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Purpose: To assess the frequency and prognostic relevance of germinal centres (GC) in cancer-free axillary lymph nodes (ALNs), in relation to stromal tumour-infiltrating lymphocytes (sTILs) and the presence of tertiary lymphoid structures (TLS) in the primary carcinoma, in LN-positive Hormone Receptor (HR)-negative invasive breast cancer patients. Patients and methods: A cohort of 161 patients with HR-negative invasive breast cancer of no special type (NST) and LN-positive status treated between 2005-10 at Tianjin Medical University (China) was identified. sTILs and TLS at the primary tumour site and GC in 2,841 involved and cancer-free ALNs were evaluated on H&E stained sections. Markers were tested for prognostic value for invasive Disease-Free Survival (iDFS), distant Disease-Free Survival (dDFS) and Overall Survival (OS), using Cox regression models adjusted for clinico-pathological factors. Results: Among the 161 HR-negative breast cancers, 47% (n=75) had >=20% sTILs and 24% (n=38) peritumoural TLS. 75% (121/161) and 76% (122/161), respectively, displayed GCs in their cancer-free and involved ALNs. Significantly higher numbers of GCs were seen in both cancer-free and involved ALNs when the primary tumours showed >=20% sTILs. The presence of TLS was significantly associated with increased GC numbers in involved but to a lesser extend in cancer-free ALNs (Kruskal-Wallis rank sum test, p Using an iterative process to determine an optimal cut off point by a minimal P value approach, a non-monotonic relationship between the frequency of GC in cancer-free ALNs and all endpoints was observed. Patients with >2 GC but less than the top 5% (= 62 GC) in cancer-free ALNs showed improved iDFS, dDFS and OS, whilst patients with 62 GC across all assessed cancer-free ALNs had poorer iDFS, dDFS and OS (see Table). In the multivariate models, the frequency of GC in cancer-free ALNs added independent prognostic information for all endpoints across all patients. In patients with >=20% sTILS, cancer-free ALNs within the top 5% for GC remained associated with a worse dDFS and iDFS (see Table). Cancer-free ALNs with Five-year iDFS, dDFS and OS in patients with 2 GC whose five-year iDFS, dDFS and OS were 65%, 65% and 69%, respectively. Conclusions: The prognostic importance of GC assessment in cancer-free ALNs in women with LN-positive HR-negative breast cancers is demonstrated. A better outcome in patients with GC formation in their cancer-free ALNs, despite low sTILs in the primary carcinoma, may suggest a systemic anticancer immune response. High sTILs but with extreme GC formation in the cancer-free ALNs could potentially reflect an overdriving but less effective immune response. The assessment of the combination of primary and nodal immune response in HR-negative breast cancers is imperative in these high-risk patients. Table 1. Univariate and Multivariate Cox Regression Analysis of Outcome by GC in Cancer-Free ALNsiDFSUnivariateAll cases=20% sTILTotal GCs number - binnedModel PHRCIModel PHRCIModel PHRCIGC624.631.75 - 12.41.930.26 - 14.5612.793.34 - 48.97MultivariateCorrected for: pNstage, sTILS & TLSCorrected for: pTstage & TLSCorrected for: pNstageTotal GCs number - binnedCovariate PModel PHRCICovariate PModel PHRCICovariate PModel PHRCIGC624.15E-059.583.25 - 28.201.94E-014.070.49 - 33.876.62E-038.861.83 - 42.82dDFSUnivariateAll cases=20% sTILTotal GCs number - binnedModel PHRCIModel PHRCIModel PHRCIGC625.872.14 - 16.082.490.32 - 19.0718.434.04 - 84.03MultivariateCorrected for: pTstage, pNstage, sTILS & TLSCorrected for: Age, pTstage, TLSCorrected for: pNstageTotal GCs number - binnedCovariate PModel PHRCICovariate PModel PHRCICovariate PModel PHRCIGC622.49E-0615.304.91 - 47.635.19E-028.610.98 - 75.553.11E-0313.302.39 - 73.89OSUnivariateAll cases=20% sTILTotal GCs number - binnedModel PHRCIModel PHRCIModel PHRCIGC624.131.18 - 14.42.570.33 - 19.8311.891.65 - 85.45MultivariateCorrected for: Age, pTstage, pNstage, sTILS & TLSCorrected for: Age & TLSCorrected for: pNstage, LVI & TLSTotal GCs number - binnedCovariate PModel PHRCICovariate PModel PHRCICovariate PModel PHRCIGC628.62E-0516.064.02 - 64.247.01E-027.050.85 - 58.342.80E-012.970.41 - 21.43*too few events Citation Format: Fangfang Liu, Thomas Hardiman, Patrycja Gazinska, Jelmar Quist, Sarah Pinder, Anita Grigoriadis. The formation of GCs in cancer-free ALNs, a non-monotonic prognostic factor in HR-negative invasive breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-36.

The level of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative (TNBC) and HER2-positive breast cancers convey prognostic information. The importance of systemic immunity to local immunity is unknown in breast cancer. We previously demonstrated that histological alterations in axillary lymph nodes (LNs) carry clinical relevance. Here, we capture local immune responses by scoring TILs at the primary tumor and systemic immune responses by recording the formation of secondary follicles, also known as germinal centers, in 2,857 cancer-free and involved axillary LNs on haematoxylin and eosin (H&E) stained sections from a retrospective cohort of 161 LN-positive triple-negative and HER2-positive breast cancer patients. Our data demonstrate that the number of germinal center formations across all cancer-free LNs, similar to high levels of TILs, is associated with a good prognosis in low TILs TNBC. This highlights the importance of assessing both primary and LN immune responses for prognostication and for future breast cancer research.

Background: Although preclinical data suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance, results from randomized clinical trials have failed to identify a subgroup of patients that derive a substantial benefit. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib can increase the anti-tumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. Methods: In this randomized, open-label, phase 2 study, 167 postmenopausal women with newly diagnosed, operable, ER-positive, HER2-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to two-weeks of preoperative treatment with anastrozole 1 mg once daily or the combination of anastrozole 1mg with pictilisib 260 mg once daily. The primary endpoint 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. Secondary endpoints include induction of apoptosis (Caspase3) and safety. Comprehensive biomarkers analyses included targeted NGS of a comprehensive cancer panel of >400 genes (Ampliseq Comprehensive Cancer panel), copy number variation analyses, and pre- and post-treatment reverse-phase protein arrays (RPPA) and RNA profiling (NanoString nCounter platform). Results:There was significantly greater geometric mean Ki67 suppression of 82.5% (90% CI, 78.3%-85.8%) for the combination vs 70.7% (61.0%-78.0%) for anastrozole [geometric mean ratio (combination/ anastrozole) 0.60 (0.58-0.85);p=0.01]. Higher baseline Ki67, Luminal B status and/or negative PR status were associated with increased benefit from adding pictilisib. A significant interaction was observed between PIK3CA mutation subtypes [helical domain mutations (HD), kinase domain mutations (KD), wildtype (WT)] and mean Ki67 suppression; the combination/anastrozole geometric mean ratio of Ki67 suppression was 0.48 (0.27-0.84; p=0.02) for patients with HD mutations and 0.63 (0.39–1.0; p=0.05) for patients with PIK3Ca WT, compared to 1.17 (0.57–2.41; p=0.64) for patients with KD mutations. This was largely due to patients with HD mutations showing a particularly poor response to anastrozole alone [mean Ki67 suppression 53.9% (9.5%-76.5%)], that was reversed by the addition of pictilisib [mean Ki-67 suppression 78.1% (71.0%-83.4%)]. On the other hand, patients with KD mutations responded well to anastrozole alone [mean Ki-67 suppression 77.7% (57.0%-88.4%)] and showed no benefit from the addition of pictilisib [mean Ki-67 suppression 73.9% (59.8%-83.0%)]. There was no significant difference in induction of apoptosis between treatment groups. Comprehensive pre- and post-treatment biomarkers analyses will be presented. Conclusions: Adding pictilisib to anastrozole significantly increases the anti-proliferative response to preoperative treatment with anastrozole. A significant interaction was observed between PIK3CA mutation subtypes, with patients with helical domain mutations showing a particularly poor response to anastrozole alone that was reversed by the addition of pictilisib. Citation Format: Schmid P, Pinder S, Wheatley D, Zummit C, Macaskill EJ, Hu J, Price R, Bundred N, Hadad S, Shia A, Sarker S-J, Lim L, Mousa K, O'Brien C, Wilson TR, Lackner MR, Gendreau S, Gazinska P, Korbie D, Trau M, Mainwaring P, Thompson A, Purushotham A. Interaction of PIK3CA mutation subclasses with response to preoperative treatment with the PI3K inhibitor pictilisib in patients with estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-02.

Background A distinctive trait of triple negative breast cancer (TNBC) is the acquisition of genome wide highly aberrant copy number states, which is more evident in metastatic settings. The level of copy number alterations can be characterized by quantitative estimates of chromosomal instability, such as allelic imbalanced copy number aberrations, telomeric allelic imbalance (NtAI), homologous recombination deficiency (HRD) score, referred here as genomic scars. Several of these scars are reported as being indicative of BRCAness and potential predictive and/or prognostic biomarkers of chemotherapy response, currently mostly demonstrated in neoadjuvant settings in TNBC. Aims Using several genomic scar measures, we aim to capture chromosomal instability and test their predictive and prognostic value in metastatic or recurrent locally advanced triple negative or BRCA1/2 mutated breast cancer in the TNT trial. Methods Patients recruited to TNT (n=376) had ER-/PR-/Her2- breast cancer or were germline BRCA mutation carriers. Genome-wide copy numbers (CN) were derived from FFPE samples including primary tumours and positive lymph nodes (n=183, docetaxel=93, carboplatin=90; BRCA1 mut=25). Genomic scars were generated using ASCAT (Van loo et al., PNAS 2010) CN profiles. HRD scores were established by Myriad Genetics, Inc. assay (n=272). BRCA1-like classifier was applied as described in Schouten et al., Mol Onc 2015. Shannon diversity index was calculated using ASCAT raw CN profiles. Association of genomic scars with PAM50 subtypes and BRCA1 deficiency status were evaluated using Kruskal-Wallis test; p-values were adjusted for multiple comparisons (Dunn's test). Statistical significance was defined as p Results HRD and NtAI scores were higher in basal like samples compared to non-basal like (median diff. HRD=11.5, p=0.005; NtAI=3, p=0.04). HRD (p=2e-14) and NtAI (p=0.003) scores were also higher in BRCA1 deficient (BRCA1 germline/somatic mutant or BRCA1 methylated) samples compared to non-deficient. Using the BRCA1-like classifier, 42 out of 50 BRCA1 deficient samples and 93 out of 133 BRCA1 non-deficient/undetermined samples were identified as BRCA1-like. The Shannon diversity index, measuring CN heterogeneity, clustered samples into 3 groups. Analysis of ORR showed non-significant trends to preferential response rates with docetaxel in cluster 1 and 3. Membership of cluster 2 predicted higher ORR to carboplatin over docetaxel (interaction p=0.017). PFS indicated a treatment effect in cluster 2, but not in cluster 1 or 3; there was no evidence of interaction between subgroups and treatment (p=0.15). Conclusions Our results suggest that the overall heterogeneity of the copy number landscape is a promising area for seeking predictive/prognostic biomarkers in metastatic TNBC, and combined with other modalities of high-dimensional omics data could provide essential treatment response information. Citation Format: Sipos O, Tovey H, Quist J, Haider S, Nowinski S, Gazinska P, Kernaghan S, Toms C, Timms KM, Lanchbury JS, Linn SC, Pinder SE, Bliss JM, Tutt A, Cheang MC, Grigoriadis A, On behalf of the TNT Trial Management Group and Investigators. Characterization of chromosomal instability in the TNT trial: A randomized phase III trial of carboplatin compared with docetaxel for patients with metastatic or recurrent locally advanced triple negative or BRCA1/2 breast cancer (CRUK/07/012) [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 P1-06-07.

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo–like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. Significance: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

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.

Locoregional recurrence of breast cancer poses significant clinical problems because of frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable receptors, such as c-Met, would be of potential benefit. The aim of this study was to assess 18F-AH113804, a peptide-based molecular imaging agent with high affinity for human c-Met, for the detection of early-stage locoregional recurrence in a human basal-like breast cancer model, HCC1954. Methods: HCC1954 tumor–bearing xenograft models were established, and 18F-AH113804 was administered. Distribution of radioactivity was determined via PET at 60 min after radiotracer injection. PET and CT images were acquired 10 d after tumor inoculation, to establish baseline distribution and uptake, and then on selected days after surgical tumor resection. CT images and caliper were used to determine the tumor volume. Radiotracer uptake was assessed by 18F-AH113804 PET imaging. c-Met expression was assessed by immunofluorescence imaging of tumor samples and correlated with 18F-AH113804 PET imaging results. Results: Baseline uptake of 18F-AH113804, determined in tumor-bearing animals after 10 d, was approximately 2-fold higher in the tumor than in muscle tissue or the contralateral mammary fat pad. The tumor growth rate, determined from CT images, was comparable between the animals with recurrent tumors, with detection of tumors of low volume (3) only possible by day 20 after tumor resection. 18F-AH113804 PET detected local tumor recurrence as early as 6 d after surgery in the recurrent tumor–bearing animals and exhibited significantly higher 18F-AH113804 uptake (in comparison to mammary fatty tissue), with a target-to-background (muscle) ratio of approximately 3:1 (P < 0.01). The c-Met expression of individual resected tumor samples, determined by immunofluorescence, correlated with the respective 18F-AH113804 imaging signals (r = 0.82, P < 0.05). Conclusion: 18F-AH113804 PET provides a new diagnostic tool for the detection of c-Met–expressing primary tumor and has potential utility for the detection of locoregional recurrence from an early stage.

Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy. Experimental Design: We evaluated FRα expression in breast cancers by genomic (n = 3,414) and IHC (n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft models. Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy–residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth. Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098–111. ©2018 AACR.

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups - tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes - may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection.

The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Triple-negative breast cancers (TNBC) are characterized by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that allelic-imbalanced copy-number aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene, HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs, as well as sensitizing cancer cells to HR-targeting therapies. Our data therefore provide a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability, and an association with response to platinum-based chemotherapy in TNBC. Significance: Previous studies have shown correlation between mutational “scars” and sensitivity to platinums extending beyond associations with BRCA1/2 mutation, but do not elucidate the mechanism. Here, a novel allele-specific copy-number characterization of genome instability identifies and functionally validates the inappropriate expression of the meiotic gene HORMAD1 as a driver of HR deficiency in TNBC, acting to induce allelic imbalance and moderate platinum and PARP inhibitor sensitivity with implications for the use of such “scars” and expression of meiotic genes as predictive biomarkers. Cancer Discov; 5(5); 488–505. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 453

Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large-scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65-70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose-response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96-98%), but yielded many false positives (positive predictive value = 30-32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large-scale, quantitative scoring of immunohistochemically stained tissue microarrays available in consortia. However, continued optimization, rigorous marker-specific quality control measures and standardization of tissue microarray designs, staining and scoring protocols is needed to enhance results.

Summary Patterns of genomic evolution between primary and metastatic breast cancer have not been studied in large numbers, despite patients with metastatic breast cancer having dismal survival. We sequenced whole genomes or a panel of 365 genes on 299 samples from 170 patients with locally relapsed or metastatic breast cancer. Several lines of analysis indicate that clones seeding metastasis or relapse disseminate late from primary tumors, but continue to acquire mutations, mostly accessing the same mutational processes active in the primary tumor. Most distant metastases acquired driver mutations not seen in the primary tumor, drawing from a wider repertoire of cancer genes than early drivers. These include a number of clinically actionable alterations and mutations inactivating SWI-SNF and JAK2-STAT3 pathways., Graphical Abstract, Highlights • Metastases mostly disseminate late from primary breast tumors, keeping most drivers • Drivers at relapse sample from a wider range of cancer genes than in primary tumors • Mutations in SWI-SNF complex and inactivated JAK-STAT signaling enriched at relapse • Mutational processes similar in primary and relapse; radiotherapy can damage genome, By sequencing primary, locally relapsed, and metastatic breast cancers, Yates et al. show that clones seeding metastasis or relapse disseminate late from primary tumors but continue to acquire mutations, including clinically actionable alterations and mutations inactivating the SWI/SNF and JAK2-STAT3 pathways.

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.

Despite advancements in the use of transcriptional information to understand and classify breast cancers, the contribution of splicing to the establishment and progression of these tumours has only recently starting to emerge. Our work explores this lesser known landscape, with special focus on the basal-like breast cancer subtype where limited therapeutic opportunities and no prognostic biomarkers are currently available. Using ExonArray analysis of 176 breast cancers and 9 normal breast tissues we demonstrate that splicing levels significantly contribute to the diversity of breast cancer molecular subtypes and explain much of the differences compared with normal tissues. We identified pathways specifically affected by splicing imbalances whose perturbation would be hidden from a conventional gene-centric analysis of gene expression. We found that a large fraction of them involve cell-to-cell communication, extracellular matrix and transport, as well as oncogenic and immune-related pathways transduced by plasma membrane receptors. We identified 247 genes in which splicing imbalances are associated with clinical patients’ outcome, whilst no association was detectable at the gene expression level. These include the signaling gene TGFBR1, the proto-oncogene MYB as well as many immune-related genes such as CCR7 and FCRL3, reinforcing evidence for a role of immune components in influencing breast cancer patients’ prognosis.

It has been shown that the tumour microenvironment plays a crucial role in regulating tumour progression by a number of different mechanisms, including the remodeling of collagen fibres in tumour-associated stroma. It is still unclear, however, if these stromal changes are of benefit to the host or the tumour. We hypothesise that stromal maturity is an important reflection of tumour biology, and thus can be used to predict prognosis. The aim of this study is to develop a texture analysis methodology which will automatically classify stromal regions from images of hematoxylin and eosin-stained (H and E) sections into two categories: mature and immature. Subsequently we will investigate whether stromal maturity could be used as a predictor of survival and also as a means to better understand the relationship between the radiological imaging signal and the underlying tissue microstructure. We present initial results for 118 regions-of-interest from a dataset of 39 patients diagnosed with invasive breast cancer.

In order to identify novel tumor drivers and gene addictions in breast cancer we have recently completed an integrated in silico/in vitro analysis of gene copy-number alterations and gene-expression profiles in a Triple Negative Breast Cancer-enriched cohort. An amplified and overexpressed gene that appeared to be a driver required for breast cancer cell proliferation is GPHR/GPR89. Several lines of evidence have recently suggested GPHR to be an anion channel, resident in the Golgi, where it controls intraluminal pH. Perturbation of GPHR function leads to altered secretory protein modification and impairment of intracellular transport. Additional data obtained in non-mammalian systems suggest that the localization of GPHR is wider and includes the endoplasmic reticulum (ER). Here, we show that GPHR is amplified and highly expressed across the breast cancer subtypes in our cohort, which we validate in the publicly available TCGA and METABRIC datasets. Using orthogonal characterization by both siRNA-shRNA and CRISPR/Cas9 gene editing, we demonstrate that GPHR down regulation impairs breast cancer cell proliferation and clonogenic ability. To further exclude the possibility that the observed phenotype was due to an “off target” effect, we also performed a rescue of function experiment overexpressing a GPHR protein resistant to GPHR shRNA that confirmed specificity. Importantly, breast cancer spheroids inducibly overexpressing GPHR are shown to have an increased area compared to control spheroids, demonstrating an important role of GPHR in breast cancer cell proliferation. Furthermore, by gradient sucrose subcellular fractionations and immunofluorescence analysis, we show that GPHR is localized in the ER of breast cancer cells. Unfolded protein response (UPR) signatures based on first-degree protein-protein interactions indicate that GPHR is positively correlated with IRE1α and negatively correlated with ATF4/CHOP networks. GPHR is also positively correlated with the XBP1 gene signature. We then further demonstrate that over expression or down regulation of GPHR are able to modulate the IRE1α/XBP1 network of proteins. In summary, in order to sustain uncontrolled growth, breast cancer cells undergo significant stress due to accelerated metabolism, deficient nutrient provision and oxidative stress. UPR signaling and GPHR expression are then essential to protect cells and to allow tumor growth by reducing protein synthesis and favoring chaperonine functions. Here we demonstrate that GPHR is over expressed across the breast cancer subtypes where it controls IRE1α/XBP1 network, and, therefore, can be a novel target for breast cancer therapy as malignant breast cells overexpressing GPHR are selectively dependent on GPHR expression. Citation Format: Riccardo Ferro, Pierfrancesco Marra, Ana Mendes Pereira, Virinder Reen, Cynthia Prince, Sumi Mathew, Alessandra Facchetti, Priyanka Ghongane-Salpe, Sedigeh Kareemaghay, Patrycja Gazinska, David Robertson, Farzana Noor, Fara Braso' Maristany, Daniel Weekes, Dragomir Krastev, Tencho Tenev, Hasan Mirza, Nirmesh Patel, Anita Grigoriadis, Andrew Tutt. The anion channel GPHR/GPR89 regulates protein-folding homeostasis by regulating the IRE1α pathway in breast cancer [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 443.

The E-cadherin (CDH1) tumour suppressor gene encodes a calcium-dependent cell-cell adhesion glycoprotein, which has roles in maintaining cell polarity, differentiation, cell migration and survival. E-cadherin dysfunction is a feature common to many epithelial tumours, with the highest incidence occurring in diffuse gastric cancer (50%) and lobular breast cancer (63%) and can occur via CDH1 mutation, deletion or epigenetic silencing. Although E-cadherin dysfunction is relatively common, precision medicine approaches that exploit this pathogenic alteration are not yet available. Using genetic and small-molecule perturbation screens in breast tumour cells with CRISPR-Cas9 engineered CDH1 mutations, we identified a synthetic lethal interaction between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Using data from large-scale genetic screens in molecularly diverse breast tumour cell lines, we found that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin defective cells, phenotypes that were associated with a defect in cytokinesis and aberrant p120-catenin phosphorylation and localisation. In vivo, ROS1 inhibitors produced profound anti-tumour effects in multiple, distinct, models of E-cadherin defective breast cancer. This data therefore provides the pre-clinical rationale for assessing ROS1 inhibitors such as the licensed drug crizotinib in appropriately stratified patients. Citation Format: Ilirjana Bajrami, Rebecca Marlow, Marieke van de Ven, Rachel Brough, Helen N. Pemberton, Jessica Frankum, Fei Fei Song, Rumana Rafiq, Asha Konde, Malini Menon, James Campbell, Aditi Gulati, Rahul Kumar, Stephen J. Pettitt, Mark D. Gurden, Marta Llorca Cardenosa, Irene Chong, Patrycja Gazinska, Fredrik Wallberg, Elinor J. Sawyer, Lesley-Ann Martin, Mitch Dowsett, Spiros Linardopoulos, Colm Ryan, Patrick W. Derksen, Jos Jonkers, Andrew N.J. Tutt, Alan Ashworth, Christopher J. Lord. E-cadherin/ROS1 inhibitor synthetic lethality in breast cancer [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 2986.

Background: Annexin A1 (ANXA1) is a protein related with the carcinogenesis process and metastasis formation in many tumors. However, little is known about the prognostic value of ANXA1 in breast cancer. The purpose of this study is to evaluate the association between ANXA1 expression, BRCA1/2 germline carriership, specific tumor subtypes and survival in breast cancer patients. Methods: Clinical-pathological information and follow-up data were collected from nine breast cancer studies from the Breast Cancer Association Consortium (BCAC) (n = 5,752) and from one study of familial breast cancer patients with BRCA1/2

Background TNT previously reported that patients(pts) with advanced triple negative breast cancer(TNBC) and germline BRCA1/2(gBRCA+) mutations are more likely to respond to carboplatin(C) than to docetaxel(D), and have longer progression-free survival with C. Here, we report results from a pre-planned biological analysis to test whether BRCA silencing or methylation status confer sensitivity to platinum. Methods Pts eligible for TNT had ER-, PgR-, HER2- BC or were known gBRCA+(any ER/PgR/HER2). Pts were randomized to C(AUC 6 q3wk) or D(100mg/m2 q3wk) for 6-8 cycles or until disease progression if sooner, with crossover possible on progression. Blood and archival tissue samples were requested for BRCA1/2 genotyping, central analysis of TN and DNA repair deficiency biomarkers. Primary endpoint was intention-to-treat objective response rate(ORR) up to cycle 6. BRCA1 mRNA level was estimated from whole-genome total RNA sequencing(RNA-Seq). BRCA1 methylation analysis was quantified by bisulfite sequencing. A methylation score for the sample was computed as percentage of methylated reads relative to total number of reads that were either methylated or not methylated for CpG sites. A sample with a score >10% was considered methylated(BRCA1meth)? a BRCA1 mRNA level "silenced" subgroup(BRCA1 silencing) was defined as value of log2(mRNA) Results 212 and 191 pts' samples were assessed for BRCA1 methylation and mRNA levels. 33 pts (15.6%) were found to be BRCA1meth. Pts with BRCA1meth had a better response to D (42%, 95%CI: 20, 64) than C (21%, 95%CI: -0.1, 43)? absolute difference (C-D) -21% (95%CI: -52, 10)? p=0.28), but not statistically significant. There was no evidence of a difference when gBRCA+ tumours were excluded. 31 pts (16%) had BRCA1 silencing. Concordant with BRCA1meth, pts with BRCA1 silencing had a better response to D (65%, 95%CI: 42, 87) than C (29%, 95%CI: 4.9, 52)? absolute difference (C-D) -36% (95%CI: -69, -3.3)? p=0.07), with an odds ratio 0.22 (95%CI: 0.035, 1.2) in favor of D, interaction with treatment not significant (p=0.066). 19 pts with BRCA1meth and BRCA1 silencing had a better response to D (60%, 95%CI: 30, 90) than C (22%, 95%CI: -5.0, 49)? absolute difference (C-D) -38% (95%CI: -79, 2.9) in favor of D (p=0.17), and the interaction was not significant (p=0.15). Further exploratory analyses examining any relationship between a response to C and new cutpoints for BRCA1meth or BRCA1 mRNA level did not suggest any evidence of a signal. Conclusion Our data did not support a priori hypotheses that BRCA1 methylation or silencing would be associated with a greater response to C than D, which is consistent with other reports in ovarian cancer. As BRCA1 methylation will not always lead to significant silencing or be the only cause of low BRCA1 gene expression, exploratory analysis is investigating the interaction of BRCA silenced group with D sensitivity. RNA-seq is ongoing and results from the final database will be presented. Citation Format: Tutt A, Cheang MCU, Kilburn L, Tovey H, Gillett C, Pinder S, Lanchbury J, Abraham J, Barrett S, Barrett-Lee P, Chan S, Gazinska P, Grigoriadis A, Kernaghan S, Hoadley K, Gutin A, Harper-Wynne C, Hatton M, Owen J, Parker P, Roylance R, Shaw A, Smith I, Thompson R, Timms K, Wardley A, Wilson G, Harries M, Ellis P, Ashworth A, Perou C, Bliss J, Rahman N, Brown R, On Behalf of the TNT Trial Management Group and Investigators. BRCA1 methylation status, silencing and treatment effect in the TNT trial: A randomized phase III trial of carboplatin compared with docetaxel for patients with metastatic or recurrent locally advanced triple negative or BRCA1/2 breast cancer (CRUK/07/012) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S6-01.

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.

Triple negative breast cancers (TNBCs) lack oestrogen (ER), progesterone (PR) and human epidermal growth factor 2 (HER2) receptors, and have limited targeted treatment options. Large scale genomic and transcriptomic studies have advanced our understanding of the changes which occur in TNBCs. However, the substantial number of copy number and gene expression alterations present in TNBCs makes it difficult to identify putative drivers, biomarkers and/or therapeutic targets of the disease. To overcome this problem, we have carried out an integrative computational and RNAi based approach to identify genes required for proliferation of TNBC. Copy number and gene expression alterations were analysed using Affymetrix Human Exon HTS1.0 and SNP6.0 data of 152 primary breast tumours enriched for a TNBC phenotype and 9 normal breast epithelium. These analyses revealed 141 candidate genes whose upregulated gene expression is copy number driven in TNBC. The functional dependence on each of these genes was subsequently examined using RNAi in an array of 17 breast cancer and non-malignant cell lines using 6-8 cell lines per gene covering the widest possible range of expression levels for that gene. We validated a malignant cell specific functional dependence on 37 of the 141 genes using this method. STRING analysis of validated hits reveals a subset of genes involved in the process of cell division and mitosis including the previously characterised mitotic kinase TTK. Of these, we further validated KIFC1 (HSET) which is known to play a role in clustering supernumerary centrosomes, a common occurrence in breast cancer. We show that siRNA and shRNA mediated depletion of KIFC1 decreases cell viability and clonogenic ability specifically in centrosome amplified cell lines, which can be rescued upon introduction of an si/shRNA resistant KIFC1. KIFC1 depletion also produces a high level of catastrophic multipolar mitoses in centrosome amplified but not in non-amplified cell lines. Furthermore, in-vivo studies show that inducible depletion of KIFC1 suppresses tumour growth of centrosome amplified cell line xenografts. Our work has identified and functionally validated novel drivers, and potential therapeutic targets in TNBC. The data presented here shows KIFC1, a druggable kinesin motor protein is a promising target for therapeutic intervention being expressed through gene copy gain in a significant proportion of TNBCs. We validate its role in cancer-specific amplified centrosome clustering showing KIFC1 plays an essential role in aiding the survival of breast cancer cells that have supernumerary centrosomes in both in-vitro and in-vivo contexts. Citation Format: Nirmesh S. Patel, Konstantinos Drosopoulos, Daniel Weekes, Elodie Noel, Hasan Mirza, Mamunur Rashid, Emanuele de Rinaldis, Fara Brasó Maristany, Sumi Mathew, Erika Francesch Domenech, Patrycja Gazinska, Farzana Noor, Jelmar Quist, Rebecca Marlow, Anita Grigoriadis, Spiros Linardopoulos, Andrew N. Tutt. An integrated copy number and gene expression genomics analysis and RNAi approach identifies and validates the KIFC1 kinesin as a malignant cell selective target in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 67.

Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene. This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an “un-targetable” oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis. We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs. Citation Format: Fara Braso-Maristany, Simone Filosto, Steven Catchpole, Rebecca Marlow, Jelmar Quist, Erika Francesch Domenech, Gianmaria Liccardi, Leila Zakka, Violeta Serra, Albert Gris, Maggie Cheang, Nirmesh Patel, Anna Perdrix Rosell, Patrycja Gazinska, Elodie Noel, Jonathan Watkins, Pierfrancesco Marra, Anita Grigoriadis, Andrew Tutt. PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 178.

Basal-like invasive breast cancer is an important clinical group because of its association with a triple-negative phenotype defined by the lack of expression of estrogen, progesterone and human epidermal growth factor receptors 2, relative lack of therapeutic options and poor prognosis. However, depending on the method used to define these lesions, morphological assessment, immunohistochemical markers or gene expression, a different set of tumors is captured. The aim of this study was to investigate the consequences of using different methodological approaches to define basal-like lesions among triple-negative breast carcinomas with regard to their clinicopathological features and patient outcome. The cohort consisted of 142 invasive breast cancers with a triple-negative receptor status. First, each was reviewed histologically and those with morphological basal-like features were characterized as 'Path-Basal'. Second, the 'Core Basal' immunohistochemical lesions, defined as cytokeratin 5/6 and/or epidermal growth factor receptor 1 positive, within the triple-negative breast cancers were identified, and third their classification based on gene expression profiling was retrieved and those in the molecular 'PAM50 basal-like' subtype recorded. A total of 116 basal-like breast cancers were identified among the 142 triple-negative breast cancers by at least one of these three classifications (80%), but only 13 samples were defined as basal-like with all three methods. None of these 13 tumors were associated with lymphovascular invasion. The 34 morphological 'Path-Basal' lesions were significantly associated with a lack of nodal metastases. Comparing the estimates of death in the three classifications, the highest risk of death was seen for the 'Core Basal' group. In this study, we highlight that the definition of basal-like breast cancer based on different methodologies varies significantly and does not identify the same lesions. This incomplete overlap of cases emphasizes the need for consistent or new approaches to improve precise identification.

Approximately 25% of triple-negative breast cancers display increased copy number in the region of 9p24. Since several genes mapping to this genomic locus have been shown to be involved in the biology of triple-negative breast cancers, a copy number-dependent transcriptional influence of genes at 9p24 on the malignant phenotypes has been suggested. Given the limited options of targeted therapy for triple-negative breast cancer patients, further studies of genes located at this locus could therefore provide potential novel targets or companion biomarkers for this disease. By integrating microarray-based DNA copy number and gene expression of genes located on 9p24 in 111 triple-negative breast cancers, we identified among a wider set of genes three hypothetical genes, KIAA0020, KIAA1432 and KIAA2026 whose expression was highly correlated with their copy number status. External validation through analysis of several comprehensive breast cancer cohorts, confirmed KIAA0020 as being highly abundant in basal-like/ triple-negative breast cancers. Involvement of KIAA0020 in PARP1 activity and the DNA damage response has previously been suggested. To elucidate KIAA0020's functional involvement in cell growth, cell cycle progression, apoptosis and DNA damage response in this subtype of breast cancer, breast cancer cell lines with and without increased 9p24 copy number levels were used as in vitro models. In cell lines with increased DNA copy number at 9p24, depletion of KIAA0020 expression selectively impaired growth causing an accumulation in S-phase and a decrease in cell proliferation. Furthermore, KIAA0020-silencing in such cell lines resulted in decreased repair of hydroxyurea induced replication associated DNA damage, accumulation of DNA double strand breaks and decreased occurrence of RAD51 and PAR foci, all pointing to a decreased repair of inactivated replication forks. Taken together, our data supports the notion that genes of unknown function, residing in the 9p24 copy number aberrant region are involved in DNA repair and may thereby also contribute to the tumourigenesis of triple-negative breast cancers. A subset of such triple-negative tumours seemed to have developed a dependency on the expression of KIAA0020, a PARP1 interacting gene, for replication fork associated repair. Thus, greater understanding of KIAA0020's molecular function may provide additional information for patient selection with regards to DNA damaging chemotherapeutics or PARP inhibitors within triple-negative breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-05.

A. Grigoriadis1, D. Weekes1, J.A. Watkins1, P. Gazinska1, J. Frankum2, C. Lord2, A. Ashworth2, J.M. Ford3, M. Telli3, A. Tutt4 Breakthrough Breast Cancer Research Unit, King’s College London Guy’s Hospital, London, UK Cancer Genomics and Therapeutics, Institute of Cancer Research ICR, London, UK Division of Oncology, Stanford University School of Medicine, Stanford, CA, USA Breakthrough Breast Cancer Research Unit, King’s College London School of Medicine, London, UK

Introduction: Subgroups within sporadic triple negative breast cancers (TNBCs) appear to share impaired DNA damage response mechanisms with BRCA1/2 mutation-associated breast cancers. This has been hypothesised to confer particular sensitivity to DNA-damaging platinum chemotherapy. The TNT trial, a randomized phase III trial in women with metastatic or recurrent locally advanced TNBC or BRCA1/2 mutation-associated breast cancer, aimed to test this hypothesis and examine treatment effect in biological subgroups. Patients & Methods: Eligible patients had either ER-, PR-, HER2- breast cancer or were known BRCA1/2 carriers (any ER/PR/HER2). Patients were randomized (1:1) to receive either C (AUC 6 q3wk) or D (100mg/m2 q3wk) for 6-8 cycles or until disease progression if sooner and could cross over to the alternative treatment on confirmed progression. Ineligible patients included those who had ECOG performance status >2, received adjuvant taxane therapy in the last 12 mths, any previous treatment with a platinum chemotherapy, or previous non-anthracycline chemotherapy for metastatic disease. For consenting patients a blood sample and archived tissue samples were obtained for BRCA1/2 genotyping and central biomarker analysis (primary tumour, lymph nodes and recurrent tumour biopsy if available) of subtypes within TNBC and biomarkers of DNA repair deficiency. The primary endpoint was RECIST objective tumour response up to cycle 6 of randomised treatment. Secondary endpoints included toxicity, progression free survival (PFS), time to progression and overall survival. TNT aimed to detect a 15% improvement in ORR with C compared to D, with planned target sample size range of 370-450 depending on assumed ORR in D patients (2-sided α=0.05, power=90%). 376 (188 C, 188 D) were recruited from 74 UK centres between Apr 08 and Mar 14. Results: A snapshot of the data was taken on 30/5/14 at which point 336 (89.4%) patients had experienced a PFS event, with overall median PFS time of 4.4 mths. Median age of patients was 55 yrs (IQR 48-63). 366/376 (97%) patients had TNBC of whom 18 were also known BRCA1/2 mutation carriers, with the remaining 10 patients receptor +ve and BRCA1/2 carriers. 338/376 (90%) had metastatic and 38/376 (10%) recurrent locally advanced disease. 53% had liver or lung metastases affecting the parenchyma and 34% had received previous adjuvant taxane therapy. Median time from initial diagnosis to entering TNT was 2.2 yrs (IQR 1.5-3.5). Primary tumour tissue has currently been received for 277 patients, blood from 286 patients and recurrent tumour tissue from 85 patients. Discussion: TNT will report evidence on the activity of single agent platinum chemotherapy compared with single agent taxane in patients with TNBC and BRCA1/2 associated breast cancer. Correlative analyses of BRCA1/2 mutation status, subtypes and DNA repair biomarkers will also be reported. TNT will be the first randomised trial to report the activity of platinum compared with standard chemotherapy within TNBC subtypes and in relation to BRCA1/2 mutation status and DNA repair biomarkers. Safety, tolerability and response to crossover treatment will also be presented. Citation Format: Andrew Tutt, Paul Ellis, Lucy Kilburn, Cheryl Gilett, Sarah Pinder, Jacinta Abraham, Sophie Barrett, Peter Barrett-Lee, Stephen Chan, Maggie Cheang, Mitch Dowsett, Lisa Fox, Patrycja Gazinska, Anita Grigoriadis, Alexander Gutin, Catherine Harper-Wynne, Matthew Hatton, Sarah Kernaghan, Jerry Lanchbury, James Morden, Julie Owen, Jyoti Parikh, Peter Parker, Nazneen Rahman, Rebecca Roylance, Adam Shaw, Ian Smith, Rose Thompson, Kirsten Timms, Holly Tovey, Andrew Wardley, Gregory Wilson, Mark Harries, Judith Bliss. The TNT trial: A randomized phase III trial of carboplatin (C) compared with docetaxel (D) for patients with metastatic or recurrent locally advanced triple negative or BRCA1/2 breast cancer (CRUK/07/012) [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 S3-01.

Introduction: The histopathological absence of ER, PR, and HER2 defines a subgroup of breast cancers named triple negative, characterized by aggressive behavior and poor prognosis. These tumors represent a significant clinical challenge as little is known about the genetic alterations underlying their development and, as a consequence, no specific targeted therapies are yet available. Using high-resolution SNP6.0 Affymetrix technology, we evaluated genetic copy number alteration events in a collection of 107 fine-needle microdissected breast cancers, including 82 triple-negative and 25 non-triple negative (17 ER+/ HER2- and 8 ER-/HER2+) samples. 18 blood samples from matching patients were also profiled and their pool was used as a baseline. Results: Upon application of circular binary segmentation algorithm, 36 and 190 chromosomal regions could be identified for being respectively frequently amplified and deleted in triple-negative tumors (frequency > 30%, p-value < 0.001). Our analysis confirms many regions, such as 1 p36, 3q26, 5q11, 8p23,17p13, already known to be amplified or deleted from previous studies. Using two- dimensional hierarchical clustering based on copy number changes of segmented DNA regions in all tumor samples, no particular chromosomal alteration appeared to classify the group of triple- negative breast cancer. However, through supervised analysis, specific DNA deletions could be identified that occur at significantly higher frequency in triple-negative tumors as compared to the non-triple negatives, mainly involving chromosomal regions 3p14, 4p15, 5q11, 5q12 and 5q13. Interestingly, these regions harbour a number of cancer-related miRNAs, such as miR-135a, miR-7g and miR-95. Conversely, regions 15q13,16q22,16q23 and 16q24 were observed to be preferentially deleted in non-triple negative tumors. We explored the biological pathways affected by genomic aberrations in triple negative and non-triple negative tumors, using a comparative gene set enrichment approach. The analysis was run on genes frequently amplified/deleted (> 30% of samples) in the two tumor groups. Among others, the “immunological response” pathway emerged as more frequently affected by events of amplification in triple-negative breast tumors (p-value < 0.001). In order to characterize functional dependencies between different genomic aberration events, we ran a co-occurrence analysis of genomic regions frequently altered in triple- negative tumors (>20%). A number of chromosome regions were identified for being frequently co-amplified. These include regions on chromosome 1q (with each other), 10p (with each other), 6q11 with 7p12 and 19q13,19q and 5p. Similarly, regions of co-deletions could be identified, such as: 8p21 (with each other), 4p15 (with each other), 17p13 and 5q11,6q26, and 17p12. Conclusions: We have performed a comprehensive survey of copy number variations in triple-negative breast tumors, pinpointing genomic regions and related genes and pathways specifically altered in this class of tumors. Dependencies between individual events of genomic amplification or deletion have been identified, suggesting they are resulting from selective pressure and their coordinated genomic alteration is required for the survival of the cancer cells involved. Our analyses shed further light on the genomically complex and heterogeneous nature of triple-negative tumors in relation to other subgroups of breast cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B3.

BACKGROUND: This study focuses on the analysis of miRNAs expression data in a cohort of 181 well characterised breast cancer samples composed primarily of triple-negative (ER/PR/HER2-negative) tumours with associated genome-wide DNA and mRNA data, extensive patient follow-up and pathological information. RESULTS: We identified 7 miRNAs associated with prognosis in the triple-negative tumours and an additional 7 when the analysis was extended to the set of all ER-negative cases. miRNAs linked to an unfavourable prognosis were associated with a broad spectrum of motility mechanisms involved in the invasion of stromal tissues, such as cell-adhesion, growth factor-mediated signalling pathways, interaction with the extracellular matrix and cytoskeleton remodelling. When we compared different intrinsic molecular subtypes we found 46 miRNAs that were specifically expressed in one or more intrinsic subtypes. Integrated genomic analyses indicated these miRNAs to be influenced by DNA genomic aberrations and to have an overall influence on the expression levels of their predicted targets. Among others, our analyses highlighted the role of miR-17-92 and miR-106b-25, two polycistronic miRNA clusters with known oncogenic functions. We showed that their basal-like subtype specific up-regulation is influenced by increased DNA copy number and contributes to the transcriptional phenotype as well as the activation of oncogenic pathways in basal-like tumours. CONCLUSIONS: This study analyses previously unreported miRNA, mRNA and DNA data and integrates these with pathological and clinical information, from a well-annotated cohort of breast cancers enriched for triple-negative subtypes. It provides a conceptual framework, as well as integrative methods and system-level results and contributes to elucidate the role of miRNAs as biomarkers and modulators of oncogenic processes in these types of tumours.