A novel age-related gene expression signature associates with proliferation and disease progression in breast cancer

Background and objective Breast cancer (BC) diagnosed at ages <40 years presents with more aggressive tumour phenotypes and poorer clinical outcome compared to older BC patients. Here, we explored transcriptional BC alterations to gain a better understanding of age-related tumour biology, also subtype-stratified. Methods We studied publicly available global BC mRNA expression (n = 3999) and proteomics data (n = 113), exploring differentially expressed genes, enriched gene sets, and gene networks in the young compared to older patients. Results We identified transcriptional patterns reflecting increased proliferation and oncogenic signalling in BC of the young, also in subtype-stratified analyses. Six up-regulated hub genes built a novel age-related score, significantly associated with aggressive clinicopathologic features. A high 6 Gene Proliferation Score (6GPS) demonstrated independent prognostic value when adjusted for traditional clinicopathologic variables and the molecular subtypes. The 6GPS significantly associated also with disease-specific survival within the luminal, lymph node-negative and Oncotype Dx intermediate subset. Conclusions We here demonstrate evidence of higher tumour cell proliferation in young BC patients, also when adjusting for molecular subtypes, and identified a novel age-based six-gene signature pointing to aggressive tumour features, tumour proliferation, and reduced survival—also in patient subsets with expected good prognosis.publishedVersio

CD47 and CD68 expression in breast cancer is associated with tumor-infiltrating lymphocytes, blood vessel invasion, detection mode, and prognosis

CD47 expressed on tumor cells binds to signal regulatory protein alpha on macrophages, initiating inhibition of phagocytosis. We investigated the relationships between tumor expression of CD47 and CD68 macrophage content, subsets of tumor-infiltrating lymphocytes (TILs), and vascular invasion in breast cancer. A population-based series of 282 cases (200 screen detected and 82 interval patients) from the Norwegian Breast Cancer Screening Program was examined. Immunohistochemical staining for CD47 and CD68 was evaluated on tissue microarray (TMA) slides. For CD47 evaluation, a staining index was used. CD68 tumor-associated macrophages were counted and dichotomized. TIL subsets (CD45, CD3, CD4, CD8, and FOXP3) were counted and dichotomized using immunohistochemistry on TMA slides. Vascular invasion (both lymphatic and blood vessel) was determined on whole tissue slides. High CD47 tumor cell expression or high counts of CD68 macrophages were significantly associated with elevated levels of all TIL subsets (p < 0.02), CD163 macrophages (p < 0.001), blood vessel invasion (CD31 positive) (p < 0.01), and high tumor cell Ki67 (p < 0.004). High CD47 expression was associated with ER negativity (p < 0.001), HER2 positive status (p = 0.03), and interval-detected tumors (p = 0.03). Combined high expression of CD47–CD68 was associated with a shorter recurrence-free survival (RFS) by multivariate analysis (hazard ratio [HR]: 2.37, p = 0.018), adjusting for tumor diameter, histologic grade, lymph node status, and molecular subtype. Patients with luminal A tumors showed a shorter RFS for CD47–CD68 high cases by multivariate assessment (HR: 5.73, p = 0.004). This study demonstrates an association of concurrent high CD47 tumor cell expression and high CD68 macrophage counts with various TIL subsets, blood vessel invasion (CD31 positive), other aggressive tumor features, and interval-presenting breast cancer. Our findings suggest a link between CD47, tumor immune response, and blood vessel invasion (CD31 positive). Combined high expression of CD47–CD68 was an independent prognostic factor associated with poor prognosis in all cases, as well as in the luminal A category.publishedVersio

Detection and significance of small and low proliferation breast cancer

Objectives To determine the frequency and discuss possible implications of early breast cancer with particularly good prognosis and defined by tumor diameter and cell proliferation. Setting Detection of small and slowly growing tumors presents a challenge in breast cancer management, due to the risk of over-treatment. Here, we attempted to define a group of such tumors by combining small diameter (≤10 mm, T1ab tumors) with low tumor cell proliferation (≤10% Ki67 expression rate). These tumors were termed small low proliferation cancers (SLPC). Methods Two population-based cohorts were studied: a small research series (n = 534), and a nation-wide registry-based series of prospectively collected routine data (n = 8433). In the latter, we stratified by detection mode; screen-detected, interval, and breast cancers detected outside of screening. Patients were treated according to national guidelines at time of their diagnosis. For both cohorts, we compared tumor histopathology and risk of breast cancer death using a log-rank test for cases with SLPC versus non-SLPC. Results In the research series (median follow-up 151 months), the frequency of SLPC was 10% (54/534), with one breast cancer death compared with 78 among the remaining 480 cases of non-SLPC (p = 0.008). In the registry series (median follow-up 42 months), the frequency of SLPC was 10% (854/8433), with five deaths compared to 187 among the remaining 7579 cases (p = 0.0004). Conclusions SLPC was associated with very low risk of breast cancer death. Prospective randomized trials are needed to clarify whether less aggressive treatment could be a safe option for women with such early breast cancers.acceptedVersio

Fibulin-2 expression associates with vascular invasion and patient survival in breast cancer

Stromal elastosis is related to good prognosis in breast cancer and fibulin-2 helps to stabilize elastic fibers in basement membranes. Here, we examined the level of perivascular fibulin-2 expression in relation to elastosis content, vascular invasion, molecular subtypes, tumour detection mode, and patient prognosis in breast cancer. We performed a population based retrospective study of invasive breast cancers from the Norwegian Breast Screening Program (Vestfold County, 2004–2009) including 200 screen-detected and 82 interval cancers. Perivascular fibulin-2 staining was semi-quantitatively graded based on immunohistochemistry (1–3) and dichotomized as high expression (grade 2–3) and low expression (grade 1). Elastosis content was graded on a 4-tiered scale and dichotomized as high (score 3) and low (score 0–2) expression, whereas lymphatic (LVI) and blood vessel invasion (BVI) were recorded as absent or present by immunohistochemistry. High perivascular fibulin-2 expression was strongly related to stromal elastosis (p<0.001), and inversely associated with BVI and LVI (p<0.001 for both). High fibulin-2 was associated with luminal breast cancer subgroups (p<0.001) and inversely with interval cancers compared with screen-detected tumours (p<0.001). By univariate analysis, low perivascular fibulin-2 was associated with reduced recurrence-free survival (p = 0.002) and disease specific survival (p = 0.019). Low perivascular fibulin-2 expression was strongly related to vascular invasion, low stromal elastosis, non-luminal breast cancer subtypes, interval presentation, and adverse prognosis.publishedVersio

Fatal and non-fatal breast cancers in women targeted by BreastScreen Norway: a cohort study

Background - Many breast cancer survivors experience anxiety related to dying from their disease even if it is detected at an early stage. We aimed to increase knowledge about fatal and non-fatal breast cancer by describing how histopathological tumour profiles and detection modes were associated with 10-year breast cancer-specific survival. Methods - This cohort study included data from women targeted by BreastScreen Norway (aged 50–69) and diagnosed with invasive breast cancer during 1996–2011. Breast cancer was classified as fatal if causing death within 10 years after diagnosis and non-fatal otherwise. We described histopathologic characteristics of fatal and non-fatal cancers, stratified by mode of detection. Recursive partitioning identified subgroups with differing survival profiles. Results - In total, 6.3% of 9954 screen-detected cancers (SDC) were fatal, as were 17.4% of 3205 interval cancers (IC) and 20.9% of 3237 cancers detected outside BreastScreen Norway. Four to five subgroups with differing survival profiles were identified within each detection mode. Women with lymph node-negative SDC or Grade 1–2, node-negative IC without distant metastases had the highest 10-year survival (95–96%). Conclusions - Two subgroups representing 53% of the cohort had excellent (95–96%) 10-year breast cancer-specific survival. Most women with SDC had excellent survival, as did nearly 40% of women diagnosed with IC

The epithelial–mesenchymal transition regulators Twist, Slug, and Snail are associated with aggressive tumour features and poor outcome in prostate cancer patients

The prognostic importance of transcription factors promoting epithelial–mesenchymal transition (EMT) and angiogenesis has not been well explored in prostate cancer patients with long follow-up, nor the interplay between these factors. The objective of this study was to assess the individual protein expression and co-expression of Twist, Slug (Snai2), Snail (Snai1), and hypoxia-inducible factor-1 alpha (Hif-1α) in prostate cancer in relation to EMT, angiogenesis, hypoxia, tumour features, disease recurrence, and patient survival. Immunohistochemical staining was performed on tissue microarray sections from 338 radical prostatectomies with long follow-up. In addition, 41 cases of prostatic hyperplasia, 33 non-skeletal metastases, 13 skeletal metastases, and 33 castration-resistant prostate carcinomas were included. Our findings were validated in external gene expression data sets. Twist was overexpressed in primary prostate cancer and markedly reduced in distant metastases (p < 0.0005). Strong expression of Twist and Slug was associated with Hif-1α in localised prostate cancer (p ≤ 0.001), and strong Twist was associated with Hif-1α in castration-resistant carcinomas (p = 0.044). Twist, Slug, and increased Snail at the tumour stromal border were associated with vascular factors (p ≤ 0.045). Each of the three EMT-regulating transcription factors were associated with aggressive tumour features and shorter time to recurrence and cancer-specific death. Notably, the co-expression of factors demonstrated an enhanced influence on outcome. In the subgroup of E-cadherinlow carcinomas, strong Slug was associated with shorter time to all end points and was an independent predictor of time to multiple end points, including cancer-specific death (hazard ratio 3.0, p = 0.041). To conclude, we demonstrate an important relation between EMT, hypoxia, and angiogenesis and a strong link between the investigated EMT regulators and aggressive tumour features and poor patient outcome in prostate cancer. Despite the retrospective nature of this long-term study, our findings could have a significant impact on the future treatment of prostate cancer, where tailored therapies might be directed simultaneously against epithelial–mesenchymal phenotypes, angiogenesis, and tumour hypoxia.publishedVersio

FOXC2 expression and epithelial–mesenchymal phenotypes are associated with castration resistance, metastasis and survival in prostate cancer

Epithelial–mesenchymal transition (EMT) is important for tumour cell invasion and metastasis and is a feature of aggressive carcinomas. EMT is characterised by reduced E‐cadherin and increased N‐cadherin expression (EN‐switch), and increased expression of the EMT‐regulating transcription factor Forkhead box protein C2 (FOXC2) has been associated with progression and poor prognosis in various malignancies. FOXC2 was recently highlighted as a novel therapy target in prostate cancer, but survival data on FOXC2 are lacking. This study evaluates the expression of FOXC2, E‐cadherin and N‐cadherin in different prostatic tissues focusing on EMT, clinico‐pathological phenotype, recurrence and patient survival. Tissue microarray sections from 338 radical prostatectomies (1986–2007) with long and complete follow‐up, 33 castration resistant prostate cancers, 33 non‐skeletal metastases, 13 skeletal metastases and 41 prostatic hyperplasias were stained immunohistochemically for FOXC2, E‐cadherin and N‐cadherin. FOXC2 was strongly expressed in primary carcinomas, including castration resistant tumours and metastatic lesions as compared to benign prostatic hyperplasia. A hybrid epithelial–mesenchymal phenotype, with co‐expression of E‐cadherin and N‐cadherin, was found in the majority of skeletal metastases and in a substantial proportion of castration resistant tumours. In localised carcinomas, the EN‐switch was associated with adverse clinico‐pathological variables, such as extra‐prostatic extension, high pathological stage and lymph node infiltration. In univariate survival analyses of the clinically important, large subgroup of 199 patients with Gleason score 7, high FOXC2 expression and EN‐switching were significantly associated with shorter time to clinical recurrence, skeletal metastases and cancer specific death. In multivariate Cox' survival analysis, high FOXC2 and the EN‐switch, together with Gleason grade group (GG3 versus GG2), were independent predictors of time to these end‐points. High FOXC2 gene expression (mRNA) was also related to patient outcome, validating our immunohistochemical findings. FOXC2 and factors signifying EMT or its intermediate states may prove important as biomarkers for aggressive disease and are potential novel therapy targets in prostate cancer.publishedVersio

Prognostic and predictive impact of stroma cells defned by PDGFRb expression in early breast cancer: results from the randomized SweBCG91RT trial

Purpose Predictive biomarkers are needed to aid the individualization of radiotherapy (RT) in breast cancer. Cancer-associated fibroblasts have been implicated in tumor radioresistance and can be identified by platelet-derived growth factor receptor-beta (PDGFRb). This study aims to analyze how PDGFRb expression affects RT benefit in a large randomized RT trial. Methods PDGFRb was assessed by immunohistochemistry on tissue microarrays from 989 tumors of the SweBCG91RT trial, which enrolled lymph node-negative, stage I/IIA breast cancer patients randomized to RT after breast-conserving surgery. Outcomes were analyzed at 10 years for ipsilateral breast tumor recurrence (IBTR) and any recurrence and 15 years for breast cancer specific death (BCSD). Results PDGFRb expression correlated with estrogen receptor negativity and younger age. An increased risk for any recurrence was noted in univariable analysis for the medium (HR 1.58, CI 95% 1.11–2.23, p = 0.011) or PDGFRb high group (1.49, 1.06–2.10, p = 0.021) compared to the low group. No differences in IBTR or BCSD risk were detected. RT benefit regarding IBTR risk was significant in the PDGFRb low (0.29, 0.12–0.67, p = 0.004) and medium (0.31, 0.16–0.59, p < 0.001) groups but not the PDGFRb high group (0.64, 0.36–1.11, p = 0.110) in multivariable analysis. Likewise, risk reduction for any recurrence was less pronounced in the PDGFRb high group. No significant interaction between RT and PDGFRb-score could be detected. Conclusion A higher PDGFRb-score conferred an increased risk of any recurrence, which partly can be explained by its association with estrogen receptor negativity and young age. Reduced RT benefit was noted among patients with high PDGFRb, however without significant interaction.publishedVersio

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.publishedVersio

Human organotypic airway and lung organoid cells of bronchiolar and alveolar differentiation are permissive to infection by influenza and SARS-CoV-2 respiratory virus

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.publishedVersio