was then created. A score of 0 was assigned to tumours where both components were low, 1 to those where only one component was high, and 2 to those where both were high. Multivariate cox regression analysis was carried out for cancer specific survival (CSS). Results: A breast cancer cohort of 1301 patients was utilised, from which 1186 H&E slides were scored for necrosis, TSP and tumour budding. Median follow up was 158 months (26-183) and there were 234 breast cancer deaths. In the full cohort, necrosis (p Conclusions: A combined score of tumour necrosis and budding shows promise as a readily-available prognostic tool to aid treatment decision making in primary operable breast cancer, both by stratifying risk in ER negative disease, and by identifying a high-risk group in ER positive, node negative disease. Citation Format: Morrow ES, Gujam F, Mohammed Z, McMillan DC, Horgan PG, Roseweir AK, Edwards J. A combined score of tumour budding and tumour necrosis has prognostic value for cancer specific survival in both ER positive and ER negative primary operable breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-23.

Background There are limited treatments for triple negative breast cancer (TNBC) patients and an unmet need for targeted approaches in these patients. In the last 4-5 years, the prevalence of high androgen receptor (AR) expression in TNBCs has been noted in up to 50% of tumors suggesting it has clinical relevance. The non-canonical NF-kB pathway is also upregulated in this patient group and it has been reported that that there is crosstalk between these pathways. Therefore the aim of this study was to examine the expression of IKKα and AR in breast cancer tissue samples, to assess if combining these markers increased prognostic power. Methods Immunohistochemistry was performed on tissue microarray of 410 patients to assess proteins level of IKK alpha and AR. Protein expression levels were assessed using the weighted histoscore (WHS) method. The median was employed as the cut off for IKK alpha and 1% as cut off for AR. Expression was analyzed for associations with cancer-specific survival (CSS) and recurrence-free survival (RFS). Results In a cohort of 370 breast cancers nether AR nor IKK alpha alone or combined were associated with CSS or RFS. Stratifying patients by ER status did not impact CSS or RFS. However, in TNBC patients (n=82) high expression of AR was associated with shorter CSS (HR 2.55 95 CI 1.61-5.59, p=0.013). To assess if combining AR and IKK alpha increased prognostic power, AR and IKK alpha were combined into a single score: 0= low expression of both or high expression of one and 1= high expression of both. In the full cohort or when stratified by ER status the score was not associated with CSS or RFS, however in TNBC the combined score potentiated the effect observed with AR alone, (HR 1.68 95 CI 1.20-2.33, p=0.001). Patient CSS was stratified from 11.5 years to 4.6 years and was independently associated with CSS when compared with common clinicopathological factors (HR 1.56 95CI 1.11-2.21, p=0.011). In addition, the combined score was associated with decrease radiotherapy use (p=0.032), increased recurrence rate (p=0.014), decreased cytotoxic T cells (p=0.007), B cells (p=0.043) and macrophages (p=0.037). Conclusions A combined AR and IKK alpha score is an independent prognostic classification for patients with TNBC. Patients with high expression of both AR and IKK alpha a significantly reduced survival and were immune cell cold. This study suggests that this patient group will not benefit from immunotherapy but dual targeting with anti-androgens and IKK alpha selective inhibitors could offer a novel therapeutic strategy for this patient group. Citation Format: Roseweir AK, Khongthong P, Dickson K, Bennett L, Edwards J. Dual targeting of androgen receptor and IKK alpha is a potential therapeutic strategy for triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-13.

Over the last 15 years there has been a change in how we understand the impact of the interaction between the tumour and the host on cancer outcomes. From the simplistic view that the make-up of tumours cells largely determines their aggressiveness to a more complex view that the interaction between the products of tumour and host cell signal transduction pathways is crucial in determining whether the tumour cell is eliminated or survives in the host. Of the host cells, those with an immune/inflammatory function are most well documented to inhibit or promote tumour cell proliferation and dissemination. It is only in the last few years that there has been greater recognition of the impact of intracellular, cellular and systemic immune/inflammatory phenotypes on patient outcomes independent of current tumour staging and that these phenotypes are useful in informing oncological research and practice. In the present review we will examine the importance of inflammatory phenotypes at the intra-cellular, cellular and systemic levels on outcomes in patients with gastrointestinal cancer with focus on colorectal cancer. Based on these phenotypes we will examine and discuss the prospects for therapeutic intervention.

Tumour cell anaerobic metabolism has been reported to be a prognostic factor in colorectal cancer. The present study investigated the association between monocarboxylate transporter (MCT) 1, MCT 2, lactate dehydrogenase (LDH) 1 and LDH 5, the tumour microenvironment, and outcome in patients with colorectal cancer. A cohort of 150 patients with stage I-III CRC were utilised to assess tumour cell expression of MCT-1, MCT-2, LDH-1 and LDH-5 by immunohistochemistry. Expression levels were dichotomised and associations with tumour factors, the tumour microenvironment and survival analysed. Nuclear LDH-5 associates with poor prognosis (HR 1.68 95% CI 0.99-2.84, p = 0.050) and trends toward increased tumour stroma percentage (TSP, p = 0.125). Cytoplasmic MCT-2 also trends toward increased TSP (p = 0.081). When combined into a single score; nuclear LDH-5 + TSP significantly associated with decreased survival independent of stage (HR 2.61 95% CI 1.27-5.35, p = 0.009), increased tumour budding (p = 0.002) and decreased stromal T-lymphocytes (p = 0.014). Similarly, cytoplasmic MCT-2 + TSP significantly associated with decreased survival (HR 2.32 95% CI 1.31-4.11, p = 0.003), decreased necrosis (p = 0.039), and increased tumour budding (p = 0.004). The present study reports that the combination of TSP and nuclear LDH-5 was significantly associated with survival, increased tumour budding, and decreased stromal T-lymphocytes. This supports the hypothesis that increased stromal invasion promotes tumour progression via modulation of tumour metabolism. Moreover, MCT-2 and LDH-5 may provide promising therapeutic targets for patients with stromal-rich CRC.

Cancer treatments often reach a refractory period leading to treatment failure and patients developing disease recurrence. This can be due to tumour cells escaping the immune response and creating an immunosuppressive microenvironment enhancing cancer progression. Immunotherapy has become a promising tool for cancer treatment as it restores the anti-tumour response of the patient’s immune system. Immune checkpoint inhibitors are the most widely studied immunotherapies worldwide and are now approved for multiple cancers. However, chimeric antigen receptor (CAR)-T cell therapy has also shown promise by targeting T lymphocytes that are genetically modified ex vivo to express CARs and this is now approved to treat some haematological cancers. Although immunotherapy has shown successful treatment outcomes in multiple cancers, some patients do not respond to this treatment. Therefore, approaches to enhance the efficacy of immunotherapies are likely to be the key to improve their effectiveness. Therefore, combination therapies of checkpoint inhibitors +/− chemotherapy are at the forefront of current research. Furthermore, biomarkers that predict treatment response are now beginning to emerge. Additionally, utilising nanoparticles as a newly targeted drug delivery system to enhance CAR-T cell therapy may enhance the efficacy of the cells when re-infused within the patient. Even if efficacy is enhanced, severe immune-related adverse events (irAEs) occur that are life-threatening and could lead to therapy being stopped. Therefore, predictive biomarkers for toxicity are also needed to improve both the patient’s quality of life and treatment outcomes. This review will look at the current immunotherapies in clinical trials and discuss how to enhance their efficacy.

Background:\ud As the survival of castration‐resistant prostate cancer (CRPC) remains poor, and the nuclear factor‐κB (NF‐κB) pathways play key roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF‐κB pathway through generating inhibitory κB kinase subunit α (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p‐IKKα S180) and threonine 23 (p‐IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome.\ud \ud Methods:\ud A cohort of 115 patients with hormone‐naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'I survival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth.\ud \ud Results:\ud High levels of cytoplasmic IKKα were associated with a higher cancer‐specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log‐rank, 0.11‐0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27‐5.33; P = .01) and cytoplasmic p‐IKKα S180 (HR, 2.10; 95% CI, 1.17‐3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T‐cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate‐specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF‐κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF‐κB and Akt pathways correlated with members of the canonical NF‐κB pathway in CRPC.\ud \ud Conclusion:\ud The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p‐IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.

Background The Glasgow Microenvironment Score (GMS) combines peritumoural inflammation and tumour stroma percentage to assess interactions between tumour and microenvironment. This was previously demonstrated to associate with colorectal cancer (CRC) prognosis, and now requires validation and assessment of interactions with adjuvant therapy. Methods Two cohorts were utilised; 862 TNM I–III CRC validation cohort, and 2912 TNM II–III CRC adjuvant chemotherapy cohort (TransSCOT). Primary endpoints were disease-free survival (DFS) and relapse-free survival (RFS). Exploratory endpoint was adjuvant chemotherapy interaction. Results GMS independently associated with DFS (p = 0.001) and RFS (p p p = 0.001). In TransSCOT, chemotherapy type (pinteraction = 0.013), but not duration (p = 0.64) was dependent on GMS. Furthermore, GMS 0 significantly associated with improved DFS in patients receiving FOLFOX compared with CAPOX (HR 2.23 95% CI 1.19–4.16, p = 0.012). Conclusions This study validates the GMS as a prognostic tool for patients with stage I–III colorectal cancer, independent of TNM, with the ability to stratify both low- and high-risk disease. Furthermore, GMS 0 could be employed to identify a subset of patients that benefit from FOLFOX over CAPOX.

It is increasingly appreciated that host factors within the tumor center and microenvironment play a key role in dictating colorectal cancer (CRC) outcomes. As a result, the metastatic process has now been defined as a result of epithelial–mesenchymal transition (EMT). Establishment of the role of EMT within the tumor center and its effect on the tumor microenvironment would be beneficial for prognosis and therapeutic intervention in CRC. The present study assessed five immunohistochemical EMT markers within the tumor center on a 185 Stage II/III CRC patient tissue microarray. In 185 patients with CRC, cytoplasmic snail (HR 1.94 95% confidence interval [CI] 1.15–3.29, p = 0.012) and a novel combined EMT score (HR 3.86 95% CI 2.17–6.86, p < 0.001) were associated with decreased cancer‐specific survival. The combined EMT score was also associated with increased tumor budding (p = 0.046), and systemic inflammation (p = 0.007), as well as decreased memory T‐cells within the stroma (p = 0.030) and at the invasive margin (p = 0.035). Furthermore, the combined EMT score was associated with cancer‐specific survival independent of TNM‐stage (HR 4.12 95% CI 2.30–7.39, p < 0.001). In conclusion, a novel combined EMT score stratifies patient's survival in Stage II/III CRC and associates with key factors of tumor metastasis. Therefore, the combined EMT score could be used to identify patients at risk of micrometastases and who may benefit from standard adjuvant therapy, potentially in combination with EMT blockade.

// Antonia K. Roseweir 1, 2 , Pamela McCall 1 , Alison Scott 1 , Benjamin Liew 1 , Zhi Lim 1 , Elizabeth A. Mallon 3 , Joanne Edwards 1 1 Unit of Experimental Therapeutics, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, United Kingdom 2 Academic Unit of Surgery, School of Medicine, University of Glasgow, Royal Infirmary, Glasgow, United Kingdom 3 Department of Pathology, Southern General Hospital, Glasgow, United Kingdom Correspondence to: Antonia K. Roseweir, email: antonia.roseweir@glasgow.ac.uk Keywords: androgen receptor, phosphorylation, MAPK, breast cancer, triple negative Received: October 29, 2016 Accepted: March 10, 2017 Published: March 21, 2017 ABSTRACT 1.7 million cases of breast cancer are diagnosed every year with 522,000 deaths. Molecular classifications of breast cancer have resulted in improved treatments. However, treatments for triple negative breast cancer (TNBC) are lacking. Analysis of molecular targets for TNBC is a priority. One potential candidate is androgen receptor (AR) phosphorylation. This study assessed the role of AR phosphorylation at ser81/ser515 and their two upstream effectors, cyclin-dependent kinase 1 (pCDK1) and extracellular-regulated kinase 1/2 (pERK1/2) in 332 ductal breast cancer patients by immunohistochemistry. pERK1/2 combined with AR-515 associated with improved cancer-specific survival (CSS, p = 0.038), decreased size ( p = 0.001), invasive grade ( p < 0.001), necrosis ( p = 0.003), b-lymphocytes ( p = 0.020), molecular subtype ( p < 0.001) and estrogen receptor (ER)/progesterone receptor (PR)-status ( p < 0.001). The cohort was therefore stratified into ER+ve and ER-ve patients. In ER+ve tumours, pERK1/2 combined with AR-515 associated with improved CSS ( p = 0.038), smaller size ( p = 0.004), invasive grade ( p = 0.001), decreased b-lymphocytes ( p = 0.013) and increased plasma cells ( p = 0.048). In contrast, in TNBC patients, phosphorylation of AR-515 associated with poorer CSS ( p = 0.007). pERK1/2 combined with AR-515 associated with decreased inflammation ( p = 0.003), increased tumour stroma ( p = 0.003) and tumour budding ( p = 0.011), with trends towards decrease CSS ( p = 0.065) and macrophage levels ( p = 0.093). In Conclusions, AR-515 may be an important regulator of inflammation in breast cancer potential via ERK1/2 phosphorylation. AR-515 is a potential prognostic marker and therapeutic target for TNBC.

Background\ud\udIn colorectal cancer (CRC), inflammatory responses have been reported to associate with patient survival. However, the specific signalling pathways responsible for regulating inflammatory responses are not clear. Src family kinases (SFKs) impact tumourigenic processes, including inflammation.\ud\ud\udMethods\ud\udThe relationship between SFK expression, inflammatory responses and cancer specific survival (CSS) in stage I-III CRC patients was assessed using immunohistochemistry on a 272 patient discovery cohort and an extended 822 patient validation cohort.\ud\ud\udResults\ud\udIn the discovery cohort, cytoplasmic FGR associated with improved CSS (P = 0.019), with membrane HCK (p = 0.093) trending towards poorer CSS. In the validation cohort membrane FGR (p = 0.016), membrane HCK (p = 0.019), and cytoplasmic HCK (p = 0.030) all associated with poorer CSS. Both markers also associated with decreased proliferation and cytotoxic T-lymphocytes (all p

Background: Efforts to prognostically stratify colorectal cancer (CRC) patients using histological samples has led to the development of the Glasgow Microenvironment Score (GMS). However, the subjective nature of the GMS assessment criteria makes reproducing the score challenging. Therefore, the aim of the current study was to assess the clinical utility of an image analysis-based approach. Methods: In two cohorts of patients (a test cohort of 297 Stage II/III CRC patients from Norway and a validation cohort of 233 Stage II/III CRC patients from Scotland), one H&E section representing the deepest point of tumour invasion was assessed for Tumour Stroma Percentage (TSP) and the Klintrup-Makinen (KM) local immune grade to produce the GMS. A decision forest-based classifier was built to segment the tumour and stroma compartments, having been trained on a hand annotated subset of the test cohort. A UNET convolutional neural network was trained on the same training set as the decision forest algorithm to segment and quantify the tumour and stroma components. Each section was also annotated for the invasive margin and a manually built, threshold-based lymphocyte detection algorithm applied to quantify the local immune grade. Results: The decision forest classifier was able to stratify the test cohort patients for cancer specific survival (P = 0.009) based on the patients TSP, however failed to stratify patients from the validation cohort (P = 0.055) for TSP without retraining, therefore the deep learning approach was used for this cohort. Individually, the TSP (P = 0.001) generated from the UNET based classifier and the quantification of the local immune grade (P = 0.005), both significantly stratified validation cohort patients for survival. When the individual scores were combined into a digital version of the GMS, not only did the image analysis approach significantly stratify patient survival (P < 0.0001), it also significantly correlated with the manual scores both statistically (Chi Square = 32, P = 1.5 × 10−8) and in terms of survival estimates per patient group (GMS 0 - manual = 159 months vs digital = 159 months; GMS 1 – manual = 136 months vs digital = 139 months ; GMS2 – manual 101 months vs 97 months). To negate the need for any annotation prior to analysis, the lymphocyte detection algorithm was applied to the stroma areas determined by the UNET algorithm. This fully automated digital version of the GMS was able to stratify validation cohort patients into the 3 prognostic groups based on CSS (P < 0.001), however, GMS 0 had a lower survival estimate (automated = 148 vs annotated = 159 months). Conclusion: An H&E based image analysis approach to the GMS, on a single H&E slide, holds prognostic significance for CRC patients and correlates significantly with manual pathological assessment, making the assessment of the GMS more time efficient, reproducible, and clinically relevant. Citation Format: Christopher J. Bigley, John Waller, Alison Bigley, Karin Oien, Joanne Edwards, Antonia Roseweir. Deep learning-based image analysis of the histological Glasgow Microenvironment Score in patients with colorectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-001.

Background: Colorectal cancer (CRC), is an inflammatory disease, with a mix of lymphoid and myeloid cells found within the tumor microenvironment. These immune cells can function as antitumorigenic or protumorigenic; therefore, it is important to evaluate all immune cell types within a patient’s tumor to understand the effects of each immune landscape on patient survival. Therefore, this study investigated the immune landscape of patients with stage I-III CRC to assess the effect of individual immune cell types and different immune landscapes on prognosis. Method: Immunohistochemistry for lymphoid and myeloid immune cells (CD3, CD8, FoxP3, CD45RO, CD68, CD80, CD163, and CD66b) was performed in 930 patients with stage I-III CRC on tissue microarrays. Cell counts were performed at three hotspots within the tumor nests and stroma separately and an average count was taken. Receiver operated curve (ROC) analysis was utilized to define cut-offs for weak and strong expression. Immune cells and immune landscapes were analyzed for associations with cancer-specific survival (CSS). Results: For tumor-infiltrating and stromal lymphoid cells, high CD3+ (both p Conclusion: The results of this study confirm that different immune cell subtypes associate with different prognosis in patients with stage I-III CRC. Elevated lymphoid cells always associate with improved prognosis independent of the presence of myeloid cells, whereas patients with only elevated myeloid cells have a poorer prognosis. Overall, the results suggest that assessing different immune landscapes utilizing broad markers for T lymphocytes, macrophages, and granulocytes could aid clinicians with patient prognosis and help target appropriate patients for immunotherapy. Citation Format: Jitwadee Inthagard, Donald McMillan, Joanne Edwards, Antonia Roseweir. The prognostic impact of different immune landscapes in patients with stage I-III colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A102.

Background: Endocrine therapy is the standard of care for patients with estrogen receptor (ER)-positive breast cancer. However, endocrine therapy resistance is still a significant challenge for clinical practice. Nuclear factor Kappa B (NF-KB) is one of the most extensively studied pathways that has been demonstrated as a critical component in the endocrine resistance mechanism. In addition, NF-KB is a crucial factor that induces PD-L1 stabilization, resulting in enhancement of tumor immune evasion and potentially leading to endocrine therapy resistance. Studies that investigate correlations between NF-KB pathway and immunologic markers are limited. Therefore, the present study aims to examine the association between IKKα, a critical kinase in the NF-KB pathway, PD-1/PD-L1, and clinical outcomes in patient with ER-positive tamoxifen-treated breast cancer. Methods: Immunohistochemistry was performed to examine IKKα, IKKα phosphorylated at threonine 23 (p-IKKαT23) or serine 176 (p-IKKαS176), PD-L1, and PD-1 on a tissue microarray of the patients with early-stage ER-positive breast cancer. The median value was used as the cut-off point to determine low or high expression. Kaplan-Meier curves and log-rank analysis were used to analyze an association between these markers and clinical outcomes in terms of cancer-specific survival (CSS), recurrence-free survival (RFS), and recurrence on tamoxifen (RoT). Result: A total of 392 patients were included. High cytoplasmic IKKα was significantly associated with decreased CSS (p=0.012), RFS (p=0.005), and RoT (p=0.010). High nuclear p-IKKα S176 was significantly associated with decrease RoT (p=0.010). High nuclear p-IKKαT23 was significantly associated with decreased CSS (p=0.023), RFS (p=0.036), and RoT (p=0.015). High PD-L1 on tumor cells was significantly associated with decreased CSS (p Conclusion: Increased expression of cytoplasmic and phosphorylated IKKα, as well as PD-L1 was significantly associated with poor clinical outcomes. However, increased PD-1 was associated with improved outcomes. In addition, combined high expression of cytoplasmic IKKα or nuclear p-IKKαT23 and PD-L1 was also significantly associated with poor clinical outcomes, indicating that in the tumor cell, activation of NF-kB pathway together with activation of PD-L1 provide additional prognosis that associates with decreased clinical outcomes and therefore could be employed as a prognostic marker. Citation Format: Phungern Khongthong, Linsay Bennett, Jean Quinn, Elizabeth Mallon, Antonia Roseweir, Joanne Edwards. The association between IKKα, immunologic markers, and clinical outcomes in patients with ER-positive tamoxifen-treated breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B49.

213 Background: The tumour microenvironment is an important determinant of survival in patients with colon cancer. Although the generalised inflammatory infiltrate as assessed by Klintrup-Mäkinen (KM) grade is a stage-independent prognostic marker, immunohistochemical staining for specific immune cell populations, such as T-cells, may have greater prognostic value. The present study examines the clinical utility of combined assessment of KM grade in addition to cytotoxic (CD8) and regulatory (FoxP3) T-cells. Methods: KM, CD8 and FoxP3 were assessed retrospectively in a cohort of 520 patients with stage I-III colon cancers. The relationship between KM grade, T-cell density and cancer-specific survival was examined. Results: KM was high in 33% of patients, of which: 16% had high FoxP3, 12% high CD8 and 56% both FoxP3 and CD8, while the remaining 15% were high KM alone. Conversely, 67% had low KM and of these: 47% had high CD8 alone or with FoxP3; 27% were high for FoxP3 alone and 25% had no inflammatory infiltrate. KM in the presence of other T-cells resulted in good outcome (Table), whereas CD8 with low KM with or without FoxP3 resulted in intermediate outcome. FoxP3 alone with low KM resulted in poor outcome. Similarly, KM alone without T-cells resulted in poor outcome. When no inflammatory cells were present, outcomes were comparable to KM or FoxP3 alone. Conclusions: A co-ordinated immune response results in good outcome and this can be assessed using a combination of a simple H&E based method (KM) in addition to T-cell markers. Where KM is low, despite the presence of cytotoxic T-cells, survival outcome is worse. Conversely, high KM in the absence of T-cells, or FoxP3 alone, are associated with poor outcome, comparable to no inflammatory response. [Table: see text]

205 Background: Histological phenotypic subtypes have been proposed that stratify survival in a discovery cohort of patients with stage I-III colorectal cancer (CRC). However, clinical utility has not been validated nor associations with recurrence and chemotherapy assessed. Therefore, this study assessed prognostic value in patients with stage I-III CRC as well as predictive value for recurrence and chemotherapy response. Methods: Two independent stage I-III CRC patient cohorts were utilized to assess associations between phenotypic subtypes, survival, and recurrence. Stage II-III patients, from the SCOT adjuvant chemotherapy trial, were utilized to assess associations between phenotypic subtypes and adjuvant chemotherapy response. Log rank analysis compared immune and stromal subtypes. Results: In an 867-patient internal cohort, phenotypic subtype stratified patients by disease-free survival (DFS) (HR 2.18 95% CI 2.26-4.47, p < 0.001); independent of stage and location. The stromal subtype also predicted increased local and distant recurrence (p < 0.001). In a 146-patient external validation cohort, phenotypic subtype significantly stratified patients by DFS (HR 3.43 95% CI 1.60-7.35, p = 0.001). In 1343 SCOT trial patients, phenotypic subtype significantly stratified patients by DFS (HR 1.59 95% CI 1.13-2.25, p = 0.010). Furthermore, there was evidence that the effect of regimen depended on phenotypic subtype (p = 0.048), only significantly stratifying DFS in patients receiving FOLFOX (HR 3.73 95% CI 1.58-8.81, p = 0.003) but not CAPOX (HR 0.84 95% CI 0.56-1.26, p = 0.396) adjuvant chemotherapy. Interestingly, the immune subtype associated with improved DFS in patients receiving FOLFOX compared to CAPOX adjuvant chemotherapy (HR 3.40 95% CI 1.41-8.19, p = 0.006). Whereas patients with a stromal subtype trended towards improved DFS in patients receiving CAPOX compared to FOLFOX adjuvant chemotherapy (HR 0.72 95% CI 0.50-1.05 p = 0.088). Conclusions: Histological phenotypic subtypes are an effective independent prognostic classification for patients with stage I-III CRC that can predict response to FOLFOX adjuvant chemotherapy as well as the presence of local and distant recurrence.

206 Background: The Glasgow Microenvironment Score (GMS), comprised of assessment of the tumour inflammatory cell infiltrate (using Klintrup-Mäkinen (KM) grade) and tumour-associated stroma (TSP), has been reported as a stage-independent prognostic score in patients with colorectal cancer. The present study aims to validate the GMS and examines its prognostic utility in the context of stage and MMR status. Methods: Patients who had undergone resection of stage I-III colon cancer were included ( n= 495). GMS was scored by combining KM and TSP as follows: high KM scores 0; low KM with low TSP scores 1; low KM and high TSP scores 2. Cancer specific (CSS) and overall survival (OS) were primary endpoints. Subgroup analysis was performed to assess the utility of GMS according to TNM, venous invasion and MMR status. Results: There were 30% of patients with GMS 0, 56% with GMS 1 and 14% with GMS 2. Five-year survival for GMS 0, 1 and 2 across the whole cohort were 89%, 74% and 66%, respectively. GMS was associated with age, mode of presentation, TNM, venous invasion and MMR status. On multivariate analysis, GMS was independently associated with CSS (HR 1.35, 95% CI: 1.02-1.79, p= 0.04) and OS (HR 1.23, 95% CI: 1.02-1.48, p= 0.03); this was independent of emergency presentation ( p< 0.01), T-stage ( p= 0.03) and N-stage ( p< 0.001) for CSS. Subgroup analysis found that GMS was able to stratify CSS regardless of node-negative or node-positive disease (both p< 0.01), venous invasion ( p< 0.05), elective presentation ( p< 0.01) and MMR-proficient tumours ( p< 0.001), although it was not able to stratify emergency presentation ( n= 154) or MMR-deficient disease ( n= 102) due to small sample size. Universally, the prognosis for GMS 0 was good, but was poor for GMS 2. The prognosis for GMS 1 varied depending on MMR and nodal status. Conclusions: This study validates the use of the GMS as an independent prognostic pathology-based tool for stratification of colon cancer. It could be readily applied to routine clinical practice and may be used to aid decision making regarding adjuvant treatments in colorectal cancer. It should be further validated in prospective randomised trials.

221 Background: 5-year survival of patients with resectable colorectal liver metastases is 25-40%. Mechanisms of disease progression are heterogenous and do not follow a clearly defined pathway from genotype to phenotype. In stage I-III colorectal cancer (CRC), patients with high tumor stroma exhibit poor prognosis, while those with high immune cell infiltrate do well following resection. We hypothesise that stromally-dense phenotypes lead to T cell exclusion, myeloid cell accumulation and aggressive metastatic progression. Here, we examine relationships between histological tumor phenotype, cellular infiltrate and outcomes in metastatic CRC. Methods: A unique cohort of synchronously resected primary CRC and matched liver metastases (n = 46) were assessed for immune cell infiltration (CD3, CD4, CD8, CD68, CD66b), inflammatory signalling (CXCR2, PDL-1, MMP9) and hypoxia (CAIX) using immunohistochemistry. Tumors were phenotypically subtyped using immune infiltrate (Klintrup-Makinen Grade (KM)), stromal invasion (tumor-stroma percentage (TSP)) and proliferation (Ki67). Results: Phenotypic subtype of primary tumors was predictive of metastatic subtype (rho = 0.522, p = 0.003). Immune phenotypes were associated with good prognosis and stromal phenotypes with poor prognosis (p = 0.004). Infiltration of macrophages and granulocytes associated with poor outcomes (p = 0.018) and increased CXCR2 expression (p = 0.03) at both sites. Increased CXCR2+ cells and macrophages at both sites associated with stromal phenotype (p = 0.02), tumour budding (p = 0.002), low KM grade (p = 0.05) and poor prognosis (p = 0.002). Macrophage and MMP9 levels increased in metastases compared to primary tumour, but no changes were seen in lymphocyte infiltration, CXCR2 and CD66b. Conclusions: Density of immune cell infiltrate, in the primary and metastatic niche, conferred good prognosis. In contrast, stromal, myeloid rich tumors convey poor prognosis. This clinically relevant and histologically efficient process permits segregation of disease and supports further study of relationships in the tumour microenvironment of CRC in the context of chemotherapy to better target therapeutics to individual patients.

Tumour cell anaerobic metabolism has been reported to be a prognostic factor in colorectal cancer. The present study investigated the association between monocarboxylate transporter (MCT) 1, MCT 2, lactate dehydrogenase (LDH) 1 and LDH 5, the tumour microenvironment, and outcome in patients with colorectal cancer. A cohort of 150 patients with stage I-III CRC were utilised to assess tumour cell expression of MCT-1, MCT-2, LDH-1 and LDH-5 by immunohistochemistry. Expression levels were dichotomised and associations with tumour factors, the tumour microenvironment and survival analysed. Nuclear LDH-5 associates with poor prognosis (HR 1.68 95% CI 0.99-2.84, p = 0.050) and trends toward increased tumour stroma percentage (TSP, p = 0.125). Cytoplasmic MCT-2 also trends toward increased TSP (p = 0.081). When combined into a single score; nuclear LDH-5 + TSP significantly associated with decreased survival independent of stage (HR 2.61 95% CI 1.27-5.35, p = 0.009), increased tumour budding (p = 0.002) and decreased stromal T-lymphocytes (p = 0.014). Similarly, cytoplasmic MCT-2 + TSP significantly associated with decreased survival (HR 2.32 95% CI 1.31-4.11, p = 0.003), decreased necrosis (p = 0.039), and increased tumour budding (p = 0.004). The present study reports that the combination of TSP and nuclear LDH-5 was significantly associated with survival, increased tumour budding, and decreased stromal T-lymphocytes. This supports the hypothesis that increased stromal invasion promotes tumour progression via modulation of tumour metabolism. Moreover, MCT-2 and LDH-5 may provide promising therapeutic targets for patients with stromal-rich CRC.

Background: In the United Kingdom, 8,000 cases of renal cancer are diagnosed each year, with a 5-year survival rate of 50%. Treatment options are limited; a potential therapeutic target is the non-canonical nuclear factor-kappa B (NF-κB) pathway. This pathway plays a role in multiple oncogenic processes in solid tumors. The aim of this study was to investigate the non-canonical nuclear factor pathway in renal cell carcinoma (RCC). Materials and Methods: NIK, IKKα, and RelB were investigated via immunohistochemistry in a cohort of 192 patients with clear cell renal cancer. Results: High cytoplasmic NIK was associated with poorer cancer-specific survival (p = 0.006) and 10-year survival stratified from 85% (low) to 65% (high, p = 0.005). Similarly, high cytoplasmic RelB was associated with poorer cancer-specific survival (p = 0.041) and 10-year survival stratified from 88% (low) to 73% (high, p = 0.030). When clinicopathological characteristics were assessed, cytoplasmic NIK was associated with survival (p = 0.014), whereas cytoplasmic RelB was associated with increased tumor grade (p = 0.020) and decreased inflammation (p = 0.019). Upon multivariate analysis, it was found that cytoplasmic NIK was independently associated with cancer-specific survival (p = 0.009). Conclusions: The non-canonical NF-κB pathway is associated with poorer cancer-specific survival in RCC patients, making it a viable target for therapeutic intervention. Furthermore, cytoplasmic NIK is a potential prognostic biomarker for this disease.

Background\ud \ud In colorectal cancer (CRC), BRAF mutations influence tumour progression. In mismatch repair-deficient (dMMR) tumours, BRAF mutations are associated with a good prognosis, whereas in MMR-competent tumours, they are detrimental. The differential expression of the downstream MAPK pathway members, which are constitutively activated in BRAF mutant patients, may account for these differences.\ud \ud Methods\ud \ud Phosphorylation of ERK, p38MAPK and JNK was assessed by immunohistochemistry, utilising CRC tissue microarrays. A discovery cohort (n = 187) and a validation cohort (n = 801) were analysed for associations with BRAF mutations, clinicopathological characteristics and cancer-specific survival (CSS).\ud \ud Results\ud In 801 CRC patients, nuclear ERK phosphorylation (HR 0.65 95% CI 0.48–0.88, p = 0.004) and the combined nuclear pERK/p-p38 score (HR 0.61 95% CI 0.45–0.82, p = 0.001) were independently associated with CSS, and were further associated with increased BRAF mutations (p = 0.003 and p = 0.002). When stratified for BRAF status, only MMR-competent patients harbouring the mutation and a strong combined nuclear pERK/p-p38 score (HR 0.49 95% CI 0.27–0.89, p = 0.016) demonstrated improved CSS. This improvement in CSS was specific to stage III CRC (HR 0.25 95% CI 0.10–0.64, p = 0.002).\ud \ud Conclusions\ud \ud MMR-competent stage III tumours harbouring BRAF mutations have an improved prognosis when strong nuclear phosphorylation of both ERK and p38MAPK is present.

Background:\ud Aromatase inhibitors improve disease-free survival compared with tamoxifen in postmenopausal women with hormone receptor–positive breast cancer. The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial compared exemestane monotherapy with sequential therapy of tamoxifen followed by exemestane. The trial failed to show a statistically significant difference between treatment arms. A robust translational program was established to investigate predictive biomarkers.\ud \ud Methods:\ud A tissue microarray was retrospectively constructed using a subset of patient tissues (n = 4631) from the TEAM trial (n = 9766). Immunohistochemistry was performed for biomarkers, classed into three groups: MAPK pathway, NF-kappa B pathway, and estrogen receptor (ER) phosphorylation. Expression was analyzed for association with relapse-free survival (RFS) at 2.5 and 10 years and treatment regimen using Kaplan-Meier curves and log-rank analysis. All statistical tests were two-sided.\ud \ud Results:\ud In univariate analysis, ER167 (hazard ratio [HR] = 0.71, 95% confidence interval [CI] = 0.59 to 0.85, P < .001), IKKα (HR = 0.74, 95% CI = 0.60 to 0.92, P = .005), Raf-1338 (HR = 0.64, 95% CI = 0.52 to 0.80, P < .001), and p44/42 MAPK202/204 (HR = 0.77, 95% CI = 0.64 to 0.92, P = .004) were statistically significantly associated with improved RFS at 10 years in patients receiving sequential therapy. Associations were strengthened when IKKα, Raf-1338, and ER167 were combined into a cumulative prognostic score (HR = 0.64, 95% CI = 0.52 to 0.77, P

Gammadelta T (γδT) lymphocytes have provoked interest in oncology, particularly as regards their potential use in immunotherapy, because of their unique ability to recognise antigens without a requirement for major histocompatibility complex antigen presentation, and to quickly activate an anti-tumour response. However, work in some cancers has suggested that they also have pro-tumourigenic activity. Their role in breast cancer is unclear. This review outlines the evidence to date in in vitro studies, in vivo mouse models and in human studies regarding the role of γδT lymphocytes in breast cancer. We describe the seemingly opposing roles of the predominantly circulating Vγ9Vδ2+ subtype, which can suppress tumour growth through direct cytotoxicity, induction of apoptosis and inhibition of angiogenesis, and the predominantly tumour-infiltrating γδ1+ subtype which can promote tumour growth and spread through immunosuppressant effects. We summarise the evidence in breast cancer for the mechanisms of action of γδT lymphocytes and describe how factors in the tumour microenvironment may affect their function, polarising them towards a pro-tumourigenic, immune-suppressing role. We also describe the experience to date of γδT lymphocytes in immunotherapy for breast cancer and suggest the direction of work going forward, particularly as regards different breast cancer subtypes.

Kisspeptin signalling is indispensable for fertility, stimulating gonadotropin-releasing hormone (GnRH) secretion and mediating gonadal steroid feedback on GnRH neurons. Moreover, kisspeptin neurons have been implicated in other non-reproductive neuroendocrine roles. Kisspeptin appears to also regulate growth hormone secretion but much of the data appear contradictory. We sought to clarify a potential role of kisspeptin in growth hormone (GH) regulation by examining the effect of kisspeptin antagonists on GH secretion in ewes under various physiological conditions. Our data show clear and robust increases in GH secretion following lateral ventricle or third ventricle infusion of kisspeptin antagonists p-234 and p-271 in either ovariectomized or anestrous ewes. Central infusion of kisspeptin-10 had no effect on GH secretion. To determine the level at which kisspeptin may influence GH secretion, we examined expression of the cognate kisspeptin receptor, GPR54, in pituitary cells and showed by immunocytochemistry that the majority of somatotropes express GPR54 while expression was largely negative in other pituitary cells. Overall, we have demonstrated that blocking kisspeptin signalling by antagonists stimulates GH secretion in ewes and that this is likely mediated by inhibiting endogenous kisspeptin activation of GPR54 expressed on somatotropes. The findings suggest that endogenous kisspeptin inhibits GH secretion through GPR54 expressed on somatotropes.

STAT1 loss has previously been implicated in cell line studies to modify prostate cancer cell growth and survival, however the clinical significance of this has not previously been established. This study investigated if STAT1 loss was associated with patient outcome measures and the phenotypic consequence of STAT1 silencing. STAT1 expression was assessed in two patient cohorts with localised (n = 78) and advanced prostate cancer at initial diagnosis (n = 39) by immunohistochemistry (IHC). Impact of STAT1 silencing on prostate cancer cells lines was assessed using Cell Death detection ELISA, TLDA gene signature apoptosis arrays, WST-1 assay, xCELLigence system, clonogenic assay, and wound healing assay. In the localised patient cohort, low expression of STAT1 was associated with shorter time to disease recurrence (3.8 vs 7.3 years, P = 0.02) and disease specific survival (6.6 vs 9.3 years, P = 0.05). In the advanced patient cohort, low expression was associated with shorter time to disease recurrence (2.0 vs 3.9 years, P = 0.001). When STAT1 was silenced in PC3 cells (AR negative) and LNCaP cells (AR positive) silencing did not influence levels of apoptosis in either cell line and had little effect on cell viability in the LNCaP cells. In contrast, STAT1 silencing in the PC3 cells resulted in a pronounced increase in cell viability (WST-1 assay: mock silenced vs STAT1 silenced, P

It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.

Colorectal cancer (CRC) is the second most common cause of cancer death in Europe. Although outcomes have improved, it is clear that from a genomic standpoint CRC is not one disease, but a heterogeneous group of malignancies that arise within one organ. Given that different subtypes have different outcomes, the ability to subtype tumours in the clinic would be highly favourable, enabling optimal treatment for individual patients. In 2015, a consortium proposed four consensus subtypes for CRC (MSI immune, canonical, metabolic, and mesenchymal) based on six classifications systems reported to have prognostic value. However, genomic assessment of tumours is not readily translated into routine pathology with a need for standardisation and reproducibility of assessment. Immunohistochemistry is widely used in routine pathology, and would present a more readily translatable method for subtyping CRC tumours. Therefore, the literature was reviewed to characterise the genomic and phenotypic features associated with each subtype, with the aim of enabling subtyping of CRC to be taken forward into routine clinical practice.

707 Background: Although inextricably linked, both comorbidity and systemic inflammatory responses have been shown to determine survival in patients undergoing surgery for colorectal cancer (CRC). The present study examines the interrelationships between comorbidity (ASA grade) and systemic inflammation (modified Glasgow Prognostic Score (mGPS)) in patients from the ScotScan dataset. Methods: Clinicopathological characteristics and outcome of consecutive patients undergoing potentially curative resection of TNM I-III CRC in Glasgow Royal Infirmary (Scotland) and Sørlandet Hospital (Norway) were prospectively collected. ASA grade and mGPS (0-CRP ≤ 10mg/L, 1-CRP > 10mg/L, 2-CRP > 10mg/L and albumin < 35g/L) prior to surgery was recorded and relationship with overall survival (OS) examined. Results: 2,295 patients (Scotland: n = 1,234 , Norway: n = 1,061) were included. Patients from Norway were more likely to be older, female and have higher ASA grade (all P < 0.001), and more likely to have colon cancer (76% vs. 67%, P < 0.001). Patients from Norway were less likely to be systemically inflamed (mGPS = 0: 72% vs. 65%, P < 0.001), even after propensity score matching ( n = 736, OR 0.36 95%CI0.25-0.51, P < 0.001). ASA grade and mGPS were significantly associated; 21% of ASA 1 patients had mGPS ≥ 1 compared to 41% of ASA four patients ( P < 0.001). In the propensity-matched cohort, both increasing ASA (HR 1.98 95% CI1.57-2.49, P < 0.001) and mGPS (HR 1.20 95% CI1.02-1.41, P = 0.027) were associated with OS independent of age, N stage and adjuvant therapy use; results in the whole cohort were similar. The combination of ASA grade and mGPS was examined with respect to OS in patients with stage II-III CRC (Table 1). In patients with stage II disease, 3-year OS was stratified from 96% (ASA 1, mGPS0) to 67% (ASA 3, mGPS2) ( P < 0.001); in patients with stage II disease, 3-year OS was stratified from 84% to 44% ( P < 0.001). Conclusions: Using a large, prospectively collected dataset of patients undergoing resection of CRC in two countries, the results of the present study confirm the independent prognostic value of measures of comorbidity and systemic inflammation prior to surgery.

// Antonia K. Roseweir 1, 2 , Arfon G.M.T. Powell 2, 3 , Lindsay Bennett 2 , Hester C. Van Wyk 1 , James Park 1 , Donald C McMillan 1 , Paul G. Horgan 1 , Joanne Edwards 2 1 Academic Unit of Surgery, School of Medicine, University of Glasgow, Royal Infirmary, Glasgow, United Kingdom 2 Unit of Experimental Therapeutics, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, United Kingdom 3 Division of Cancer and Genetics, Cardiff University, Heath Park, Cardiff, United Kingdom Correspondence to: Antonia K. Roseweir, email: antonia.roseweir@glasgow.ac.uk Keywords: colorectal cancer, PTEN, Akt, COX-2, inflammation Received: June 30, 2016 Accepted: September 04, 2016 Published: September 20, 2016 ABSTRACT In patients with colorectal cancer (CRC), local and systemic inflammatory responses have been extensively reported to associate with cancer survival. However, the specific signalling pathways responsible for inflammatory responses are not clear. The PTEN/Akt pathway is a plausible candidate as it may play a role in mediating inflammation via COX-2, and has been associated with cancer progression. This study therefore examined the relationship between tumour PTEN/Akt/COX-2 expression, inflammatory responses and survival in CRC patients using a tissue microarray. In 201 CRC patients, activation of tumour-specific PTEN/Akt significantly associated with poorer CSS (12.0yrs v 7.3yrs, P =0.032), poorer differentiation ( P =0.032), venous invasion ( P =0.008) and peritoneal involvement ( P =0.004). Patients were stratified for peri-nuclear expression of COX-2 to examine associations with inflammatory responses. In patients with absent peri-nuclear COX-2 expression, activation of tumour-specific PTEN/Akt significantly associated with poorer CSS (11.9yrs v 5.4yrs , P =0.001), poorer differentiation ( P =0.018), venous invasion ( P =0.003) and peritoneal involvement ( P =0.001). However, no associations were seen with either the local or systemic inflammatory responses. In CRC patients, tumour-specific PTEN/Akt pathway activation was significantly associated with poorer CSS, particularly when peri-nuclear COX-2 expression was absent. However, activation of the PTEN/Akt pathway appears not to be responsible for the regulation of inflammatory responses.

Background: 8000 cases of renal cancer are diagnosed each year in the UK, with a five-year survival rate of 50 %.\ud Treatment options are limited; a potential therapeutic target is the Src family kinases (SFKs). SFKs have roles in\ud multiple oncogenic processes and promote metastases in solid tumours. The aim of this study was to investigate\ud SFKs as potential therapeutic targets for clear cell renal cell carcinoma (ccRCC).\ud Methods: SFKs expression was assessed in a tissue microarray consisting of 192 ccRCC patients with full clinical\ud follow-up. SFK inhibitors, dasatinib and saracatinib, were assessed in early ccRCC cell lines, 786-O and 769-P and a\ud metastatic ccRCC cell line, ACHN (± Src) for effects on protein expression, apoptosis, proliferation and wound\ud healing.\ud Results: High nuclear expression of Lyn and the downstream marker of activation, paxillin, were associated with\ud decreased patient survival. Conversely, high cytoplasmic expression of other SFK members and downstream marker\ud of activation, focal adhesion kinase (FAK) were associated with increased patient survival. Treatment of non-metastatic\ud 786-O and 769-P cells with dasatinib, dose dependently reduced SFK activation, shown via SFK (Y419) and FAK (Y861)\ud phosphorylation, with no effect in metastatic ACHN cells. Dasatinib also increased apoptosis, while decreasing\ud proliferation and migration in 786-O and 769-P cell lines, both in the presence and absence of Src protein.\ud Conclusions: Our data suggests that nuclear Lyn is a potential therapeutic target for ccRCC and dasatinib\ud affects cellular functions associated with cancer progression via a Src kinase independent mechanism.

Kisspeptin inhibits cancer cell metastasis and placental trophoblast cell migration. Kisspeptin gene expression in the placenta and circulating kisspeptin levels change during normal pregnancy and they are altered in preeclampsia. We therefore assessed the effect of kisspeptin-10 on the in vitro migration of a human placental cell line derived from first trimester extravillious trophoblasts (HTR8SVneo). HTR8SVneo cells specifically bound 125 I-Kisspeptin-10 but kisspeptin-10 did not induce inositol phosphate production. Cell migration was inhibited by kisspeptin-10 with a maximal inhibition at 100 nM. The signaling pathways involved in inhibition of cell migration were examined. Treatment with kisspeptin-10 elicited phosphorylation of GSK3 beta at Ser 9 (which inhibits activity), with a 3-fold increase at 5 min. Transient phosphorylation of ERK1/2 and p38MAPK peaked at 10 min. Phosphorylation of focal adhesion kinase (FAK) at Tyr 925 increased 3-fold at 10 min. Inhibition of GSK3 beta correlated with release of beta-catenin into the cytoplasm. These signaling events were differentially blocked by inhibitors of G q/11 , Src, EGFR, PI(3)K, PKC and MEK. The data suggest that kisspeptin/GPR54 EGF-receptor transactivation leads to phosphorylation of ERK1/2, causing activation of p90rsk which in turn inhibits GSK3 beta via Ser 9 phosphorylation. Inactivation of GSK3 beta results in release of beta-catenin into the cytoplasm, affecting cell–cell adhesion and Tyr 925 phosphorylation of FAK, which increases phosphorylation of ERK1/2 via RAS/Raf-1 creating a feedback loop to enhance the effects on migration. These findings indicate that kisspeptin-10 inhibits the migration of human placental trophoblast-derived HTR8SVneo cells by stimulating complex ERK1/2–p90rsk–GSK3 beta–FAK feedback interactions.

Kisspeptins stimulate GnRH and thus gonadotropin secretion. Kisspeptin-10 is the minimal kisspeptin sequence with full intrinsic bioactivity, but it has not been studied in man.We investigated our hypothesis that kisspeptin-10 increases GnRH and thus LH pulse frequency.The dose response of kisspeptin-10 was investigated by administering iv bolus doses (0.01-3.0 μg/kg) and vehicle to healthy men. Effects on LH pulse frequency and size were determined by deconvolution analysis during infusion of kisspeptin-10 for up to 22.5 h.Intravenous bolus kisspeptin-10 resulted in a rapid and dose-dependent rise in serum LH concentration, with maximal stimulation at 1 μg/kg (4.1 ± 0.4 to 12.4 ± 1.7 IU/liter at 30 min, P0.001, n = 6). Administration of 3 μg/kg elicited a reduced response vs. 1 μg/kg (P0.05). Infusion of kisspeptin-10 at 4 μg/kg · h for 22.5 h elicited an increase in LH from a mean of 5.4 ± 0.7 to 20.8 ± 4.9 IU/liter (n = 4; P0.05) and serum testosterone increased from 16.6 ± 2.4 to 24.0 ± 2.5 nmol/liter (P0.001). LH pulses were obscured at this high rate of secretion, but a lower dose infusion of kisspeptin-10 (1.5 μg/kg · h) increased mean LH from 5.2 ± 0.8 to 14.1 ± 1.7 IU/liter (n = 4; P0.01) and increased LH pulse frequency from 0.7 ± 0.1 to 1.0 ± 0.2 pulses/h (P0.05) and secretory burst mass from 3.9 ± 0.4 to 12.8 ± 2.6 IU/liter (P0.05).Kisspeptin-10 boluses potently evoke LH secretion in men, and continuous infusion increases testosterone, LH pulse frequency, and pulse size. Kisspeptin analogues have therapeutic potential as regulators of LH and thus testosterone secretion.

Kisspeptins are the product of the Kiss1 gene and potently stimulate GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the arcuate nucleus (ARC) and dorsal-lateral preoptic area and both appear to mediate the positive feedback effect of estradiol to generate the preovulatory GnRH/LH surge. To determine the role of kisspeptin in transmitting estrogen-positive feedback in the hypothalamus, we administered the kisspeptin antagonist p-271 to ewes subjected to an estradiol benzoate-induced LH surge. Kisspeptin antagonist treatment significantly attenuated these LH surges. We further examined the response to kisspeptin treatment prior to the LH surge. Kisspeptin significantly stimulated GnRH secretion into the hypophysial portal system, but the response to kisspeptin was similar in luteal and late-follicular phase ewes. Kiss1r mRNA expression in GnRH neurons was also similar across the estrous cycle. To examine alternative pathways for kisspeptin stimulation of GnRH neurons, we examined the origin of kisspeptin neuronal fibers in the external zone of the median eminence (ME) using neuronal tracing and immunohistochemical techniques. ARC populations of kisspeptin neurons project fibers to the ME. Finally, we showed kisspeptin stimulates GnRH release from ovine ME-cultured explants. This suggests direct kisspeptin to GnRH terminal-to-terminal communication within the ME. Overall, these data indicate an essential role for kisspeptin in receiving stimulatory estrogen signals and generating the full positive feedback GnRH/LH surge. Kisspeptin neurons of the ARC project to the external zone of the ME and kisspeptin acts upon the GnRH fibers at this level.

The canonical NF-κB pathway regulates the transcription of many genes which may be involved in processes such as inflammation and proliferation, suggesting a potential link between the tumor, the tumor microenvironment and cancer-specific survival in colorectal cancer patients. Immunohistochemistry was used to assess the expression of the upstream kinase TAK1/pTAK1 and four members of the canonical NF-κB pathway in tissue microarrays. Protein expression was determined using the weighted histoscore method. BRaf status, tumor stroma percentage, local inflammation, systemic inflammation and cancer-specific survival were examined in patients with colorectal cancer. Cytoplasmic IKKβ was significantly associated with the inflammatory cell infiltrate (p=0.0015), systemic inflammation (p=0.03) and cancer-specific survival (p=0.046). In contrast, no significant association was found with the other three members. Cytoplasmic pTAK1 was associated with the tumor microenvironment (p=0.045) and cancer-specific survival (p=0.032). On multivariate analysis only cytoplasmic IKKβ associated with survival (HR1.75, 95%CI 1.05-291, p=0.033) and this was independent of TNM stage and markers of the tumor microenvironment. When cytoplasmic IKKβ was stratified with BRaf status (wild type (n=151) or mutant (n=48)), it was no longer associated with cancer-specific survival. However, when cytoplasmic pTAK1 was stratified with BRaf status its association with cancer-specific survival was strengthened. Cytoplasmic pTAK1 was significantly associated with cancer-specific survival in patients with wild type BRaf (p=0.014). In patients with BRaf mutations there was no significant association with cancer-specific survival (p=0.105), however the numbers were low and this is currently being investigated in a larger cohort. The results of the present study show that high expression of cytoplasmic IKKβ was associated with decreased cancer-specific survival and with markers of the tumor microenvironment in patients with colorectal cancer. Expression of cytoplasmic pTAK1 was associated with cancer-specific survival and this was enhanced in patients with tumors expressing wild type BRaf. Citation Format: Jean A. Quinn, Lindsay Bennett, Meera Patel, Lynette Loi, Mikaela Frixou, Antonia Roseweir, James H. Park, Paul G. Horgan, Donald C. McMillan, Joanne Edwards. The relationship between members of the canonical NF-κB pathway, components of the tumor microenvironment and cancer-specific survival in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4070.