Stromal oncostatin M cytokine promotes breast cancer progression by reprogramming the tumor microenvironment.

The tumor microenvironment (TME) is reprogrammed by cancer cells and participates in all stages of tumor progression. The contribution of stromal cells to the reprogramming of the TME is not well understood. Here, we provide evidence of the role of the cytokine oncostatin M (OSM) as central node for multicellular interactions between immune and nonimmune stromal cells and the epithelial cancer cell compartment. OSM receptor (OSMR) deletion in a multistage breast cancer model halted tumor progression. We ascribed causality to the stromal function of the OSM axis by demonstrating reduced tumor burden of syngeneic tumors implanted in mice lacking OSMR. Single-cell and bioinformatic analysis of murine and human breast tumors revealed that OSM expression was restricted to myeloid cells, whereas OSMR was detected predominantly in fibroblasts and, to a lower extent, cancer cells. Myeloid-derived OSM reprogrammed fibroblasts to a more contractile and tumorigenic phenotype and elicited the secretion of VEGF and proinflammatory chemokines CXCL1 and CXCL16, leading to increased myeloid cell recruitment. Collectively, our data support the notion that the stromal OSM/OSMR axis reprograms the immune and nonimmune microenvironment and plays a key role in breast cancer progression.

Diagnosis of the sentinel lymph node in breast cancer: a reproducible molecular method: a multicentric Spanish study.

AIMS: Standardization of the sentinel node (SN) as a diagnostic tool has not yet been achieved, because the protocol for histopathological study is highly variable between centres. We compared the results of a new method with conventional histological tests and evaluated its feasibility for intra-operative evaluation, and propose it as a method to standardize the sentinel node evaluation procedure. METHODS AND RESULTS: Trial 1 included 181 cases; in parallel, 2-mm-thick sections of the SN were processed alternately for histological analysis and for the one-step nucleic acid amplification (OSNA) procedure. A final concordance of 99.45% was observed in the first trial of our study. For trial 2, the timing of every procedural step was recorded in an electronic database in order to discern the time spent for each step, the total SN evaluation time and to identify areas of improvement. In the second trial, after a learning period and feedback on data recorded, we spent a mean of 31 min for the entire SN evaluation procedure. CONCLUSION: Our multi-centric trial using the OSNA assay for sentinel node evaluation in breast cancer demonstrates that this is a highly sensitive, specific and reproducible technique that allows for standardization of the SN diagnostic procedure, a necessary, and until now unresolved, issue.

DNA methylation epigenotypes in breast cancer molecular subtypes.

INTRODUCTION: Identification of gene expression based breast cancer subtypes is considered as a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and there is only a limited understanding of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and deliver specific epigenotypes associated with particular breast cancer subtypes. METHODS: Using a microarray approach we analyzed DNA methylation in regulatory regions of 806 cancer related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biological validation by Pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A and 48 luminal B paired breast cancer/adjacent tissues. Using all-subset selection method, we identified the most subtype predictive methylation profiles in multivariable logistic regression analysis. RESULTS: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identify novel subtype specific epigenotypes which clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. CONCLUSIONS: Our results provide evidence that well defined DNA methylation profiles enables breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases.

Background: The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods: Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results: The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions: GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making.

Luminal B breast cancer subtype displays a dicotomic epigenetic pattern.

Luminal B breast tumors have aggressive clinical and biological features, and constitute the most heterogeneous molecular subtype, both clinically and molecularly. Unfortunately, the immunohistochemistry correlate of the luminal B subtype remains still imprecise, and it has now become of paramount importance to define a classification scheme capable of segregating luminal tumors into clinically meaningful subgroups that may be used clinically to guide patient management. With the aim of unraveling the DNA methylation profiles of the luminal subtypes currently being most used in the clinical setting, we have quantified the DNA methylation level of 27,578 CpG sites in 17 luminal B (ER+, Ki67 ≥ 20 % or PgR < 20 % and HER2-), 8 luminal A (ER+ and Ki67 > 20 %) and 4 luminal B-HER2+ (ER+ and HER2+) breast cancer samples by using the Illumina Infinium methylation microarray approach. Unsupervised hierarchical clustering revealed that DNA methylation stratifies luminal B samples in two categories with differing epigenetic and clinical features. One subgroup of luminal B samples showed a methylator phenotype and clustered with the lumB-HER tumors, while the other showed less methylated events, clustered with the luminal A. A 3 CpG marker panel capable of discriminating methylator versus non-methylator luminal B samples was identified and further validated in an independent cohort of patients. Our results provide evidence that DNA methylation and, more specifically, a panel of 3 CpG markers, enables the stratification of luminal B samples in two categories with differing epigenetic and clinical features and support the utilization of this panel for therapeutic stratification of patients with luminal breast cancer.

p53 and cyclin D1 as prognostic factors in squamous cell carcinoma of the larynx.

OBJECTIVES/HYPOTHESIS: Proteins p53 and cyclin D1 play a crucial role in cell cycle control. Protein p53 mutations are one of the most common genetic alterations in human cancer, and cyclin D1 gene amplification has been found to be associated with poor prognosis in different types of tumors. Functional alterations of these proteins may play an important role both in the carcinogenesis of squamous carcinomas of the head and neck and in the clinical evolution of these tumors. The objective of the present study was to evaluate whether the presence of p53 and/or cyclin D1 proteins (detected by immunohistochemical analysis) could serve as relevant variables for the assessment of the prognosis of laryngeal squamous cell carcinoma. STUDY DESIGN: Retrospective multicentric study. METHODS: Paraffin-embedded biopsy specimens from 62 human epidermoid laryngeal carcinomas were randomly selected. The expression of p53 and cyclin D1 was examined by means of immunohistochemical analysis with a view to evaluating whether there is a correlation between the aberrant expression of these proteins and disease prognosis. RESULTS: In the sample, the presence of immunohistochemically detectable p53 is associated with shorter survival and disease-free intervals, as shown in Kaplan-Meier survival curve analysis. Indeed, multivariate statistical analysis revealed that the accumulation of p53 is an independent prognostic factor, which is associated with shorter survival. This association was not evident in the case of cyclin D1. CONCLUSION: A more precise prognosis of patients with laryngeal epidermoid carcinomas could be achieved by taking into account the presence of p53 (as assayed by immunohistochemical analysis) in biopsy tissue

The PLGA implant as an antimitotic delivery system after experimental trabeculectomy.

PURPOSE: To investigate the effect of poly (lactic-co-glycolic acid) (PLGA) implants loaded with mitomycin C (MMC) and with different adjuvant treatments after glaucoma filtration surgery (GFS), in comparison to standard treatments. METHODS: Forty-two New Zealand White rabbits underwent bilateral GFS and received different treatments: topical MMC (group 1); topical 5-fluorouracil (5-FU; group 2); PLGA implant (group 3); MMC-loaded and -coated PLGA implant (group 4); MMC-loaded and 5-FU-coated PLGA implant (group 5); subconjunctival bevacizumab (group 6); MMC-loaded PLGA implant and subconjunctival bevacizumab (group 7); and no treatment (right eye of all animals; control group). Intraocular pressure (IOP) and filtering bleb were evaluated on different days after GFS. Histology was performed to examine the conjunctiva, sclerotomy, filtering bleb, and persistence of the implant. RESULTS: The best hypotensive results were achieved in the MMC-loaded and -coated PLGA implant group, which presented the lowest IOP values on days 1, 5, 7, 14, and 28 after GFS. Excluding the implant groups, in which the bleb could not be properly measured, bleb survival was superior to controls in groups 1, 2 and lower in group 6. Group 7 presented greater extension, height, and vascularization of the bleb. Epithelial thinning and lymphoplasmacytic infiltrate were observed in groups 1, 2, 4, 5, and 7. The rates of closure of the sclerotomy and bleb were 100% and 76%, respectively, and implant persistence was 95%. CONCLUSIONS: MMC-loaded and -coated implants have optimal surgical results, followed by topical MMC application. In this experimental model, bevacizumab could interact with MMC.

mRNA in situ hybridization (HistoSonda): a new diagnostic tool for HER2-status in breast cancer-a multicentric Spanish study.

Monoclonal therapies could represent baseline-personalized medicine for patients with neoplasia. One of the most successful examples is Trastuzumab, a humanized antibody against epidermal growth factor receptor 2. Human epidermal growth factor receptor 2 (HER2) is a trans-membrane tyrosine kinase coded by the gene HER2/neu and overexpressed in approximately 12% to 20% of infiltrating breast carcinomas. The overexpression of HER2 is an independent adverse prognostic factor in relation to survival and is also predictive of response to treatment. Therefore, the correct evaluation of HER2 status is essential for the management of infiltrating breast carcinoma to determine the response to Trastuzumab. The most common evaluation technique is immunohistochemistry, which is confirmed by fluorescent or chromogenic monochrome or dual-gene in situ hybridization in ambiguous cases (immunohistochemical 2+). Our objective was to evaluate the diagnostic value of a new technique on the basis of HER2 mRNA in situ hybridization (HistoSonda) and study its correlation with immunohistochemistry and dual-chromogenic in situ hybridization (DUO-CISH) in 403 cases of infiltrating breast carcinoma. The percentage of DUO-CISH amplification was 25.8%, HistoSonda positivity was 31.2%, and positivity for Hercep-Test was 48.1%, including (+2) and (+3). Comparisons were made of each of the techniques, HistoSonda to IHQ and HistoSonda to DUO-CISH. The overall concordance between DUO-CISH and HistoSonda was 89%. Our data support the consistency of HistoSonda as a useful tool to determine HER2 status in breast cancer.