EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells.

INTRODUCTION: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model. METHODS: SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. RESULTS: The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo. CONCLUSIONS: Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC

Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas.

BACKGROUND: Enumeration of circulating tumor cells (CTCs) isolated from the peripheral blood of breast cancer patients holds promise as a clinically relevant, minimally invasive diagnostic test. However, CTC utility has been limited as a prognostic indicator of survival by the inability to stratify patients beyond general enumeration. In comparison, histological biopsy examinations remain the standard method for confirming malignancy and grading malignant cells, allowing for cancer identification and then assessing patient cohorts for prognostic and predictive value. Typically, CTC identification relies on immunofluorescent staining assessed as absent/present, which is somewhat subjective and limited in its ability to characterize these cells. In contrast, the physical features used in histological cytology comprise the gold standard method used to identify and preliminarily characterize the cancer cells. Here, we superimpose the methods, cytologically subtyping CTCs labeled with immunohistochemical fluorescence stains to improve their prognostic value in relation to survival. METHODS: In this single-blind prospective pilot study, we tracked 36 patients with late-stage breast cancer over 24 months to compare overall survival between simple CTC enumeration and subtyping mitotic CTCs. A power analysis (1-β = 0. 9, α = 0.05) determined that a pilot size of 30 patients was sufficient to stratify this patient cohort; 36 in total were enrolled. RESULTS: Our results confirmed that CTC number is a prognostic indicator of patient survival, with a hazard ratio 5.2, p = 0.005 (95 % CI 1.6-16.5). However, by simply subtyping the same population based on CTCs in cytological mitosis, the hazard ratio increased dramatically to 11.1, p \u3c 0.001 (95 % CI 3.1-39.7). CONCLUSIONS: Our data suggest that (1) mitotic CTCs are relativity common in aggressive late-stage breast cancer, (2) mitotic CTCs may significantly correlate with shortened overall survival, and (3) larger and more defined patient cohort studies are clearly called for based on this initial pilot study

Phase II evaluation of dasatinib in the treatment of recurrent or persistent epithelial ovarian or primary peritoneal carcinoma: a Gynecologic Oncology Group study.

OBJECTIVES: Preclinical data suggest an important role for the sarcoma proto-oncogene tyrosine kinase (SRC) in the oncogenesis of epithelial ovarian cancer (EOC) or primary peritoneal carcinoma (PPC). The Gynecologic Oncology Group (GOG) conducted a Phase II trial to evaluate the efficacy and safety of dasatinib, an oral SRC-family inhibitor in EOC/PPC, and explored biomarkers for possible association with clinical outcome. METHODS: Eligible women had measurable, recurrent or persistent EOC/PPC and had received one or two prior regimens which must have contained a platinum and a taxane. Patients were treated with 100mg orally daily of dasatinib continuously until progression of disease or adverse effects prevented further treatment. Primary endpoints were progression-free survival (PFS)≥6months and response rate. Serial plasma samples were assayed for multiple biomarkers. Circulating free DNA was quantified as were circulating tumor and endothelial cells. RESULTS: Thirty-five (35) patients were enrolled in a two-stage sequential design. Of the 34 eligible and evaluable patients, 20.6% (90% confidence interval: 10.1%, 35.2%) had a PFS≥6months; there were no objective responses. Grade 3-4 toxicities were gastrointestinal (mostly nausea and emesis; n=4), pulmonary (dyspnea and/or pleural effusion; n=4) and pain (n=5), and infrequent instances of anemia, malaise, insomnia, rash, and central nervous system hemorrhage. Lack of clinical activity limited any correlation of biomarkers with outcome. CONCLUSION: Dasatinib has minimal activity as a single-agent in patients with recurrent EOC/PPC

Immune phenotype of patients with stage IV metastatic inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The inflammatory nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies. METHODS: Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients. RESULTS: IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4+ T cells, whereas reductions in CD8+ T cells were more concentrated in memory subsets. Immature cytokine-producing CD56bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4+ T cells and CD56dim NK cells, and higher numbers of CD8+ effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20+ B cells in the tumor. CONCLUSIONS: Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC

Mutational patterns in chemotherapy resistant muscle-invasive bladder cancer

Despite continued widespread use, the genomic effects of cisplatin-based chemotherapy and implications for subsequent treatment are incompletely characterized. Here, we analyze whole exome sequencing of matched pre- and post-neoadjuvant cisplatin-based chemotherapy primary bladder tumor samples from 30 muscle-invasive bladder cancer patients. We observe no overall increase in tumor mutational burden post-chemotherapy, though a significant proportion of subclonal mutations are unique to the matched pre- or post-treatment tumor, suggesting chemotherapy-induced and/or spatial heterogeneity. We subsequently identify and validate a novel mutational signature in post-treatment tumors consistent with known characteristics of cisplatin damage and repair. We find that post-treatment tumor heterogeneity predicts worse overall survival, and further observe alterations in cell-cycle and immune checkpoint regulation genes in post-treatment tumors. These results provide insight into the clinical and genomic dynamics of tumor evolution with cisplatin-based chemotherapy, suggest mechanisms of clinical resistance, and inform development of clinically relevant biomarkers and trials of combination therapies

Circulating tumor cells: silent predictors of metastasis [version 1; referees: 2 approved]

Circulating tumor cells (CTCs) were added to the arsenal of clinical testing in 2004 for three cancer types: metastatic breast, prostate, and colorectal cancer. CTCs were found to be an independent prognostic indicator of survival for these three diseases. Multiple enrichment/isolation strategies have been developed and numerous assay applications have been performed using both single and pooled captured/enriched CTCs. We have reviewed the isolation techniques and touched on many analyses. The true utility of a CTC is that it acts as a “silent” predictor of metastatic disease. The mere presence of a single CTC is an indication that disease has spread from the primary site. Comments and suggestions have been set forth for CTCs and cell-free DNA to be used as a screening panel for the early detection of disease recurrence and metastatic spread, providing the opportunity for early intervention with curative intent to treat metastatic disease

Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the CellSearch(®) CTC test.

Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc