BACKGROUND:Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. PATIENTS AND METHODS:Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. RESULTS:BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by in vitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. CONCLUSIONS:These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Background: Identification of targetable biology in breast cancer is an unmet need, particularly for triple negative breast cancer (TNBC) where patient outcome is poor and there are few clinically approved targeted therapies. Here, we identify the anion channel GPR89, to be a novel breast cancer oncogene relevant in TNBC and other breast cancer subtypes. Materials and Methods: We analyzed GPR89 subcellular localization using immunofluorescence, subcellular fractionation, and electro-microscopy techniques. We developed reporter cell lines to determine the pH of the endoplasmic reticulum (ER) and used assays to define ion homeostasis. We used siRNA- shRNA- and CRISPRn- mediated gene perturbation to determine the function of GPR89 in malignant and non-malignant cells and breast cancer organoids. We used a genetically engineered mouse model of breast cancer to study the effects of GPR89 on tumor development in vivo and mass spectrometry to analyze GPR89 associated tumor proteomes and the GPR89 interactome. Results: We found that whilst GPR89 is localized in the Golgi apparatus in non-malignant cells, localization extends to the ER in breast tumor cells. Tumor cells with ER localization also displayed addiction to GPR89, whereas those with Golgi-only expression did not. In delineating the underlying biology of this relationship, we found that MYC activation changes the localization of GPR89 from Golgi-only to Golgi plus ER. When located in the ER, GPR89 modulated ER luminal pH and facilitated the unfolded protein response, limiting the otherwise deleterious effects on cell fitness of MYC-driven ER stress. Using electron microscopy, we also found that GPR89 is localized to the ER-side of mitochondria associated membranes in tumor cells, where it interacts with the voltage dependent anion channel VDAC2 and drives pro-tumor fitness by rewiring the metabolism towards glycolysis. Finally, we found that GPR89 overexpression accelerates TNBC tumorigenesis in murine mammary tumors with ectopic expression of Myc, suggesting that GPR89 facilitates Myc-driven oncogenesis. Conclusions: The voltage-dependent anion channel GPR89 is a novel breast cancer oncogene that co-operates with Myc to support tumorigenesis. Selective GPR89 dependency is associated with extended localization of GPR89 to the ER, revealing a novel drug target and potential patient selection biomarker. No conflict of interest.

Background: As only a minor portion of the information present in histological sections is accessible by eye, recognition and quantification of complex patterns and relationships among constituents relies on digital image analysis. In this study, our working hypothesis was that, with the application of digital image analysis technology, visually unquantifiable breast cancer microarchitectural features can be rigorously assessed and tested as prognostic parameters for invasive breast carcinoma of no special type. Methods: Digital image analysis was performed using public domain software (ImageJ) on tissue microarrays from a cohort of 696 patients, and validated with a commercial platform (Visiopharm). Quantified features included elements defining tumour microarchitecture, with emphasis on the extent of tumour-stroma interface. The differential prognostic impact of tumour nest microarchitecture in the four immunohistochemical surrogates for molecular classification was analysed. Prognostic parameters included axillary lymph node status, breast cancer-specific survival, and time to distant metastasis. Associations of each feature with prognostic parameters were assessed using logistic regression and Cox proportional models adjusting for age at diagnosis, grade, and tumour size. Results: An arrangement in numerous small nests was associated with axillary lymph node involvement. The association was stronger in luminal tumours (odds ratio (OR) = 1.39, p = 0.003 for a 1-SD increase in nest number, OR = 0.75, p = 0.006 for mean nest area). Nest number was also associated with survival (hazard ratio (HR) = 1. 15, p = 0.027), but total nest perimeter was the parameter most significantly associated with survival in luminal tumours (HR = 1.26, p = 0.005). In the relatively small cohort of triple-negative tumours, mean circularity showed association with time to distant metastasis (HR = 1.71, p = 0.027) and survival (HR = 1.8, p = 0.02). Conclusions: We propose that tumour arrangement in few large nests indicates a decreased metastatic potential. By contrast, organisation in numerous small nests provides the tumour with increased metastatic potential to regional lymph nodes. An outstretched pattern in small nests bestows tumours with a tendency for decreased breast cancer-specific survival. Although further validation studies are required before the argument for routine quantification of microarchitectural features is established, our approach is consistent with the demand for cost-effective methods for triaging breast cancer patients that are more likely to benefit from chemotherapy.

Introduction/ Background Breast cancer is the most common cancer in theUK. Although 10 year survival has increased over last decades, significant improvement is still needed. Clinical management decisions are largely dependent on assessment of histological features. The traditional approach to histopathological assessment has been the expert manual reporting of cases as viewed with a light microscope and has remained virtually unchanged since 1928, with minor modifications that have led to the current routinely applied semi-quantitative tumour grading system. However, the abundance of information within the tumour microenvironment is not reflected in the traditionally evaluated histological features, and there remain morphological features with prognostic potential that have previously been beyond investigation by traditional manual microscopic means. Tumour prognosis is closely related to metastasis, a complex process involving tumour cell migration through the stromal microenvironment before entering the lymphovascular compartment. Tumour/stromal interaction is crucial in the process and represents a potential candidate for therapeutic intervention. This interaction is partly affected by the pattern of tumour migration, revealed in the tumour architecture, and partly by the stromal response. Aims Our working hypothesis for the proposed study framework was that, with the application of digital image analysis technology, previously unquantifiable tumour architectural and microenvironmental features can be rigorously assessed in detail and tested as potential prognostic parameters. Quantified features included tumour extracellular particles at the tumour-stroma interface, tumour infiltrating lymphocytes, tumour nest perimeter, number, size and shape. The selected prognostic parameter was axillary lymph node metastasis. Methods Our initial study included diagnostic core biopsies from 19 HER2 positive breast cancers, with approximately equal number of ER strongly positive or weakly positive/ negative cases. Her2 immunohistochemistry allowed rigorous segregation of epithelial elements. Immunostained sections were digitised using a Hamamatsu scanner and x10 magnification consecutive segments from .ndpi files were captured as .jpeg files and analysed using Fiji (Image J), a public domain image processing program. The entirety of each core was examined in all cases. Several native Fiji Functions and Fiji plugins, including Trainable Weka Segmentation, Colour Segmentation and Colour Deconvolution were employed in different combinations for different types of analysis. The analysis is currently being expanded to a large set of digitised breast cancer tissue microarray (TMA) slides which have been stained with cytokeratin to highlight tumour cells. The set includes breast cancer cases from all molecular subtypes and is linked with detailed histological and outcome data. Results Increased number of extracellular particles at the tumour-stroma interface and decreased number of tumour-infiltrating lymphocytes were significantly associated with axillary lymph node metastasis (p=0.0062 and p=0.0154 respectively). Combination of the two parameters increased further the strength of the association (p=0.0011). Increased total tumour nest perimeter, tumour nest number and tumour nest shape irregularity were also significantly associated with axillary lymph node metastasis (p=0.0288, p=0.0085 and 0.0203 respectively). Data from the analysis of TMAs are currently analyzed and will be presented.

Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.

Abstract Background: HER3 is known to locate in the nucleus. Unlike HER4, nuclear HER3 translocation has not been reported to be due to a proteolytic cleavage process by ADAM17 and gamma-secretase. The mechanisms of nuclear HER3 induction and its role in relation to trastuzumab treatment and resistance for HER2-positive breast cancer is unclear. Methods: Using nuclear fractionation and confocal microscopy, nuclear HER3 localisation was investigated in response to trastuzumab with or without ADAM17 inhibitor and gamma-secretase inhibitor in a panel of HER2 expressing cell lines. We also correlated nuclear HER3 expression by immunohistochemistry with treatment response in patients who underwent window trastuzumab study as well as the survival outcome in a cohort of HER2-positive breast cancer patients using Kaplan–Meier survival curves with Log-rank test. Results: HER3 ligand heregulin and trastuzumab was found to induce nuclear HER3 translocation in HER2-positive breast cancer cell lines, including SKBR3. Nuclear HER3 was also enriched in acquired trastuzumab resistant SKBR3 cells (SKBr3-TR). Trastuzumab treatment induced several HER3 fragments and HER3100kD was found to be responsible for nuclear HER3 enrichment by fractionation. This fragment was confirmed to be a specific band of HER3 as shown by HER3 knockdown. Nuclear HER3 was reduced by inhibiting either gamma-secretase or ADAM17 inhibitor. Gamma-secretase or ADAM17 inhibitor reduced HER3100kD in both SKBr3 and SKBr3-TR cells. In HER2-positive breast cancer patients who underwent window trastuzumab study, baseline nuclear HER3 status was not a predictor of response for trastuzumab monotherapy at day 21. However, nuclear HER3 was enriched after trastuzumab treatment in a poor-responder patient. Total HER3 expression level in cytoplasm positively was correlated with poor response to trastuzumab monotherapy in HER2-positive patients (r = 0.67, p = 0.05). There was no statistically significant difference in disease-free survival between positive and negative nuclear HER3 expression but the number of patients was small (n = 87). Further validation to assess nuclear HER3 expression as a prognostic and predictive biomarker in HER2-positive breast cancer patients undergoing trastuzumab treatment will be assessed in randomized tumour samples from FinHER study. Conclusion: Heregulin and trastuzumab treatment seems to induce nuclear HER3 translocation in some of the HER2 positive breast cancer cells. This may be due to proteolytic cleavage of HER3 as it is reduced by ADAM17 or gamma-secretase inhibitor. Enriched nuclear localisation of HER3 seems to be one possible mechanism of acquired resistance to trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-08.

Over-expression of the HER family has been implicated in various cancers. Its phosphorylation activates intracellular signaling cascades that regulate proliferation and survival. Although many tyrosine kinase inhibitors (TKI) and antibodies have been developed to target the HER family specifically, resistance to these drugs is still a major issue in cancer treatment. Several recent studies have shown that re-activation of mainly HER3 could play an important role in this resistance. Protein tyrosine phoshatases are enzymes that remove phosphates from phosphorylated tyrosine residues in proteins. Yuan et al. showed that PTPN9 inhibits EGF-evoked signalling by direct dephosphorylation of EGFR and HER2, but the effect on HER3 has not been intensely studied. In this study we identify that PTP9 reduces phosphorylation of HER3 and may therefore be an important player in the development of resistance to targeted therapy. When we inhibited Akt directly using an Akt inhibitor (Akti), we showed a rapid decrease in HER3 phosphorylation as well as an increase in PTPN9. Knocking-out PTPN9 using siRNAs blocked the dephosphorylation of HER3 after Akti treatment, further suggesting the role of PTPN9 in dephosphorylation of HER3. We went on to show that a loss of PTPN9 leads to increased resistance to two commonly used HER-targeting treatments, gefitinib and trastuzumab, in HER2 positive breast cancer cells. Using tumour microarrays with samples from patients with HER2-overexpressing breast cancer we investigated the correlation between levels of PTPN9 and survival. Low levels of PTPN9 correlated with a poor overall survival as well as disease free survival in these patients. Therefore, the preliminary data indicates that PTPN9 could act as a biomarker to predict response to HER-targeted therapy, although the results will need to be verified in a larger sample population. As higher levels of PTPN9 appear to be beneficial to a good clinical response, increasing PTPN9 could also be a new area for drug discovery to focus on.