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Tumour-associated macrophages (TAMs) play an important role in tumour progression, which is facilitated by their ability to respond to environmental cues. Here we report, using murine models of breast cancer, that TAMs expressing fibroblast activation protein alpha (FAP) and haem oxygenase-1 (HO-1), which are also found in human breast cancer, represent a macrophage phenotype similar to that observed during the wound healing response. Importantly, the expression of a wound-like cytokine response within the tumour is clinically associated with poor prognosis in a variety of cancers. We show that co-expression of FAP and HO-1 in macrophages results from an innate early regenerative response driven by IL-6, which both directly regulates HO-1 expression and licenses FAP expression in a skin-like collagen-rich environment. We show that tumours can exploit this response to facilitate transendothelial migration and metastatic spread of the disease, which can be pharmacologically targeted using a clinically relevant HO-1 inhibitor. The relationship between wound healing and cancer is intricate and complex. Here, the authors show that in breast cancer models an IL-6 driven co-expression of FAP and HO-1 in tumour-associated macrophages, similar to the wound healing response, facilitates migration and metastatic spread.

PIK3CA mutations are reported to be present in approximately 25% of breast cancer (BC), particularly the estrogen receptor—positive (ER+) and HER2-overexpressing (HER2+) subtypes, making them one of the most common genetic aberrations in BC. In experimental models, these mutations have been shown to activate AKT and induce oncogenic transformation, and hence these lesions have been hypothesized to render tumors highly sensitive to therapeutic PI3K/mTOR inhibition. By analyzing gene expression and protein data from nearly 1,800 human BCs, we report that a PIK3CA mutation—associated gene signature (PIK3CA-GS) derived from exon 20 (kinase domain) mutations was able to predict PIK3CA mutation status in two independent datasets, strongly suggesting a characteristic set of gene expression—induced changes. However, in ER+/HER2—BC despite pathway activation, PIK3CA mutations were associated with a phenotype of relatively low mTORC1 signaling and a good prognosis with tamoxifen monotherapy. The relationship between clinical outcome and the PIK3CA-GS was also assessed. Although the PIK3CA-GS was not associated with prognosis in ER — and HER2+BC, it could identify better clinical outcomes in ER+/HER2— disease. In ER+ BC cell lines, PIK3CA mutations were also associated with sensitivity to tamoxifen. These findings could have important implications for the treatment of PIK3CA-mutant BCs and the development of PI3K/mTOR inhibitors.

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.

The tumor microenvironment (TME) is highly heterogeneous and can dictate the success of therapeutic interventions. Identifying TMEs that are susceptible to specific therapeutic interventions paves the way for more personalized and effective treatments. In this study, using a spontaneous murine model of breast cancer, we characterize a TME that is responsive to inhibitors of the heme degradation pathway mediated by heme oxygenase (HO), resulting in CD8+ T cell- and NK cell-dependent tumor control. A hallmark of this TME is a chronic type I interferon (IFN) signal that is directly involved in orchestrating the antitumor immune response. Importantly, we identify that similar TMEs exist in human breast cancer that are associated with patient prognosis. Leveraging these observations, we demonstrate that combining a STING agonist, which induces type I IFN responses, with an HO inhibitor produces a synergistic effect leading to superior tumor control. This study highlights HO activity as a potential resistance mechanism for type I IFN responses in cancer, supporting a therapeutic rationale for targeting the heme degradation pathway to enhance the efficacy of STING agonists.

Breast cancer has a long natural history. Established and emerging biologic markers address overall risk but not necessarily timing of recurrence. 346 adjuvant naïve breast cancer cases from Guy’s Hospital with 23 years minimum follow-up and archival blocks were recut and reassessed for hormone-receptors (HR), HER2-receptor and grade. Disease-specific survival (DSS) was analyzed by recursive partitioning. To validate insights from this analysis, gene-signatures (proliferative and HR-negative) were evaluated for their ability to predict early versus late metastatic risk in 683 node-negative, adjuvant naïve breast cancers annotated with expression microarray data. Risk partitioning showed that adjuvant naïve node-negative outcome risk was primarily partitioned by tumor receptor status and grade but not tumor size. HR-positive and HER2-negative (HRpos) risk was partitioned by tumor grade; low grade cases have very low early risk but a 20% fall-off in DSS 10 or more years after diagnosis. Higher grade HRpos cases have risk over >20 years. Triple-negative (Tneg) and HER2-positive (HER2pos) cases DSS events occurred primarily within the first 5 years. Among node-positive cases, only low grade conferred late risk, suggesting that proliferative gene signatures that identify proliferation would be important for predicting early but not late recurrence. Using pooled data from four publicly available data sets for node-negative tumors annotated with gene expression and outcome data, we evaluated four prognostic gene signatures: two proliferation-based and two immune function-based. Tumor proliferative capacity predicted early but not late metastatic risk for HRpos cases. The immune function or HRneg specific signatures predicted only early metastatic risk in Tneg and HER2pos cases. Breast cancer prognostic signatures need to inform both risk and timing of metastatic events and may best be applied within subsets. Current signatures predict for outcome risk within 5 years of diagnosis. Predictors of late risk for HR positive disease are needed.

Background Metastases from primary breast cancers can involve single or multiple organs at metastatic disease diagnosis. Molecular risk factors for particular patterns of metastastic spread in a clinical population are limited. Methods A case-control design including 1357 primary breast cancers was used to study three distinct clinical patterns of metastasis, which occur within the first six months of metastatic disease: bone and visceral metasynchronous spread, bone-only, and visceral-only metastasis. Whole-genome expression profiles were obtained using whole genome (WG)-DASL assays from formalin-fixed paraffin-embedded (FFPE) samples. A systematic protocol was developed for handling FFPE samples together with stringent data quality controls to identify robust expression profiling data. A panel of published and novel gene sets were tested for association with these specific patterns of metastatic spread and odds ratios (ORs) were calculated. Results Metasynchronous metastasis to bone and viscera was found in all intrinsic breast cancer subtypes, while immunohistochemically (IHC)-defined receptor status and specific IntClust subgroups were risk factors for visceral-only or bone-only first metastases. Among gene modules, those related to proliferation increased the risk of metasynchronous metastasis (OR (95% CI) = 2.3 (1.1–4.8)) and visceral-only first metastasis (OR (95% CI) = 2.5 (1.2–5.1)) but not bone-only metastasis (OR (95% CI) = 0.97 (0.56–1.7)). A 21-gene module ( BV ) was identified in estrogen-receptor-positive breast cancers with metasynchronous metastasis to bone and viscera (area under the curve = 0.77), and its expression increased the risk of bone and visceral metasynchronous spread in this population. BV was further orthogonally validated with NanoString nCounter in primary breast cancers, and was reproducible in their matched lymph nodes metastases and an external cohort. Conclusion This case-control study of WG-DASL global expression profiles from FFPE tumour samples, after careful quality control and RNA selection, revealed that gene modules in the primary tumour have differing risks for clinical patterns of metasynchronous first metastases. Moreover, a novel gene module was identified as a putative risk factor for metasynchronous bone and visceral first metastatic spread, with potential implications for disease monitoring and treatment planning.

The prognostic importance of lymph node (LN) status and tumour‐infiltrating lymphocytes (TILs), is well established, particularly TILs in triple negative breast cancers (TNBCs). So far, few studies have interrogated changes in involved and uninvolved LNs and evaluated if their morphological patterns add valuable information for the prediction of disease progression in breast cancer. In a cohort of 309 patients enriched for TNBCs (170/309), we histologically characterised immune and stromal features in primary tumours and associated involved and uninvolved axillary LNs on routine haematoxylin and eosin stained sections. Of the 309 patients, 143 had LN‐positive disease. Twenty‐five histopathological features were assessed, including the degree of TIL presence, quantitative and qualitative assessment of germinal centres (GCs) and sinus histiocytosis. Multivariate and cross‐validated proportional hazard regression analyses were used to identify optimal covariate sets for prediction of distant metastasis‐free survival (DMFS). The degree of intratumoural and peritumoural immune infiltrate was associated with architectural changes in both uninvolved and involved LNs. By including clinicopathological characteristics as well as tumour and LN histopathological features in L2‐regularised proportional hazard models, the prediction of 5‐year DMFS was improved by 3–15% over the baseline in all cancers and in TNBCs. In LN‐positive cancers, the combination of Salgado's classification, lymphocytic lobulitis, size and number of GCs in the uninvolved LNs and location of GCs in the involved LNs carried significant prognostic information. From these features, a multivariate cross‐validation‐stable risk signature was constructed, which identified low‐risk groups within both LN‐positive breast cancers and the LN‐positive TNBCs group with a 10‐year DMFS probability of 78 and 87%, respectively. This study illustrates that, by incorporating histopathological patterns of involved and uninvolved LNs combined with primary tumour immune and stromal features, the prediction of developing distant metastasis in LN‐positive breast cancers can be estimated more accurately.

Background Immunohistochemical assessment of proliferation may provide additional prognostic information in early breast cancer. However, due to a lack of methodological standards proliferation markers are still not routinely used for determining therapy. Even for Ki67, one of the most widely-studied markers, disagreements over the optimal cutoff exist. Improvements in digital microscopy may provide new avenues to standardise and make data more reproducible. Methods We studied the immunohistochemical expression of three markers of proliferation: Ki67, Mini-Chromosome Maintenance protein 2 and Geminin, by conventional light microscope and digital imaging on triplicate TMAs from 309 consecutive cases of primary breast cancers. Differences between the average and the maximum percentage reactivity in tumour cell nuclei from the three TMA cores were investigated to assess the validity of the approach. Time-dependent Receiver Operating Characteristic curves were utilized to obtain optimal expression level cut-offs, which were then correlated with clinico-pathological features and survival. Results High concordance between conventional and digital scores was observed for all 3 markers (Ki67: r s  = 0.87, P  < 0.001; MCM2: r s  = 0.94, P  < 0.001; and Geminin: r s  = 0.86, P  < 0.001; Spearman’s rank). There was no significant difference according to the number of TMA cores included for either Ki67 or MCM2; analysis of two or three cores produced comparable results. Higher levels of all three proliferation markers were significantly associated with higher grade ( P  < 0.001) and ER-negativity ( P  < 0.001). Optimal prognostic cut-offs for percentage expression in the tumour were 8 %, 12 and 2.33 % for Ki67, MCM2 and Geminin respectively. All 3 proliferation marker cutoffs were predictive of 15-year breast cancer-specific survival in univariable Cox regression analyses. In multivariable analysis only lymph node status (HR = 3.9, 95 % CI = 1.79-8.5, P  = 0.0006) and histological grade (HR = 1.84, 95 % CI = 1–3.38, P  = 0.05) remained significantly prognostic. Conclusions Here we show that. MCM2 is a more sensitive marker of proliferation than Ki67 and should be examined in future studies, especially in the lymph node-negative, hormone receptor-positive subgroup. Further, digital microscopy can be used effectively as a high-throughput method to evaluate immunohistochemical expression.

Cyclin D1 protein plays an important part in regulating the progress of the cell during the G1 phase of the cell cycle. The cyclin D1 gene, CCND1, is amplified in approximately 20% of mammary carcinomas, and the protein is over-expressed in approximately 50% of cases. This has led to intensive study to ascertain whether cyclin D1 is a biological marker in breast cancer; however, the clinical work has produced unexpected results. Work in cell lines and in transgenic mice indicate that CCND1 is a weak oncogene and it was expected that, like c-erbB-2, over-expression of cyclin D1 protein would be associated with a poor prognosis. Early immunohistochemical prognostic studies produced equivocal results but we, and others, have recently shown that strong staining for cyclin D1 is more likely to be seen in well differentiated, estrogen receptor positive carcinomas. Furthermore, we have found that over-expression of cyclin D1 is actually associated with a good outcome, both in terms of prognosis and response to endocrine treatment. Cyclin D1 is frequently over-expressed in ductal carcinoma in situ but not in benign breast disease, including atypical ductal hyperplasia; hence its expression appears to be closely linked with carcinogenesis. In order to help explain the apparent beneficial effects of cyclin D1 over-expression, a number of closely associated cell cycle proteins have also been evaluated, including the cyclin dependent kinase inhibitor p27, which blocks the activating effects of cyclin D1. Initial reports show that high levels of p27 are associated with a good prognosis and we have shown a positive association between p27 and cyclin D1 expression. These clinical results of cyclin D1 are an example of how information obtained from basic cell biology studies needs to be complemented by clinical studies to ascertain the true worth of a prognostic marker.

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100–200 ng, from this microdissected tissue required use of the Atlas SMART™ Probe Amplification Kit to synthesize and amplify cDNA and make 33 P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.