Age-Related Clusters and Favorable Immune Phenotypes in Breast Cancer of the Young Patients.

Breast cancer (BC) patients aged <40 years at diagnosis experience aggressive disease and poorer survival compared with women diagnosed with BC at 40 to 49 years, but the age-related biology is described to little extent. Here, we explored transcriptional alterations in BC to gain better understanding of age-related tumor biology. We studied a subset of the Bergen in-house cohort (n = 127; age range, 26-49 years) and used the NanoString Breast Cancer 360 expression panel on formalin-fixed paraffin-embedded BC tissue, and publicly available global BC messenger RNA expression data (n = 204, age range, 22-49 years), to explore differentially expressed genes between the young (age <40 years) and older (age 40-49 years) patients. Unsupervised hierarchical clustering was applied to identify gene expression-based patient clusters. We applied established computational approaches to define the PAM50 subtypes, risk of recurrence scores (ROR), and risk groups and to infer the proportions of 22 immune cell types from bulk gene expression profiles of patients aged <50 years at BC diagnosis. Differentially expressed genes and gene sets were investigated using OncoEnrichR and g:Profiler to describe functional profiles and pathway enrichment. We identified 4 age-related patient clusters presenting distinct characteristics of PAM50 subtypes and ROR profiles, which demonstrated independent prognostic value when adjusted for traditional clinicopathologic variables and the known molecular subtypes. Our findings showed better survival than expected in the basal-enriched cluster 2 and in triple-negative and basal-like BC. Deconvolution analyses of immunophenotypes indicated higher levels of M0 and M1 macrophages than M2 macrophages in subsets of young BC. Our approach identifies age-based patient clusters with distinct clinicopathologic profiles, to a large extent overlapping with the PAM50 subtypes, although with independent prognostic values in multivariate survival analyses. The patient clusters provided new insight in the immune cell distribution across tumor subtypes, potentially contributing to survival differences between the clusters and the molecular subtypes and indicating age-related mechanisms improving outcome. Our study confirms the applicability of ROR as a valid prognosticator also in a young BC cohort.

Age-related phenotypes in breast cancer: A population-based study.

Breast cancer in young (<40 years) is associated with a higher frequency of aggressive tumor types and poor prognosis. It remains unclear if there is an underlying age-related biology that contributes to the unfavorable outcome. We aim to investigate the relationship between age and breast cancer biology, with emphasis on proliferation. Clinico-pathologic information, immunohistochemical markers and follow-up data were obtained for all patients aged <50 (Bergen cohort-1; n = 355, not part of a breast screening program) and compared to previously obtained information on patients aged 50 to 69 years (Bergen cohort-2; n = 540), who participated in the Norwegian Breast Cancer Screening Program. Young breast cancer patients presented more aggressive tumor features such as hormone receptor negativity, HER2 positivity, lymph-node metastasis, the HER2-enriched and triple-negative subtypes and shorter survival. Age <40 was significantly associated with higher proliferation (by Ki67). Ki67 showed weaker prognostic value in young patients. We point to aggressive phenotypes and increased tumor cell proliferation in breast cancer of the young. Hence, tumors of young breast cancer patients may present unique biological features, also when accounting for screen/interval differences, that may open for new clinical opportunities, stratifying treatment by age.

Vessel size as a marker of survival in estrogen receptor positive breast cancer.

PURPOSE: Angiogenesis is crucial for tumor growth and is one of the hallmarks of cancer. In this study, we analyzed microvessel density, vessel median size, and perivascular a-SMA expression as prognostic biomarkers in breast cancer. METHODS: Dual IHC staining was performed where alpha-SMA antibodies were used together with antibodies against the endothelial cell marker CD34. Digital images of stainings were analyzed to extract quantitative data on vessel density, vessel size, and perivascular alpha-SMA status. RESULTS: The analyses in the discovery cohort (n = 108) revealed a statistically significant relationship between large vessel size and shorter disease-specific survival (p = 0.007, log-rank test; p = 0.01, HR 3.1; 95% CI 1.3-7.4, Cox-regression analyses). Subset analyses indicated that the survival association of vessel size was strengthened in ER + breast cancer. To consolidate these findings, additional analyses were performed on a validation cohort (n = 267) where an association between large vessel size and reduced survival was also detected in ER + breast cancer (p = 0.016, log-rank test; p = 0.02; HR 2.3, 95% CI 1.1-4.7, Cox-regression analyses). CONCLUSION: Alpha-SMA/CD34 dual-IHC staining revealed breast cancer heterogeneity regarding vessel size, vessel density, and perivascular a-SMA status. Large vessel size was linked to shorter survival in ER + breast cancer.

Global and single-cell proteomics view of the co-evolution between neural progenitors and breast cancer cells in a co-culture model.

Tumor neurogenesis, a process by which new nerves invade tumors, is a growing area of interest in cancer research. Nerve presence has been linked to aggressive features of various solid tumors, including breast and prostate cancer. A recent study suggested that the tumor microenvironment may influence cancer progression through recruitment of neural progenitor cells from the central nervous system. However, the presence of neural progenitors in human breast tumors has not been reported. Here, we investigate the presence of Doublecortin (DCX) and Neurofilament-Light (NFL) co-expressing (DCX+/NFL+) cells in patient breast cancer tissue using Imaging Mass Cytometry. To map the interaction between breast cancer cells and neural progenitor cells further, we created an in vitro model mimicking breast cancer innervation, and characterized using mass spectrometry-based proteomics on the two cell types as they co- evolved in co-culture. Our results indicate stromal presence of DCX+/NFL+ cells in breast tumor tissue from a cohort of 107 patient cases, and that neural interaction contribute to drive a more aggressive breast cancer phenotype in our co-culture models. Our results support that neural involvement plays an active role in breast cancer and warrants further studies on the interaction between nervous system and breast cancer progression.

PRSS2 remodels the tumor microenvironment via repression of Tsp1 to stimulate tumor growth and progression.

The progression of cancer from localized to metastatic disease is the primary cause of morbidity and mortality. The interplay between the tumor and its microenvironment is the key driver in this process of tumor progression. In order for tumors to progress and metastasize they must reprogram the cells that make up the microenvironment to promote tumor growth and suppress endogenous defense systems, such as the immune and inflammatory response. We have previously demonstrated that stimulation of Tsp-1 in the tumor microenvironment (TME) potently inhibits tumor growth and progression. Here, we identify a novel tumor-mediated mechanism that represses the expression of Tsp-1 in the TME via secretion of the serine protease PRSS2. We demonstrate that PRSS2 represses Tsp-1, not via its enzymatic activity, but by binding to low-density lipoprotein receptor-related protein 1 (LRP1). These findings describe a hitherto undescribed activity for PRSS2 through binding to LRP1 and represent a potential therapeutic strategy to treat cancer by blocking the PRSS2-mediated repression of Tsp-1. Based on the ability of PRSS2 to reprogram the tumor microenvironment, this discovery could lead to the development of therapeutic agents that are indication agnostic.

Stathmin expression associates with vascular and immune responses in aggressive breast cancer subgroups.

Studies indicate that stathmin expression associates with PI3K activation in breast cancer, suggesting stathmin as a marker for targetable patient subgroups. Here we assessed stathmin in relation to tumour proliferation, vascular and immune responses, BRCA1 germline status, basal-like differentiation, clinico-pathologic features, and survival. Immunohistochemical staining was performed on breast cancers from two series (cohort 1, n = 187; cohort 2, n = 198), and mass spectrometry data from 24 cases and 12 breast cancer cell lines was examined for proteomic profiles. Open databases were also explored (TCGA, METABRIC, Oslo2 Landscape cohort, Cancer Cell Line Encyclopedia). High stathmin expression associated with tumour proliferation, p53 status, basal-like differentiation, BRCA1 genotype, and high-grade histology. These patterns were confirmed using mRNA data. Stathmin mRNA further associated with tumour angiogenesis, immune responses and reduced survival. By logistic regression, stathmin protein independently predicted a BRCA1 genotype (OR 10.0, p = 0.015) among ER negative tumours. Cell line analysis (Connectivity Map) implied PI3K inhibition in tumours with high stathmin. Altogether, our findings indicate that stathmin might be involved in the regulation of tumour angiogenesis and immune responses in breast cancer, in addition to tumour proliferation. Cell data point to potential effects of PI3K inhibition in tumours with high stathmin expression.

Integrin α11ß1 is expressed in breast cancer stroma and associates with aggressive tumor phenotypes.

Cancer-associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen-binding integrin α11ß1 has been proposed to be upregulated in a pro-tumorigenic subtype of cancer-associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11ß1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin-fixed paraffin-embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co-expression patterns of integrin α11 in relation to αSMA and cytokeratin-14 were also investigated. Integrin α11 was expressed to varying degrees in spindle-shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co-localized with αSMA in stromal cells, and with αSMA and cytokeratin-14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple-negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.