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PurposeObesity in children and adolescents is a significant public health concern. The World Health Organization Health Promoting Schools (HPS) framework promotes good nutrition and physical activity in school settings. While HPS is embraced globally, effective implementation and sustainable programmes are a continued challenge. This paper aims to report on the characteristics of current school interventions based on HPS and implementation barriers and enablers.Design/methodology/approachA literature search identified peer-reviewed studies of school health interventions reflective of the HPS framework focusing on obesity prevention. Studies from all countries were included, if conducted in primary and/or secondary schools; included a sufficient amount of qualitative implementation or process evaluation data to draw conclusions regarding key barriers and enablers to implementation; and were published in English.FindingsNine interventions (n = 9) from seven countries were included. Most were implemented in primary schools and focused on specific grade levels. Engaging parents, the home environment, teacher time constraints, fun interventions, student participation, teacher training, integration with the curriculum and stakeholder engagement all emerged as strong implementation themes. Teachers as role models, establishing community partnerships and policy support also emerged as common themes.Originality/valueFuture interventions may benefit from enhancing teacher and parent health promotion. Partnerships with initiatives focusing on environmental sustainability may simultaneously benefit human and planetary health while strengthening stakeholder engagement opportunities and consistent messaging throughout the community. More comprehensive evaluation data are needed, in particular, for long-term HPS initiatives.

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.

PurposeThe host inflammatory response is an important determinant of cancer outcome, however, the factor/s that regulate this response remains unclear. We aimed to determine if Human Leukocyte Antigen (HLA) class I and tumour cell proliferation are associated with CD8+ infiltration and survival in patients undergoing potentially curative resection for colorectal cancer.MethodsHLA class I expression (W6/32 and B2-microglobulin) and tumour proliferation index (Ki67) were quantified using immunohistochemistry on tissue micro arrays (TMA). The local inflammatory response at the invasive margin was assessed using the Klintrup-Makinen (K-M) score and CD8+ infiltration was assessed at the invasive margin (mCD8+), stroma (sCD8+) and cancer cell nests (cCD8+).ResultsPreserved HLA class I expression was associated with lower Dukes’ stage (p=0.032), lower T stage (p=0.040) and higher cCD8+ (p=0.003). High Ki67 was associated with a good K-M score (p+ (p=0.033), higher sCD8+ (p=0.025) and higher cCD8+ (p+ infiltration within the cancer cell nests. On multivariate survival analysis, preserved HLA class I expression was associated with disease free (HR 0.47 95%CI (0.25–0.89),p=0.020) and cancer specific survival (HR 0.52 95%CI (0.28–0.97),p=0.038).ConclusionThis study suggests that a pronounced local inflammatory response is independently associated with both, HLA class I expression and tumour proliferation.

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.

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico-pathological characteristics. After siRNA-mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA-MB-231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease-free survival (p = 0.029) and recurrence-free survival on tamoxifen (p

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

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics' glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

// Lindsay Bennett 1, 3 , Elizabeth A. Mallon 2 , Paul G. Horgan 3 , Andrew Paul 4 , Donald C. McMillan 3 and Joanne Edwards 1 1 Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom 2 Department of Pathology, Queen Elizabeth University Hospital, Glasgow, Scotland, United Kingdom 3 Academic Unit of Surgery, School of Medicine, University of Glasgow, Glasgow Royal Infirmary, Glasgow, Scotland, United Kingdom 4 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, United Kingdom Correspondence to: Joanne Edwards, email: joanne.edwards@glasgow.ac.uk Keywords: breast cancer, NF-κB, tumour microenvironment, survival Received: November 17, 2016 Accepted: February 12, 2017 Published: March 09, 2017 ABSTRACT The aim of the present study was to examine the relationship between tumour NF-κB activation, tumour microenvironment including local inflammatory response (LIR) and cancer-specific survival in patients with operable ductal breast cancer. Immunohistochemistry (tissue microarray of 376 patients) and western blotting (MCF7 and MDA-MB-231 breast cancer cells) was performed to assess expression of key members of the canonical NF-κB pathway (inhibitory kappa B kinase (IKKβ) and phosphorylated p65 Ser-536 (p-p65)). Following silencing of IKKβ, cell viability and apoptosis was assessed in both MCF7 and MDA-MB-231 cell lines. P-p65 was associated with cancer-specific survival (CSS) (nuclear P=0.042 and total P=0.025). High total p-p65 was associated with increase grade tumour grade (P=0.010), ER positivity (P=0.023), molecular subtype (P=0.005), lower Klintrup-Makinen grade (P=0.013) and decreased CD138 count (P=0.032). On multivariate analysis, total p-p65 expression independently associated with poorer CSS (P=0.020). In vitro work demonstrated that the canonical NF-κB pathway was inducible by exposure to TNFα in ER-positive MCF7 cells and to a lesser extent in ER-negative MDA-MB-231 cells. Reduction of IKKβ expression by siRNA transfection increased levels of apoptosis and reduced cell viability in both MCF7 (P= 0.001, P=0.002, respectively). This is consistent with the hypothesis that canonical IKKβ-NF-κB signalling drives tumour survival. These results suggest that activation of the canonical NF-κB pathway is an important determinant of poor outcome in patients with invasive ductal breast cancer.

// 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: Cell line models suggest that activation of NFκB is associated with progression of prostate cancer. This pathway may be a therapeutic target if these observations translate to clinical specimens. Methods: Immunohistochemistry measured NFκBp65 (p65), NFκBp65 nuclear localisation signal (NLS), NFκBp65 phosphorylated at ser 276 (p65ser276), NFκBp65 phosphorylated at ser 536 (p65ser536), IκBα phosphorylated at ser 32/36 (pIκBαser32/36) and MMP-9 protein expression in 61 matched hormone naive prostate cancer (HNPC) and castrate-resistant prostate cancer (CRPC) tumours. Animal and cell models were used to investigate the role of NFκB inhibition in prostate carcinogenesis. Results: In HNPC tumours, NLS expression significantly associated with a shorter time to disease recurrence and disease-specific death. In CRPC tumours p65, pIκBαser32/36 and MMP-9 expression significantly associated with shorter time to death from disease recurrence and shorter disease-specific death. MMP-9 and pIκBαser32/36 expression significantly associated with metastases at recurrence and were independent of Gleason sum and prostate-specific antigen at recurrence. Expression of phosphorylated Akt was associated with increased p65 activation in mouse models and inhibition of NFκB in LNCaP cells significantly reduced cellular proliferation and induced apoptosis. Conclusion: These results provide further evidence that the NFκB pathway could be exploited as a target for CRPC.

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.

631 Background: In patients with colorectal cancer (CRC) the local and systemic inflammatory responses (LIR and SIR) are important determinants of disease progression. The present study examines the association of transcription factor RelB, a key member of the non-canonical NF-κB pathway and its association with LIR and SIR in patients undergoing resection of CRC. Methods: Patients with stage I-III CRC who underwent curative resection in a single institution and were in a previously constructed tissue microarray were studied. IHC was utilised to examine cytoplasmic and nuclear RelB expression. The relationship between RelB, clinicopathological characteristics, LIR (Klintrup-Mäkinen (KM) grade, CD3+ and CD8+T-cell density), SIR and cancer-specific survival (CSS) was examined. Results: 208 patients were included in the analysis. Cytoplasmic RelB (cyto-RelB) was associated with nuclear RelB ( p=0.006). High expression of cyto-RelB was associated with MMR competence ( p=0.010) but not with TNM stage ( p=0.468), venous invasion ( p=0.973), tumour budding ( p=0.068), tumour necrosis ( p=0.786), tumour cell proliferation ( p=0.907), BRAF V600E mutation ( p=0.585) or administration of adjuvant chemotherapy ( p=0.853). High cyto-RelB was inversely associated with mGPS (mGPS >1: low cyto-RelB – 19% vs. high cyto-RelB – 8%, p=0.017). Also, cyto-RelB was inversely associated with tumour inflammatory cell infiltrate at the margin, Klintrup-Mäkinen grade ( p=0.059), (CD3+ p=0.010, CD8+ p=0.007) and in the tumour (CD3+ p=0.002) and a trend with tumour stroma percentage ( p=0.079). High expression of cyto-RelB was not significantly associated with CSS ( p=0.052). Conclusions: In patients undergoing CRC resection, high expression of cyto-RelB was associated with an adverse host local inflammatory response. Up-regulation of the non-canonical NF- κB pathway may be an important mechanism whereby the tumour deregulates local inflammatory responses and evades host immunosurveillance. Further investigation of inflammation based signal transduction pathways in patients with colorectal cancer is warranted.

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR94), 308 (pAR308), 650(pAR650) and 791(pAR791). No correlations with clinical parameters were observed for pAR94 or pAR650 in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR308 is significantly associated with a longer time to disease specific death (p= 0.011) and high pAR791 expression significantly associated with a longer time to disease recurrence (p= 0.018) in HNPC tumours and longer time to death from disease recurrence (p= 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p= 0.022) and low proliferating tumours (p= 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer.

The canonical NF-κB pathway regulates transcription of a wide range of genes involved in various processes including inflammation and proliferation, suggesting that it might provide a link between the systemic inflammatory response (SIR), local inflammatory response (LIR) and tumor signalling, the present study was to examine the relationship between SIR, tumor NF-κB pathway, tumor microenvironment including LIR and cancer specific survival in patients with colorectal cancer Immunohistochemistry of inhibitory kappa β kinase (IKK)-β, p65 (Rel A) and phosphorylated p65 (p-p65) was performed using colorectal cancer tissue microarray of 151 patients and the weight histoscore method was employed to assess protein expression. The relationship between these proteins, BRAF status, tumor stroma percentage, local inflammation, systemic inflammation, and cancer-specific survival were examined. In addition, western blotting was performed to assess expression of key members of the canonical NF-κB pathway following stimulation with either TNFα or IL-1 in BRAF wildtype (T84) and BRAF V600E mutated cells (HT-29). Expression of neither p65 nor p-p65, at any cellular location, was associated with clinical outcome measures. However a significant association between cancer specific survival and cytoplasmic IKKβ (p = 0.015), was observed and 10 year survival stratified from 78% to 59%. In addition, when patients were stratified by BRAF status the association with cancer specific survival and cytoplasmic IKKβ was upheld in those patients with wild type BRAF (p = 0.014) but negated in those with mutated BRAF (p = 0.287). Cytoplasmic IKKβ was significantly associated with venous invasion (p = 0.024), immunoscore (p = 0.023), Klintrup-Makinen grade (p = 0.050) and Glasgow microenvironment score. Total cell IKKβ expression was also associated with cancer specific survival, (p = 0.015) and 10 year survival was stratified from 73% to 53%. Total cell IKKβ was significantly associated with TNM (p = 0.004), tumor differentiation (p = 0.001), venous invasion (p = 0.011), necrosis (p = 0.003) and tumor stoma percentage (p = 0.048). In vitro, the canonical NF-κB pathway was induced in colon cancer cell lines T84 and HT-29 by exposure to either TNFα or IL-1 (p-p65 levels increased at 15 min), IKKβ expression remained constant. In colorectal cancer patients, cytoplasmic and total cell IKKβ expression is associated with cancer specific survival and this was related to tumor microenviroment and LIR but not the SIR. Interactions between stromal and NF-κB pathway may be an important method by which disease progression occurs. Citation Format: Jean A. Quinn, Lindsay Bennett, 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 microenvironment and survival in patients with colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B125.

588 Background: Although breast cancer morality rates have fallen, resistance to treatment remains a major problem. The NF-κB pathway regulates transcription of a wide range of genes involved in inflammation, proliferation and apoptosis and are hypothesized to play a role in tumor progression. Methods: Immunohistochemistry was employed to assess expression of members of the NF-κB pathway in a cohort of 544 breast cancer specimens with full clinical follow-up. Antibodies for the p65 subunit, phosphorylated p65 at the serine 536 residue (p-p65 S536), nuclear localization signal of p65 (p65 NLS), IKKα and IKKb had specificity confirmed by both a single band of appropriate size on a western blot and appropriate expression patterns in cell pellets treated with TNFα. Results: When the full cohort was considered patients with tumors that expressed high levels of cytoplasmic IKKα had significantly shorter disease free survival compared to those with low expression (P=0.015). Increased expression of nuclear p-p65 S536 was also associated with shorter disease free survival (P=0.005). By western blot analysis we observed that expression levels of p-p65 S536 were higher in MD-MBA-231 cells which are ER negative compared to MCF-7 cells that are ER positive, suggesting that NF-κB may have a prominent role in ER negative disease. However when the cohort was subdivided, by ER status the association observed for IKKα expression and shorter disease free survival was attenuated in ER positive tumors (p=0.008). In addition this attenuation with the ER positive cohort was also observed for nuclear p-p65 S536 (P=0.002). Other members of the pathway investigated (p65, p65 NLS and IKKb) were not associated with disease free survival. Conclusions: These results suggest that in the clinical setting the NF-kB canonical pathway is associated with poor prognosis in ER positive tumors and not with ER negative tumors as the cell line work would suggest. These results require validation in an independent cohort, but highlight the need to consider subtypes of breast cancer as individual diseases with increasingly different therapeutic targets.

Although therapies exist for breast cancer and mortality rates have fallen, resistance to current therapies remains a major problem. The NF-κB pathways regulate the transcription of a wide range of genes involved in the immune response, inflammation, proliferation and apoptosis. Many of these processes are hallmarks of cancer and NF-κB has been hypothesised to be a link between inflammation and tumourigenesis. The aim of the current study was to investigate the role of both NF-κB pathways in the pathogenesis and recurrence of breast cancer. Immunohistochemistry was employed to assess key components of the canonical (p65 and IKKβ) and non-canonical (NIK, RelB, IKKα) pathways on a TMA of 544 patients with full clinical follow up and clinical information including ER status, subtype (luminal A/B, triple negative, HER2 enriched), necrosis, apoptosis and angiogenesis. Nuclear expression of p-p65 S536 was observed in 81% (336/414) of patients and was associated with angiogenesis (p=0.028) and disease free survival (p=0.003). Those patients with tumours that expressed high levels of p-p65 S536 in the nuclear compartment had significantly shorter disease free survival compared to those with low expression (6.95 years vrs 7.91 years). The cohort was then subdivided by ER status and this observation was attenuated in ER positive tumours (p=0.002, 7.10 years vrs 8.34 years). When split further into subtype this was negated in luminal A tumours (p=0.120) but significance increased in luminal B tumours (p=2.54x10-4, 5.52 years vrs 8.16 years). A differing role was observed for the non-canonical pathway. Cytoplasmic expression of IKKα was observed in 97% (367/378) of patients and was associated with apoptosis (p=0.024) and necrosis (p=0.020) and a shorter disease free survival in those with high expression (p=0.014, 7.16 years vrs 7.64 years). This observation was attenuated in ER positive tumours when the cohort was subdivided by ER status (p=0.008, 7.56 years vrs 8.00 years). When split into subtype, the relationship between cytoplasmic IKKα and disease free survival was negated in all subtypes except luminal A (p=0.015, 7.92 years vrs 8.32 years). Other members of the NF-κB pathways (p65 NLS, IKKβ, NIK and RelB) were not associated with disease free survival. Our results suggest that the NF-κB pathways are involved in promoting recurrence in patients with ER positive tumours and may be involved with development of endocrine resistance. However, each pathway seems to be involved in different subtypes with the canonical pathway associating with recurrence in the luminal B subtype and the non-canonical with the luminal A subtype. Further studies in an independent cohort combined with mechanistic studies are required. These results highlight that patients with ER positive tumours may have different signalling pathways driving recurrence and therefore benefit from different therapeutic strategies. Citation Format: Lindsay Bennett, Clare Orange, Elizabeth Mallon, Julie Doughty, Paul G. Horgan, Andrew Paul, Joanne Edwards. NF-κB pathways are associated with increased recurrence in different subtypes of ER positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3468. doi:10.1158/1538-7445.AM2013-3468