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

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

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

Background: Intracellular 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. Methods: Ultracentrifugation 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. Results: Patients 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. Discussion: Our 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 state may provide a promising way forward for restoring immune competence in specific breast cancer patient populations

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

Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium

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.Peer reviewe

Annexin A1 expression in a pooled breast cancer series: Association with tumor subtypes and prognosis

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 mutations (n = 107). ANXA1 expression was scored based on the percentage of immunohistochemical staining in tumor cells. Survival analyses were performed using a multivariable Cox model. Results: The frequency of ANXA1 positive tumors was higher in familial breast cancer patients with BRCA1/2 mutations than in BCAC patients, with 48.6 % versus 12.4 %, respectively; P adj = 1.35; 95 % CI = 1.05-1.73), but the association weakened after 10 years (HRadj = 1.13; 95 % CI = 0.91-1.40). ANXA1 was a significant independent predictor of survival in HER2+ patients (10-years BCSS: HRadj = 1.70; 95 % CI = 1.17-2.45). Conclusions: ANXA1 is overexpressed in familial breast cancer patients with BRCA1/2 mutations and correlated with poor prognosis features: triple negative and poorly differentiated tumors. ANXA1 might be a biomarker candidate for breast cancer survival prediction in high risk groups such as HER2+ cases

RORγt+ innate lymphoid cells promote lymph node metastasis of breast cancers

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

Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: the TNT Trial

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

Annexin A1 expression in a pooled breast cancer series : association with tumor subtypes and prognosis

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 mutations (n = 107). ANXA1 expression was scored based on the percentage of immunohistochemical staining in tumor cells. Survival analyses were performed using a multivariable Cox model. Results: The frequency of ANXA1 positive tumors was higher in familial breast cancer patients with BRCA1/2 mutations than in BCAC patients, with 48.6 % versus 12.4 %, respectively; P <0.0001. ANXA1 was also highly expressed in BCAC tumors that were poorly differentiated, triple negative, EGFR-CK5/6 positive or had developed in patients at a young age. In the first 5 years of follow-up, patients with ANXA1 positive tumors had a worse breast cancer-specific survival (BCSS) than ANXA1 negative (HRadj = 1.35; 95 % CI = 1.05-1.73), but the association weakened after 10 years (HRadj = 1.13; 95 % CI = 0.91-1.40). ANXA1 was a significant independent predictor of survival in HER2+ patients (10-years BCSS: HRadj = 1.70; 95 % CI = 1.17-2.45). Conclusions: ANXA1 is overexpressed in familial breast cancer patients with BRCA1/2 mutations and correlated with poor prognosis features: triple negative and poorly differentiated tumors. ANXA1 might be a biomarker candidate for breast cancer survival prediction in high risk groups such as HER2+ cases.Peer reviewe

Clinically Compliant Spatial and Temporal Imaging of Chimeric Antigen Receptor T-cells

Adoptive transfer of chimeric antigen receptor (CAR) T cells has shown promising anticancer results in clinical trials. Here the authors use the human sodium iodide symporter (hNIS) as a reporter gene to image human CAR T cells in cancer-bearing mice using broadly available tracers and imaging platforms