Automated Classification of Breast Cancer Stroma Maturity from Histological Images

OBJECTIVE: The tumour microenvironment plays a crucial role in regulating tumour progression by a number of different mechanisms, in particular the remodelling of collagen fibres in tumour-associated stroma, which has been reported to be related to patient survival. The underlying motivation of this work is that remodelling of collagen fibres gives rise to observable patterns in Hematoxylin and Eosin (H&E) stained slides from clinical cases of invasive breast carcinoma that the pathologist can label as mature or immature stroma. The aim of this paper is to categorise and automatically classify stromal regions according to their maturity and show that this classification agrees with that of skilled observers, hence providing a repeatable and quantitative measure for prognostic studies. METHODS: We use multi-scale Basic Image Features (BIF) and Local Binary Patterns (LBP), in combination with a random decision trees classifier for classification of breast cancer stroma regions-ofinterest (ROI). RESULTS: We present results from a cohort of 55 patients with analysis of 169 ROI. Our multi-scale approach achieved a classification accuracy of 84%. CONCLUSION: This work demonstrates the ability of texture-based image analysis to differentiate breast cancer stroma maturity in clinically acquired H&E stained slides at least as well as skilled observers

Revealing a Pre-neoplastic Renal Tubular Lesion by p-S6 Protein Immunohistochemistry after Rat Exposure to Aristolochic Acid

Aristolochic acid (AA) has, in the last decade, become widely promoted as the cause of the Balkan endemic nephropathy and associated renal or urothelial tumours, although without substantial focal evidence of the quantitative dietary exposure via bread in specific households in hyperendemic villages. Occasional ethnobotanical use of Aristolochia clematitis might be a source of AA, and Pliocene lignite contamination of well-water is also a putative health risk factor. The aim of this study was two-fold: to verify if extracts of A. clematitis and Pliocene, or AA by itself, could induce the development of renal or urothelial tumours, and to test the utility of the ribosomal protein p-S6 to identify preneoplastic transformation. Rats were given extracts of A. clematitis in drinking water or AA I, by gavage.  After seven months, renal morphology was studied using conventional haematoxylin and eosin and immunohistochemistry for ribosomal p-S6 protein.  Plant extracts (cumulative AA approximately 1.8 g/kg b.w.) were tolerated and caused no gross pathology or renal histopathological change, with only faint diffuse p-S6 protein (except in the papilla) as in controls. Cumulative AA I (150 mg/kg b.w. given over 3 days) was also tolerated for seven months by all recipients, without gross pathology or kidney tumours. However, p-S6 protein over-expression was consistent particularly within the renal papilla. In one case given AA I, intense p-S6 protein staining of a proximal tubule fragment crucially matched the pre-neoplastic histology in an adjacent kidney section.  We briefly discuss these findings, which compound uncertainty concerning the cause of the renal or upper urinary tract tumours of the Balkan endemic nephropathy

RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma.

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities

Serum-derived extracellular vesicles from breast cancer patients contribute to differential regulation of T-cell-mediated immune-escape mechanisms in breast cancer subtypes

BackgroundIntracellular communication within the tumour is complex and extracellular vesicles (EVs) have been identified as major contributing factors for the cell-to-cell communication in the local and distant tumour environments. Here, we examine the differential effects of breast cancer (BC) subtype-specific patient serum and cell-line derived EVs in the regulation of T cell mediated immune responses. MethodsUltracentrifugation was used to isolate EVs from sera of 63 BC patients, 15 healthy volunteers and 4 human breast cancer cell lines. Longitudinal blood draws for EV isolation for patients on neoadjuvant chemotherapy was also performed. Characterization of EVs was performed by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy (TEM) and immunoblotting. CD63 staining was performed on a tissue microarray of 218 BC patients. In-house bioinformatics algorithms were utilized for the computation of EV associated expression scores within The Cancer Genome Atlas (TCGA) and correlated with tumour infiltrating lymphocyte (TIL) scores. In vitro stimulation of PBMCs with EVs from serum and cell-line derived EVs was performed and changes in the immune phenotypes characterized by flow cytometry. Cytokine profiles were assessed using a 105-plex immunoassay or IL10 ELISA. ResultsPatients with triple negative breast cancers (TNBCs) exhibited the lowest number of EVs in the sera; whilst the highest was detected in ER+HER2+ cancers; reflected also in the higher level of CD63+ vesicles found within the ER+HER2+ local tumour microenvironment. Transcriptomic analysis of the TCGA data identified that samples assigned with lower EV scores had significantly higher abundance of CD4+ memory activated T cells, T follicular cells and CD8 T cells, plasma, and memory B cells; whilst samples with high EV scores were more enriched for anti-inflammatory M2 macrophages and mast cells. A negative correlation between EV expression scores and stromal TIL counts was also observed. In vitro experiments confirmed that circulating EVs within breast cancer subtypes have functionally differing immunomodulatory capabilities, with EVs from patients with the most aggressive breast cancer subtype (TNBCs) demonstrating the most immune-suppressive phenotype (decreased CD3+HLA-DR+ but increased CD3+PD-L1 T cells, increased CD4+CD127-CD25hi T regulatory cells with associated increase in IL10 cytokine production). In depth assessment of the cytokine modulation triggered by the serum/cell line derived exosomes confirmed differential inflammatory cytokine profiles across differing breast cancer subtypes. Studies using the MDA-231 TNBC breast cancer cell-line derived EVs provided further support that TNBC EVs induced the most immunosuppressive response within PBMCs.DiscussionOur study supports further investigations into how tumour derived EVs are a mechanism that cancers can exploit to promote immune suppression; and breast cancer subtypes produce EVs with differing immunomodulatory capabilities. Understanding the intracellular/extracellular pathways implicated in alteration from active to suppressed immune may provide a promising way forward for restoring immune competence in specific breast cancer patient populations

Histological scoring of immune and stromal features in breast and axillary lymph nodes is prognostic for distant metastasis in lymph node-positive breast cancers.

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

Phase II randomized preoperative window-of-opportunity study of the PI3K inhibitor pictilisib plus anastrozole compared with anastrozole alone in patients with estrogen receptor-positive breast cancer

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

Vessel co-option mediates resistance to anti-angiogenic therapy in liver metastases

The efficacy of angiogenesis inhibitors in cancer is limited by resistance mechanisms that are poorly understood. Notably, instead of through the induction of angiogenesis, tumor vascularization can occur through the nonangiogenic mechanism of vessel co-option. Here we show that vessel co-option is associated with a poor response to the anti-angiogenic agent bevacizumab in patients with colorectal cancer liver metastases. Moreover, we find that vessel co-option is also prevalent in human breast cancer liver metastases, a setting in which results with anti-angiogenic therapy have been disappointing. In preclinical mechanistic studies, we found that cancer cell motility mediated by the actin-related protein 2/3 complex (Arp2/3) is required for vessel co-option in liver metastases in vivo and that, in this setting, combined inhibition of angiogenesis and vessel co-option is more effective than the inhibition of angiogenesis alone. Vessel co-option is therefore a clinically relevant mechanism of resistance to anti-angiogenic therapy and combined inhibition of angiogenesis and vessel co-option might be a warranted therapeutic strategy

Genomic evolution of breast cancer metastasis and relapse

A.G.L. and J.H.R.F. were supported by a Cancer Research UK Program Grant to Simon Tavaré (C14303/A17197).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.Publisher PDFPeer reviewe

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population