Comprehensive multiplexed immune profiling of the ductal carcinoma in situ immune microenvironment regarding subsequent ipsilateral invasive breast cancer risk.

BACKGROUND: Ductal carcinoma in situ (DCIS) is treated to prevent subsequent ipsilateral invasive breast cancer (iIBC). However, many DCIS lesions will never become invasive. To prevent overtreatment, we need to distinguish harmless from potentially hazardous DCIS. We investigated whether the immune microenvironment (IME) in DCIS correlates with transition to iIBC. METHODS: Patients were derived from a Dutch population-based cohort of 10,090 women with pure DCIS with a median follow-up time of 12 years. Density, composition and proximity to the closest DCIS cell of CD20+ B-cells, CD3+CD8+ T-cells, CD3+CD8- T-cells, CD3+FOXP3+ regulatory T-cells, CD68+ cells, and CD8+Ki67+ T-cells was assessed with multiplex immunofluorescence (mIF) with digital whole-slide analysis and compared between primary DCIS lesions of 77 women with subsequent iIBC (cases) and 64 without (controls). RESULTS: Higher stromal density of analysed immune cell subsets was significantly associated with higher grade, ER negativity, HER-2 positivity, Ki67 ≥ 14%, periductal fibrosis and comedonecrosis (P < 0.05). Density, composition and proximity to the closest DCIS cell of all analysed immune cell subsets did not differ between cases and controls. CONCLUSION: IME features analysed by mIF in 141 patients from a well-annotated cohort of pure DCIS with long-term follow-up are no predictors of subsequent iIBC, but do correlate with other factors (grade, ER, HER2 status, Ki-67) known to be associated with invasive recurrences.

HLA class II expression on tumor cells and low numbers of tumor-associated macrophages predict clinical outcome in oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a highly immunogenic tumor and differences in tumor microenvironment might contribute to the improved survival of HPV-positive OPSCC patient. METHODS: A comprehensive multivariate analysis with clinical and immune variables (human leukocyte antigen [HLA] I/II, programmed death ligand 1 (PD-L1), programmed death receptor 1 (PD1), T cells, and macrophages) was performed in 142 OPSCC patients. RESULTS: We found an inverse correlation between the expression of HLA class II molecules on tumor cells and CD68+ CD163+ tumor-associated macrophages (TAMs). High HLA-DP/DQ/DR expression and low number of TAMs were associated with longer disease-specific survival and disease-free survival (DFS). Furthermore, a new population of CD8+ FoxP3+ T cells was correlated with shorter DFS in multivariate analysis. CONCLUSIONS: \We identified new prognostic markers for patients with oropharyngeal cancer, which can be used for selecting patients that can benefit from immunotherapy.

Assessment of PD-L1 expression across breast cancer molecular subtypes, in relation to mutation rate, BRCA1-like status, tumor-infiltrating immune cells and survival.

To better understand the expression pattern of programmed death-ligand 1 (PD-L1) expression in different breast cancer types, we characterized PD-L1 expression in tumor and tumor-infiltrating immune cells, in relation to mutation rate, BRCA1-like status and survival. We analyzed 410 primary treatment-naive breast tumors comprising 162 estrogen receptor-positive (ER+) and HER2-, 101 HER2+ and 147 triple-negative (TN) cancers. Pathologists quantified tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells and TILs using whole slides and tissue microarray. Mutation rate was assessed by DNA sequencing, BRCA1-like status using multiplex ligation-dependent probe amplification, and immune landscape by multiplex image analyses of CD4, CD68, CD8, FOXP3, cytokeratin, and PD-L1. Half of PD-L1 scores evaluated by tissue microarray were false negatives compared to whole slide evaluations. We observed at least 1% of PD-L1-positive (PD-L1+) cells in 53.1% of ER+HER2-, 73.3% of HER2+, and 84.4% of TN tumors. PD-L1 expression was higher in ductal compared to lobular carcinomas, also within ER+HER2- tumors (p = 0.04). High PD-L1+ TILs score (> 50%) was independently associated with better outcome in TN tumors (HR = 0.27; 95%CI = 0.10-0.69). Within TN tumors, PD-L1 and TIL scores showed a modest but significant positive association with the number of silent mutations, but no association with BRCA1-like status. Multiplex image analyses indicated that PD-L1 is expressed on multiple immune cells (CD68+ macrophages, CD4+, FOXP3+, and CD8+ T cells) in the breast tumor microenvironment, independent of the PD-L1 status of the tumor cells. We found no evidence that levels of PD-L1+ TILs in TN breast cancer are driven by high mutation rate or BRCA1-like status.

Orthopedic surgery increases atherosclerotic lesions and necrotic core area in ApoE-/- mice.

BACKGROUND AND AIMS: Observational studies show a peak incidence of cardiovascular events after major surgery. For example, the risk of myocardial infarction increases 25-fold early after hip replacement. The acuteness of this increased risk suggests abrupt enhancement in plaque vulnerability, which may be related to intra-plaque inflammation, thinner fibrous cap and/or necrotic core expansion. We hypothesized that acute systemic inflammation following major orthopedic surgery induces such changes. METHODS: ApoE-/- mice were fed a western diet for 10 weeks. Thereafter, half the mice underwent mid-shaft femur osteotomy followed by realignment with an intramedullary K-wire, to mimic major orthopedic surgery. Mice were sacrificed 5 or 15 days post-surgery (n = 22) or post-saline injection (n = 13). Serum amyloid A (SAA) was measured as a marker of systemic inflammation. Paraffin embedded slides of the aortic root were stained to measure total plaque area and to quantify fibrosis, calcification, necrotic core, and inflammatory cells. RESULTS: Surgery mice showed a pronounced elevation of serum amyloid A (SAA) and developed increased plaque and necrotic core area already at 5 days, which reached significance at 15 days (p = 0.019; p = 0.004 for plaque and necrotic core, respectively). Macrophage and lymphocyte density significantly decreased in the surgery group compared to the control group at 15 days (p = 0.037; p = 0.024, respectively). The density of neutrophils and mast cells remained unchanged. CONCLUSIONS: Major orthopedic surgery in ApoE-/- mice triggers a systemic inflammatory response. Atherosclerotic plaque area is enlarged after surgery mainly due to an increase of the necrotic core. The role of intra-plaque inflammation in this response to surgical injury remains to be fully elucidated.

High prevalence of cardiovascular disease in South Asians: Central role for brown adipose tissue?

Cardiovascular disease (CVD) is the leading cause of death in modern society. Interestingly, the risk of developing CVD varies between different ethnic groups. A particularly high risk is faced by South Asians, representing over one-fifth of the world's population. Here, we review potential factors contributing to the increased cardiovascular risk in the South Asian population and discuss novel therapeutic strategies based on recent insights. In South Asians, classical ('metabolic') risk factors associated with CVD are highly prevalent and include central obesity, insulin resistance, type 2 diabetes, and dyslipidemia. A contributing factor that may underlie the development of this disadvantageous metabolic phenotype is the presence of a lower amount of brown adipose tissue (BAT) in South Asian subjects, resulting in lower energy expenditure and lower lipid oxidation and glucose uptake. As it has been established that the increased prevalence of classical risk factors in South Asians cannot fully explain their increased risk for CVD, other non-classical risk factors must underlie this residual risk. In South Asians, the prevalence of "inflammatory" risk factors including visceral adipose tissue inflammation, endothelial dysfunction, and HDL dysfunction are higher compared with Caucasians. We conclude that a potential novel therapy to lower CVD risk in the South Asian population is to enhance BAT volume or its activity in order to diminish classical risk factors. Furthermore, anti-inflammatory therapy may lower non-classical risk factors in this population and the combination of both strategies may be especially effective.

Supraclavicular skin temperature as a measure of 18F-FDG uptake by BAT in human subjects.

BACKGROUND: Brown adipose tissue (BAT) has emerged as a novel player in energy homeostasis in humans and is considered a potential new target for combating obesity and related diseases. The current 'gold standard' for quantification of BAT volume and activity is cold-induced 18F-FDG uptake in BAT. However, use of this technique is limited by cost and radiation exposure. Given the fact that BAT is a thermogenic tissue, mainly located in the supraclavicular region, the aim of the current study was to investigate whether cold-induced supraclavicular skin temperature and core body temperature may be alternative markers of BAT activation in humans. SUBJECTS/METHODS: BAT volume and activity were measured in 24 healthy lean adolescent males (mean age 24.1±0.8 years), using cold-induced 18F-FDG uptake with PET-CT. Core body temperature was measured continuously in the small intestine with use of an ingestible telemetric capsule and skin temperature was measured by eighteen wireless iButtons attached to the skin following ISO-defined locations. RESULTS: Proximal and distal (hand/feet) skin temperatures markedly decreased upon cold exposure, while supraclavicular skin temperature significantly increased (35.2±0.1 vs. 35.5±0.1°C, p = 0.001). Furthermore, cold-induced supraclavicular skin temperature positively correlated with both total (R2 = 0.28, P = 0.010) and clavicular BAT volume (R2 = 0.20, P = 0.030) and clavicular SUVmax (R2 = 0.27, P = 0.010), while core body temperature did not. CONCLUSIONS: Supraclavicular skin temperature as measured by iButtons may have predictive value for BAT detection in adult humans. This is highly desirable considering the increasing interest in pharmacological interventions to stimulate BAT in human subjects. TRIAL REGISTRATION: NTR 2473.

Brown adipose tissue volume in healthy lean south Asian adults compared with white Caucasians: a prospective, case-controlled observational study.

BACKGROUND: Individuals of south Asian origin have a very high risk of developing type 2 diabetes compared with white Caucasians. We aimed to assess volume and activity of brown adipose tissue (BAT), which is thought to have a role in energy metabolism by combusting fatty acids and glucose to produce heat and might contribute to the difference in incidence of type 2 diabetes between ethnic groups. METHODS: We enrolled Dutch nationals with south Asian ancestry and matched Caucasian participants at The Rijnland Hospital (Leiderdorp, Netherlands). Eligible participants were healthy lean men aged 18-28 years, and we matched groups for BMI. We measured BAT volume and activity with cold-induced (18)F-fluorodeoxyglucose ((18)F-FDG) PET CT scans, and assessed resting energy expenditure, non-shivering thermogenesis, and serum parameters. This study is registered with the Netherlands Trial Register, number 2473. FINDINGS: Between March 1, 2013, and June 1, 2013, we enrolled 12 participants in each group; one Caucasian participant developed hyperventilation after (18)F-FDG administration, and was excluded from all cold-induced and BAT measurements. Compared with Caucasian participants, south Asian participants did not differ in age (mean 23.6 years [SD 2.8] for south Asians vs 24.6 years [2.8] for Caucasians) or BMI (21.5 kg/m(2) [2.0] vs 22.0 kg/m(2) [1.6]), but were shorter (1.74 m [0.06] vs 1.85 m [0.04]) and lighter (65.0 kg [8.5] vs 75.1 kg [7.2]). Thermoneutral resting energy expenditure was 1297 kcal per day (SD 123) in south Asian participants compared with 1689 kcal per day (193) in white Caucasian participants (difference -32%, p=0.0008). On cold exposure, shiver temperature of south Asians was 2.0°C higher than Caucasians (p=0.0067) and non-shivering thermogenesis was increased by 20% in white Caucasians (p<0.0001) but was not increased in south Asians. Although the maximum and mean standardised uptake values of (18)F-FDG in BAT did not differ between groups, total BAT volume was lower in south Asians (188 mL [SD 81]) than it was in Caucasians (287 mL [169]; difference -34%, p=0.04). Overall, BAT volume correlated positively with basal resting energy expenditure in all assessable individuals (ß=0.44, p=0.04). INTERPRETATION: Lower resting energy expenditure, non-shivering thermogenesis, and BAT volumes in south Asian populations might underlie their high susceptibility to metabolic disturbances, such as obesity and type 2 diabetes. Development of strategies to increase BAT volume and activity might help prevent and treat such disorders, particularly in south Asian individuals. FUNDING: Dutch Heart Foundation (2009T038) and Dutch Diabetes Research Foundation (2012.11.1500).