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Background Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker. Methods We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay. Results Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses. Conclusions sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments.

Evaluation of FRalpha expression by tumor cell lines

Table of Patient Characteristics

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3–stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083–96. ©2017 AACR.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.

Supplementary Methods described

Time lapse microscopy experiment of sorted NKp46-ILC3 cells (CD3-, CD11c-, B220, NKp46-, CD127+, CD90.2+) co-cultured with MSC cells as described in Figure 2B. Low magnification (Ã-10) video shows the general clustering pattern of NKp46-ILC3 cells around the MSC cells over a duration of 10 hours.

Figure S1, Related to Figure 1 FACS and immunofluorescence validation of Lin-CD127+CD90.2+NKP46- gated ILC3 cells Figure S2, Related to Figure 2 ELISA quantification of CXCL13 and CCL21 chemokines levels in conditioned media (CM) obtained from the bone marrow derived MSC cell line (HS-5) and from the breast cancer cell line 4T1.2 cell line is shown. Figure S3, Related to Figure 2 Proliferation assay of ILC3 cells and ELISA quantification of CXCL13 and CCL21 from siRNA transfected MSC cells. Figure S4, Related to Figure 4 CXCR5 expression on MSC cells. Figure S5, Related to Figure 5 Identification of a RORγT+CD127+CD3- ILC3 cells within the tertiary lymphoid structures within breast cancers. Figure S6, Related to Figure 5 Expression of lymphoid chemokine and chemokine receptor genes in breast cancer datasets. Table S1, Related to Figure 5 Table S1: Clinico-pathological characteristics for the METABRIC sample.

Description of additional experiments and procedures carried out in this study

Supplementary Figure legends

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin‐stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time‐point zero. Furthermore, targeted gene expression analysis (NanoString PanCancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q

There has been significant interest in the prospects of chimeric antigen receptor (CAR) T-cell therapy in the treatment of solid malignancies, and multiple clinical trials are in progress1. However, the scope of these trials has been restricted by the lack of availability of tumorspecific targets to direct CAR binding. Tumor specificity is crucial as on-target off-tumor activation of CAR T-cells in healthy tissues can result in potentially lethal toxicities due to uncontrolled cytokine release syndrome2. Here we engineer a stringent hypoxia-sensing CAR T-cell system which achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by an inadequate oxygen supply. Using murine xenograft models, we demonstrate that despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate the unlimited expansion of the CAR T-cell target repertoire for treating solid malignancies.

The tumour microenvironment plays a crucial role in the growth and progression of cancer and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies show that TAMs show transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype. These TAMs can recruit and maintain neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This novel macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.

The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described. These TAMs can recruit and prolong the lifespan of neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients., Beatson et al. show that a sialylated tumour-associated glycoform of the mucin MUC1 induces the differentiation of monocytes into tumour-associated macrophages. These macrophages are found in breast cancer stroma and their presence is associated with poor prognosis.

Summary Utilizing T cells expressing chimeric antigen receptors (CARs) to identify and attack solid tumors has proven challenging, in large part because of the lack of tumor-specific targets to direct CAR binding. Tumor selectivity is crucial because on-target, off-tumor activation of CAR T cells can result in potentially lethal toxicities. This study presents a stringent hypoxia-sensing CAR T cell system that achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by inadequate oxygen supply. Using murine xenograft models, we demonstrate that, despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate unlimited expansion of the CAR T cell target repertoire for treating solid malignancies.

Purpose Preclinical data support a key role for the PI3K pathway in estrogen receptor–positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. Patients and Methods In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor–positive, human epidermal growth factor receptor 2 (HER2)–negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. Results There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination:anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P = .03); for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. Conclusion Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer.

Background: All antibodies approved for the treatment of cancer are monoclonal IgGs, and no IgE therapy has yet been tested in humans. The biology of IgE, compared with IgG, offers potential for enhanced immune surveillance and superior effector cell potency against tumor cells. IgE antibodies in preclinical cancer models are not associated with allergic toxicity even in immunocompetent animals, and in vivo efficacy compares favorably with equivalent IgGs. Methods: We conducted a first-in-human first-in-class trial of MOv18, a chimeric monoclonal IgE, in patients with solid tumors expressing folate receptor-alpha, the antigen recognized by this antibody. Antigen expression was deemed positive in the presence of >5% membrane staining of any intensity using the mouse clone BN3.2. Intravenous treatment was administered weekly for 6 weeks, then two-weekly. The trial incorporated pre-treatment skin prick testing with MOv18 IgE, and an ex vivo basophil activation test (BAT) using patient whole blood, with the aim of predicting systemic allergic toxicity and excluding patients at potential risk. Safety, efficacy, markers of immune response, and pharmacokinetics have been evaluated in 24 patients to date, at total doses ranging from 0.07 to 3.0mg. Results: Treatment was well tolerated in almost all patients. The most common toxicity was readily manageable urticaria, without systemic symptoms, signs or tryptase elevation. One patient treated at the 0.5mg dose experienced anaphylaxis, with tryptase elevation, despite a negative pre-dose skin prick test. This was the only patient in the trial with baseline circulating basophils that could be activated by ex vivo exposure to MOv18 IgE. This BAT assay was subsequently used to ensure no further patient with reactive basophils was exposed. Maximum tolerated dose has not yet been reached. Dose-dependent increases in Cmax were observed, and plasma concentrations of 70-100ng/mL achieved at the 1.5mg dose are comparable to typical levels of endogenous IgE. No consistent anti-drug antibody response has been detected. Preliminary evidence of anti-tumor activity was seen in a patient with ovarian cancer at a total MOv18 IgE dose of 0.7mg. Shrinkage of peritoneal metastases was accompanied by a tumor marker reduction meeting Gynecologic Cancer InterGroup criteria for response. Conclusions: These results support for the first time the safety of IgE as a treatment for cancer, and provide preliminary evidence for anti-tumor efficacy of this new therapeutic class. The mechanism of cutaneous toxicity is being investigated. Clinical testing of class-switched IgE versions of approved IgG-based therapeutic antibodies is warranted. Citation Format: James Spicer, Bristi Basu, Ana Montes, Udai Banerji, Rebecca Kristeleit, Gareth J. Veal, Christopher Corrigan, Stephen Till, George Nintos, Timothy Brier, Ionut G. Funingana, Joo Ern Ang, Kam Zaki, Annie Griffin, Claire Barton, Paul Jones, Sarah Mellor, Susan Brook, Katie Stoddart, Christopher Selkirk, Simon Carroll, Heike Lentfer, Natalie Woodman, Amy Pope, Giulia Pellizzari, Mano Nakamura, Kristina M. Ilieva, Atousa Khiabany, Chara Stavraka, Hannah Gould, Jitesh Chauhan, Heather Bax, Sarah Pinder, Debra Josephs, Sophia Karagiannis. Phase 1 trial of MOv18, a first-in-class IgE antibody therapy for cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT141.

Background Histological changes in the cancer-free lymph nodes (LNs) can add to the risk prediction of developing distant metastasis for LN-positive breast cancer patients. As Whole Slide Images (WSI) demonstrated suitability for detection of LN metastasis with high accuracy based on Convolutional Neural Networks (CNN), we extended the assessments to cancer-free LNs to determine whether early signs of disease progression could be identified. Methods Clinico-pathological data, including level of LNs in the axilla, were available for 143 LN-positive breast cancer patients. All resected LNs were digitised (×40 magnification), totalling to 1,054 WSI. CNN models were based on standard ResNet architecture, and trained using 224x224 sized WSI patches, to classify germinal centres (GC), follicles, sinus histiocytosis, and adipose tissue as defined by 2 independent pathologists. To increase the robustness of the CNN, training data was augmented using image rotation and colour perturbation. A weighted cross entropy was used as a training loss function to account for the imbalance in the four types of patches. Confusion matrix and one-vs-all AUC scores were computed on WSIs patches extracted from held-out test patients to assess the discriminative power of the trained CNN models. Multivariate and cross-validated L2-regularised proportional hazard regression analyses were used for outcome prediction. Results An increased number and smaller sized GCs in cancer-free LNs, the prevalence of metastatic LNs in the low axilla, the location of GCs in metastatic LNs, and lymphocytic infiltration at the tumour invasive front were all indicative of developing distant metastasis in LN-positive breast patients. Metastatic LNs were found exclusively in the low axilla in 77% of patients. Amongst the 913 H&E WSI of cancer-free LNs, 21% presented with GC. Patch level AUC scores of detecting each type of tissue region ranged between 0.82 to 0.94 on held-out patients quantifying the effectiveness of CNN to be used as topological feature detector. Conclusions Addition planned WSI analyses of large breast cancer cohorts will expand on these data, capturing histological features in LNs, with the aim of identifying high-risk and low-risk disease, and aiding in pathological prognostication. Legal entity responsible for the study The authors. Funding Breast Cancer Research Trust, Breast Cancer Now, CRUK. Disclosure All authors have declared no conflicts of interest.

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein–protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC–ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically

Mesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of three peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all three candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar in vitro potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. In vivo anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3–stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083–96. ©2017 AACR.

Malignant mesothelioma remains an incurable cancer. We demonstrated that mesotheliomas expressed EGFR (79.2%), ErbB4 (49.0%) and HER2 (6.3%), but lacked ErbB3. At least one ErbB family member was expressed in 88% of tumors. To exploit ErbB dysregulation in this disease, patient T-cells were engineered by retroviral transduction to express a panErbB-targeted chimeric antigen receptor (CAR), co-expressed with a chimeric cytokine receptor that allows interleukin (IL)-4 mediated CAR T-cell proliferation. This combination is referred to as T4 immunotherapy. T-cells from mesothelioma patients were uniformly amenable to T4 genetic modification and expansion/enrichment thereafter using IL-4. Patient-derived T4+ T-cells were activated upon contact with a panel of four mesothelioma cell lines, leading to cytotoxicity and cytokine release in all cases. Adoptive transfer of T4 immunotherapy to SCID Beige mice with an established bioluminescent LO68 mesothelioma xenograft was followed by regression or eradication of disease in all animals. Despite the established ability of T4 immunotherapy to elicit cytokine release syndrome in SCID Beige mice, therapy was very well tolerated. These findings provide a strong rationale for the clinical evaluation of intracavitary T4 immunotherapy to treat mesothelioma.

Background: Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early versus late metastatic risk prediction. The aim of our work was to identify biologically validated quantitative imaging parameters with improved correlation to clinical outcome, and to address some of the remaining obstacles for a truly robust prognostic model in clinical use. Method: We identified 4 seed proteins (ezrin/radixin/moesin-cofilin), along with several kinases as biologically relevant subnetwork of proteins that control tumor cell motility and metastasis. Patient-derived breast cancer tumour samples were used to perform a combination of imaging methods such as Fluoresecence lifetime imaging microscopy, automated segmentation and co-localisation intensity analysis. A complexity optimized Bayesian proportional hazard regression model was performed on a total of 419 breast cancer patients to validate time dependent predictions using traditional clinicopathological, genomic and our novel optical imaging-derived parameters. An independent dataset of 300 patient samples from the Leeds Institute of Molecular Medicine is currently being evaluated, representing a large cross centre validation of our integrated model. Results: We demonstrate that the traditional gold standard clinico-pathological variables are poor predictors for patients that survive long periods, and that their predictive significance (in terms of hazard ratios) varies significantly between two temporal cohorts where the adjuvant treatments are vastly different. Moreover, we investigate the predictive accuracy of a combined imaging/clinicopathological model compared with genomic/clinicopathological models. We demonstrate how to reduce over-fitting to help improve the performance of prognostic models. Results of an integrated model combining genomic and imaging parameters are still awaited. Discussion: We have produced the first optical imaging-derived multivariate tumour metastatic signature, which measures underlying key biological variables involved in regulating cancer cell motility. Using Bayesian proportional hazards regression in a time-dependent manner, we highlight the inadequacies of existing prediction tools and present a model combining the clinicopathological parameters with our imaging-based metastatic signature, as an integrative reproducible prognostic tool across different temporal cohorts. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-29.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt

Overexpression of HER2 is an important prognostic marker, and the only predictive biomarker of response to HER2-targeted therapies in invasive breast cancer. HER2-HER3 dimer has been shown to drive proliferation and tumor progression, and targeting of this dimer with pertuzumab alongside chemotherapy and trastuzumab, has shown significant clinical utility. The purpose of this study was to accurately quantify HER2-HER3 dimerisation in formalin fixed paraffin embedded (FFPE) breast cancer tissue as a novel prognostic biomarker. FFPE tissues were obtained from patients included in the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) study. HER2-HER3 dimerisation was quantified using an improved fluorescence lifetime imaging microscopy (FLIM) histology-based analysis. Analysis of 131 tissue microarray cores demonstrated that the extent of HER2-HER3 dimer formation as measured by Förster Resonance Energy Transfer (FRET) determined through FLIM predicts the likelihood of metastatic relapse up to 10 years after surgery (hazard ratio 3.91 (1.61–9.5), p = 0.003) independently of HER2 expression, in a multivariate model. Interestingly there was no correlation between the level of HER2 protein expressed and HER2-HER3 heterodimer formation. We used a mathematical model that takes into account the complex interactions in a network of all four HER proteins to explain this counterintuitive finding. Future utility of this technique may highlight a group of patients who do not overexpress HER2 protein but are nevertheless dependent on the HER2-HER3 heterodimer as driver of proliferation. This assay could, if validated in a group of patients treated with, for instance pertuzumab, be used as a predictive biomarker to predict for response to such targeted therapies.

We describe an 'open' design methodology for wide-field fluorescence, confocal and fluorescence lifetime imaging microscopy (FLIM), and how the resulting microscopes are being applied to radiation biology and protein activity studies in cells and human tissue biopsies. The design approach allows easy expansion as it moves away from the use of a monolithic microscope body to small, commercial off-the-shelf and custom made modular components. Details have been made available under an open license for non-commercial use at http://users.ox.ac.uk/~atdgroup. Two radiobiology 'end-stations' have been constructed which enable fast radiation targeting and imaging of biological material opening up completely novel studies, where the consequences of ionising radiation (signaling and protein recruitment) can be studied in situ , at short times following irradiation. One is located at Surrey University, UK, where radiation is a highly focused in beam (e.g. protons, helium or higher mass ions). The second is installed at the Gray Institute linear accelerator facility, Oxford University, which uses sub-microsecond pulses of 6 MeV electrons. FLIM capabilities have enhanced the study of protein-protein interactions in cells and tissues via Forster Resonance Energy Transfer (FRET). Extracting FRET signals from breast cancer tissue is challenging because of endogenous and fixation fluorescence; we are investigating novel techniques to measure this robustly. Information on specific protein interactions from large numbers of patient tumors will reveal prognostic and diagnostic information.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

Background: Preclinical and clinical data support a key role for the PI3K pathway in resistance to endocrine therapy in patients with ER+ breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. Short-term preoperative window of opportunity (WOO) studies are a validated strategy for novel treatments to provide proof-of-concept and define the most appropriate patient population by directly assessing treatment effects in tumour tissue before and after treatment. This is the first WOO study with the PI3K inhibitor pictilisib (GDC-0941) in combination with anastrozole (ANA). Methods: 73 postmenopausal patients (pts) have been randomized (2:1 in favour of the combination) to receive 2-week preoperative treatment with ANA plus pictilisib (n=50, "ANA+PIC" arm) or ANA alone (n=23, "ANA" arm). Pts had newly diagnosed, operable, ER+, HER2-negative breast cancer of ≥1 cm size. Pts receiving HRT were excluded. Treatment effects and correlative studies were assessed using FFPE and frozen tumour biopsies taken before and after 14 days of study treatment. The primary endpoint was inhibition of tumour-cell proliferation, as measured by change in Ki67 expression, determined centrally by 2 investigators. Secondary endpoints include induction of apoptosis (Caspase3) and safety. Comprehensive biomarkers analyses include targeted NGS of a comprehensive cancer panel of >400 genes, copy number analyses, and pre- and post-treatment reverse-phase protein arrays (RPPA) and RNA profiling. Results: Baseline (BL) disease characteristics were similar between both study arms. PAM50 analysis showed that 53% and 47% of tumors were Luminal (Lum) A and B, respectively. 65% of tumors had >14% Ki67-positive cells. Observed treatment-emergent AEs were consistent with those previously described for single-agent pictilisib and anastrozole. Mean post-treatment percentage reduction of Ki67 was 84% (95% CI, 75%-89%) for ANA+PIC and 72% (54%-87%) for ANA. Ki67-response (≥50% drop in % of Ki67+ cells) was 86% for ANA+PIC and 60% for ANA. By using the definition that pts with a natural logarithm of %Ki67+ cells of ≤1 or 1-2 have a day 15 anti-proliferative response, 93% [ln(ki67): Preplanned subgroup analyses showed a significant interaction of response to ANA+PIC with molecular subtype and Ki67 levels. Patients with LumB tumors or high BL Ki67 (>14%) had a higher Ki67 response with ANA+PIC compared to ANA (LumB, 83% vs 38%; Ki67>14%, 94% vs 55%), whereas Ki67 response was similar for both treatments for LumA tumors (ANA, 75%; ANA+PIC, 73%) or tumors with low BL Ki67. Mean post-treatment % reduction of Ki67 in LumB tumors was 87% (95% CI, 49%-96%) for ANA+PIC and 56% (16%-77%) for ANA (p=0.03). Additional data on apoptosis and comprehensive pre- and post-treatment biomarkers analyses will be presented. Conclusions: This first report of a preoperative WOO study evaluating a PI3K inhibitor in early breast cancer demonstrated addition of pictilisib to ANA was associated with increased anti-proliferative response over single-agent ANA. Citation Format: Peter Schmid, Sarah E Pinder, Duncan Wheatley, Jane Macaskill, Charles Zammit, Jennifer Hu, Robert Price, Nigel Bundred, Sirwan Hadad, Alice Shia, Louise Lim, Shah-Jalal Sarker, Patrycja Gazinska, Natalie Woodman, Darren Korbie, Matt Trau, Paul Mainwaring, Peter Parker, Arnie Purushotham, Alastair M Thompson. Preoperative window of opportunity study of the PI3K inhibitor pictilisib (GDC-0941) plus anastrozole vs anastrozole alone in patients with ER+, HER2-negative operable breast cancer (OPPORTUNE study) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S2-03.

Background Prostaglandin (PG) E 2 binds to 4 G-protein–coupled receptors designated EP 1 through EP 4 . Although PGE 2 plays an immunomodulatory role in asthma, there is little information on the expression of PGE 2 receptors in this disease. Objective We hypothesized that profiles of E-prostanoid (EP) receptor expression are altered on asthmatic bronchial inflammatory cells in vivo and further altered by allergen challenge in vivo and proinflammatory mediators in vitro . Methods The numbers and phenotypes of EP 1-4 immunoreactive induced sputum cells from atopic asthmatics (n=13; before and 24 hours after allergen inhalational challenge) and normal controls (n=9; 3 after saline challenge) and EP 1-4 expression on purified blood eosinophils from both groups (n=4 for each) before and after stimulation with LPS and/or IL-5 in vitro were measured by using single and double immunocytochemistry. Results Subsets of sputum cells of all phenotypes expressed all 4 EP receptors in both patients with asthma and controls. There were significantly greater numbers of macrophages expressing all 4 EP receptors and increased percentages of macrophages expressing EP 2 and EP 4 in patients with asthma compared with controls. Allergen bronchial challenge of patients with asthma was associated with a selective influx of eosinophils, but the percentages of these and other leukocytes expressing all 4 EP receptors were unchanged. Compared with sputum, only small percentages of peripheral blood eosinophils expressed each receptor, but this was increased by culture with exogenous IL-5 or LPS. Conclusion E-prostanoid receptor expression is increased on airway macrophages of patients with asthma at baseline and may be altered on eosinophils after allergen challenge in vivo in response to inflammatory stimuli.

We review novel, in vivo and tissue-based imaging technologies that monitor and optimize cancer therapeutics. Recent advances in cancer treatment centre around the development of targeted therapies and personalisation of treatment regimes to individual tumour characteristics. However, clinical outcomes have not improved as expected. Further development of the use of molecular imaging to predict or assess treatment response must address spatial heterogeneity of cancer within the body. A combination of different imaging modalities should be used to relate the effect of the drug to dosing regimen or effective drug concentration at the local site of action. Molecular imaging provides a functional and dynamic read-out of cancer therapeutics, from nanometre to whole body scale. At the whole body scale, an increase in the sensitivity and specificity of the imaging probe is required to localise (micro)metastatic foci and/or residual disease that are currently below the limit of detection. The use of image-guided endoscopic biopsy can produce tumour cells or tissues for nanoscopic analysis in a relatively patient-compliant manner, thereby linking clinical imaging to a more precise assessment of molecular mechanisms. This multimodality imaging approach (in combination with genetics/genomic information) could be used to bridge the gap between our knowledge of mechanisms underlying the processes of metastasis, tumour dormancy and routine clinical practice. Treatment regimes could therefore be individually tailored both at diagnosis and throughout treatment, through monitoring of drug pharmacodynamics providing an early read-out of response or resistance.

Rationale The eicosanoid prostaglandin E 2 (PGE 2 ) exerts a variety of patho/physiological effects via 4 G-protein-coupled receptors, EP 1–4 . PGE 2 plays an immunomodulatory role in asthma (for example, it inhibits bronchospasm caused by allergen inhalational challenge), but there is little information on the expression of PGE 2 receptors in this disease. Methods Using immunocytochemistry with receptor-specific antibodies, we measured the numbers and phenotypes of EP 1–4 immunoreactive cells in cytospins of induced sputum obtained from atopic asthmatics (n=13) before and 24 hrs after allergen inhalational challenge. Results Prior to challenge, 25.1 ± 2.9%, 35.3 ± 4.4%, 18.1 ± 2.5% and 40.9 ± 7.6% of all sputum cells expressed immunoreactivity for EP 1–4 , respectively. For all 4 receptors, macrophages compared 50–80%, neutrophils 10–40% and eosinophils 5–10% of total immunoreactive cells, with only a small percentage accounted for by lymphocytes, epithelial and squamous cells. Conversely, 20–80% of neutrophils, 10–30% of eosinophils, 20–40% macrophages and 2–10% of lymphocytes showed immunoreactivity for all 4 receptors. Following allergen challenge, there was a significant increase in the absolute numbers of sputum eosinophils, but not other cells, expressing all 4 receptors (p Conclusions Inflammatory leukocytes in induced sputum of atopic asthmatics express all 4 PGE 2 receptors, and allergen challenge is associated with an influx of eosinophils expressing all 4 receptors. Our further preliminary data suggest that there is a low level of expression on peripheral blood eosinophils. This provides one possible mechanism for the anti-inflammatory effects of PGE 2 in asthma.