Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

Therapeutic Implication of Genomic Landscape of Adult Metastatic Sarcoma.

PURPOSE: This study investigated therapeutic potential of integrated genome and transcriptome profiling of metastatic sarcoma, a rare but extremely heterogeneous group of aggressive mesenchymal malignancies with few systemic therapeutic options. METHODS: Forty-three adult patients with advanced or metastatic non-GI stromal tumor sarcomas of various histology subtypes who were enrolled in the Personalized OncoGenomics program at BC Cancer were included in this study. Fresh tumor tissues along with blood samples underwent whole-genome and transcriptome sequencing. RESULTS: The most frequent genomic alterations in this cohort are large-scale structural variation and somatic copy number variation. Outlier RNA expression as well as somatic copy number variations, structural variations, and small mutations together suggest the presence of one or more potential therapeutic targets in the majority of patients in our cohort. Point mutations or deletions in known targetable cancer genes are rare; for example, tuberous sclerosis complex 2 provides a rationale for targeting the mammalian target of rapamycin pathway, resulting in a few patients with exceptional clinical benefit from everolimus. In addition, we observed recurrent 17p11-12 amplifications, which seem to be a sarcoma-specific event. This may suggest that this region harbors an oncogene(s) that is significant for sarcoma tumorigenesis. Furthermore, some sarcoma tumors carrying a distinct mutational signature suggestive of homologous recombination deficiency seem to demonstrate sensitivity to double-strand DNA-damaging agents. CONCLUSION: Integrated large-scale genomic analysis may provide insights into potential therapeutic targets as well as novel biologic features of metastatic sarcomas that could fuel future experimental and clinical research and help design biomarker-driven basket clinical trials for novel therapeutic strategies.

Prognosis in patients diagnosed with loco-regional failure of breast cancer: 34 years longitudinal data from the Stockholm-Gotland cancer registry.

RATIONALE: Survival after loco-regional failure (LRF) of breast cancer was investigated at the population level. METHODS: Using the Stockholm cancer registry, 2698 patients diagnosed with LRF between 1980 and 2014 were identified and divided into three cohorts by year of LRF diagnosis. Post-relapse event-free survival (EFS) and overall survival (OS) were analyzed separately in local and loco-regional relapses and compared across the cohorts by Kaplan-Meier method. Relative survival was estimated and Poisson regression models, adjusted for clinically relevant prognostic factors, were fitted for excess mortality ratio calculation. Age-related survival trends were also explored. RESULTS: Among 1922 patients diagnosed with local relapse, 1032 (54%) EFS events and 931 (48%) deaths were registered. A significant improvement in EFS (p < 0.001) and OS (p < 0.001) was demonstrated in tumors that recurred locally in the years 1990-1999 and 2000-2014 compared with 1980-1989, regardless of age at relapse (≤ 60 years; > 60 years). In women with loco-regional relapse, 557 out of 776 (72%) experienced a post-relapse event and 522 (67%) died. Significantly longer EFS and OS were seen over time in the whole group (p < 0.001 and p = 0.003, respectively) and in younger (p < 0.001; p < 0.001) but not in older women (p = 0.55; p = 0.80). Relative survival was consistent with OS and a statistically significant decrease in mortality after loco-regional recurrence over time was seen only in women aged ≤ 60 years. CONCLUSIONS: Survival after loco-regional failure of breast cancer has improved over time, especially in younger women.

Metabolic factors, anthropometric measures, diet, and physical activity in long-term breast cancer survivors: change from diagnosis and comparison to non-breast cancer controls.

PURPOSE: We studied metabolic factors, diabetes, and anthropometric measurements at diagnosis and long-term follow-up (LTFU), mean 12.5 years post-diagnosis, in breast cancer (BC) survivors, and compared their status at LTFU to that of age-matched women without BC. Diet and physical activity were also assessed. METHOD: 535 non-diabetic BC patients treated at three University of Toronto hospitals were followed prospectively; 285 surviving patients, without distant recurrence, participated in a LTFU study. A control group of 167 age-matched women without BC was recruited from a mammogram screening program at one of the hospitals. Change over time was analyzed using paired t tests, and comparisons between BC survivors and controls used age and education (AE)-adjusted regression models. RESULTS: Median weight gain in BC survivors was 2.00 kg (p < 0.0001); BMI, glucose, insulin, homeostasis model assessment (HOMA), and total cholesterol increased modestly but significantly. Waist circumference, glucose, and triglycerides were higher in LTFU BC survivors versus controls. BC survivors had significantly greater prevalence of diabetes/pre-diabetes versus controls (33 vs. 20.4%, AE-adjusted odds ratio (OR) 1.59, p = 0.050). This effect was restricted to those with lower levels of physical activity (<56 metabolic equivalent (MET)-hours/week: OR 2.70 versus 0.94 for those with higher physical activity, interaction p = 0.034). At LTFU, BC survivors were more physically active than at diagnosis (median increase 28 MET-hours/week interquartile range -14.8 to 82), and compared to controls (median 68.2 vs. 44 MET-hours/week, p < 0.0001). CONCLUSION: The prevalence of the metabolic syndrome and diabetes/pre-diabetes was significantly higher in BC survivors than in controls group, notably in those with lower levels of physical activity. Enhanced diabetes/metabolic syndrome screening and promotion of physical activity may be warranted in BC survivors.

Insulin- and obesity-related variables in early-stage breast cancer: correlations and time course of prognostic associations.

PURPOSE: To investigate patterns of prognostic associations over time of insulin- and obesity-related variables measured at diagnosis of early breast cancer (BC), focusing on whether the prognostic associations with distant recurrence and death changed over time. PATIENTS AND METHODS: Five hundred thirty-five nondiabetic women with T1-3, N0-1, M0 invasive BC diagnosed from 1989 to 1996 were included in the study. Insulin-related variables included fasting insulin, Homeostasis Model Assessment, C-peptide, and glucose. Obesity-related variables included weight, body mass index (BMI), waist and hip circumference, and leptin. Correlations were examined using the Pearson correlation coefficient and prognostic associations using the Cox model. RESULTS: There was evidence that associations of baseline insulin-related variables with distant recurrence and death were not constant over time; univariable adverse prognostic associations were significant only during the first 5 years (eg, insulin quartile 4 v 1: hazard ratio [HR], 2.32; 95% CI, 1.39 to 3.86; P < .001 for distant disease-free survival [DDFS]; and HR, 2.85; 95% CI, 1.48 to 5.50; P = .002 for overall survival [OS], with little attenuation of this pattern in multivariable analyses). In contrast, obesity-related variables (BMI, weight, leptin) exerted significant adverse univariable associations that were constant over time (eg, BMI quartile 4 v 2: HR, 1.40; 95% CI, 1.07 to 1.82 for DDFS; P = .014; and HR, 1.50; 95% CI, 1.16 to 1.93; P < .001 for OS); prognostic associations of leptin remained significant in multivariable analyses. CONCLUSION: Baseline insulin- and obesity-related variables exert different patterns of prognostic associations over time in early BC.

Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright) cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright) iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright) iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

A phase II study of pazopanib in patients with recurrent or metastatic invasive breast carcinoma: a trial of the Princess Margaret Hospital phase II consortium.

PURPOSE: Angiogenesis is an important hallmark of breast cancer growth and progression. Pazopanib, an oral small molecule inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and KIT, has activity across a range of solid tumors. We evaluated the activity of single-agent pazopanib in recurrent or metastatic breast cancer (MBC). PATIENTS AND METHODS: Patients with recurrent breast cancer or MBC, treated with up to two prior lines of chemotherapy, were eligible to receive pazopanib, 800 mg daily until progression. The primary endpoint was the objective response rate as measured by Response Evaluation Criteria in Solid Tumors. Secondary endpoints included time to progression, the stable disease rate, and toxicity. Using a two-stage design, confirmed response in three of 18 patients was required to proceed to stage 2. RESULTS: Twenty evaluable patients were treated, with a median age of 56 years; 70% were estrogen receptor positive, all were human epidermal growth factor receptor 2 negative. The majority had one or two prior lines of chemotherapy. One patient (5%) had a partial response, 11 (55%) had stable disease (SD) [four (20%) with SD > or = 6 months], and seven (35%) had progressive disease as their best response. One (5%) was not evaluable. The median time to progression was 5.3 months. Pazopanib did not cause significant severe toxicity aside from grade 3-4 transaminitis, hypertension, and neutropenia in three patients each (14% each) and grade 3 gastrointestinal hemorrhage in one patient (5%). CONCLUSION: Pazopanib provides disease stability in advanced breast cancer. The activity seen is comparable with that of other antiangiogenic agents in this setting. Pazopanib may be of interest for future studies in breast cancer, including in combination with other systemic agents.