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Background: To assess metastatic breast cancer (MBC) patient psychological factors, perceptions, and comprehension of tumor genomic testing., Methods: In a prospective, single institution, single-arm trial, patients with MBC underwent next-generation sequencing at study entry with sequencing results released at progression. Patients who completed surveys before undergoing sequencing were included in the present secondary analysis (n = 58). We administered four validated psychosocial measures: Center for Epidemiologic Studies Depression Scale, Beck Anxiety Inventory, Trust in Physician Scale, and Communication and Attitudinal Self-Efficacy scale for Cancer. Genetic comprehension was assessed using 7-question objective and 6-question subjective measures. Longitudinal data were assessed (n = 40) using paired Wilcoxon signed rank and McNemar's test of agreement., Results: There were no significant differences between the beginning and end of study in depression, anxiety, physician trust, or self-efficacy (median time on study: 7.6 months). Depression and anxiety were positively associated with each other and both negatively associated with self-efficacy. Self-efficacy decreased from pre- to post-genomic testing (p = 0.05). Objective genetics comprehension did not significantly change from pre- to post-genomic testing, but patients expressed increased confidence in their ability to teach others about genetics (p = 0.04). Objective comprehension was significantly lower in non-white patients (p = 0.02) and patients with lower income (p = 0.04)., Conclusions: This is the only study, to our knowledge, to longitudinally evaluate multiple psychological metrics in MBC as patients undergo tumor genomic testing. Overall, psychological dimensions remained stable over the duration of tumor genomic testing. Among patients with MBC, depression and anxiety metrics were negatively correlated with patient self-efficacy. Patients undergoing somatic genomic testing had limited genomic knowledge, which varied by demographic groups and may warrant additional educational intervention., Clinical Trial Information: NCT01987726, registered November 13, 2013.

Triple Negative Breast Cancers (TNBC) is a heterogeneous disease at the molecular and clinical level with poor outcome. Molecular subclassification of TNBCs is essential for optimal use of current therapies and for development of new drugs. microRNAs (miRNA) are widely recognized as key players in cancer progression and drug resistance; investigation of their involvement in a TNBC cohort may reveal biomarkers for diagnosis and prognosis of TNBC. Here we stratified a large TNBC cohort into Core Basal (CB, EGFR and/or CK5, 6 positive) and five negative (5NP) if all markers are negative. We determined the complete miRNA expression profile and found a subset of miRNAs specifically deregulated in the two subclasses.We identified a 4-miRNA signature given by miR-155, miR-493, miR-30e and miR-27a expression levels, that allowed subdivision of TNBCs not only into CB and 5NP subgroups (sensitivity 0.75 and specificity 0.56; AUC=0.74) but also into high risk and low risk groups. We tested the diagnostic and prognostic performances of both the 5 IHC marker panel and the 4-miRNA expression signatures, which clearly identify worse outcome patients in the treated and untreated subcohorts. Both signatures have diagnostic and prognostic value, predicting outcomes of patient treatment with the two most commonly used chemotherapy regimens in TNBC: anthracycline or anthracycline plus taxanes. Further investigations of the patients’ overall survival treated with these regimens show that regardless of IHC group subdivision, taxanes addition did not benefit patients, possibly due to miRNA driven taxanes resistance. TNBC subclassification based on the 5 IHC markers and on the miR-155, miR-493, miR-30e, miR-27a expression levels are powerful diagnostic tools. Treatment choice and new drug development should consider this new subtyping and miRNA expression signature in planning low toxicity, maximum efficacy therapies.

Introduction: Circulating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology., Methods: A total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM)., Results: CD45-negative/CK-positive (CD45- CK+) populations with EpCAM + and EpCAM - expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM - populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM - events/ml than CK + EpCAM + events/ml in both the CD45- and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45- and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM - were associated with worse overall survival (P = 0.0292)., Conclusions: Metastatic breast cancer patients have atypical cells that are CK + EpCAM - circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM - circulating cells as a prognostic and predictive marker.

Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vδ2 T cell subset was shown to recognize and limit the growth of various tumors at various degrees. The differential recognition of the tumor cells by Vγ2Vδ2 T cells are yet to be defined. Our study reveals that γδ T cells limit in vitro growth of most breast tumor cells, such as SkBr7 (HER2+), MCF7 (ER+) and MDA-MB-231 (ER-) by inhibiting their survival and inducing apoptosis, except BrCa-MZ01 (PR+) cells. To investigate detail mechanisms of antineoplastic effects, we found that cell death was associated with the surface expression levels of MICA/B and ICAM1. Molecular signaling analysis demonstrated that inhibition of cell growth by γδ T cells was associated with the lower expression levels of cell survival-related molecules such as AKT, ERK and concomitant upregulation of apoptosis-related molecules, such as PARP, cleaved caspase 3 and tumor suppressor genes PTEN and P53. However, opposite molecular signaling was observed in the resistant cell line after coculture with γδ T cells. In vivo, antineoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the downregulation of survival signals, strong induction of apoptotic molecules, disruption of microvasculature and increased infiltration of tumor associated macrophages. These findings reveal that a complex molecular signaling is involved in γδ T cell-mediated antineoplastic effects., (Copyright © 2013 UICC.)

Background and Objective: Biomarkers for subtyping triple negative breast cancer (TNBC) are needed given the absence of responsive therapy and relatively poor prediction of survival. Morphology of cancer tissues is widely used in clinical practice for stratifying cancer patients, while genomic data are highly effective to classify cancer patients into subgroups. Thus integration of both morphological and genomic data is a promising approach in discovering new biomarkers for cancer outcome prediction. Here we propose a workflow for analyzing histopathological images and integrate them with genomic data for discovering biomarkers for TNBC., Materials and Methods: We developed an image analysis workflow for extracting a large collection of morphological features and deployed the same on histological images from The Cancer Genome Atlas (TCGA) TNBC samples during the discovery phase (n=44). Strong correlations between salient morphological features and gene expression profiles from the same patients were identified. We then evaluated the same morphological features in predicting survival using a local TNBC cohort (n=143). We further tested the predictive power on patient prognosis of correlated gene clusters using two other public gene expression datasets., Results and Conclusion: Using TCGA data, we identified 48 pairs of significantly correlated morphological features and gene clusters; four morphological features were able to separate the local cohort with significantly different survival outcomes. Gene clusters correlated with these four morphological features further proved to be effective in predicting patient survival using multiple public gene expression datasets. These results suggest the efficacy of our workflow and demonstrate that integrative analysis holds promise for discovering biomarkers of complex diseases.

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

It has been reported previously that: (1) normal-breast epithelial cells that are CD24-/44+ express higher levels of stem/progenitor cell-associated genes; (2) cancer cells that have undergone epithelial to mesenchymal transition display CD24-/44+ cell-surface expression, a marker for breast cancer stem cells; (3) loss of E-cadherin is a preliminary step in epithelial to mesenchymal transition; and (4) vimentin is a marker of mesenchymal phenotype. We hypothesized that stem cell subpopulations would be more frequent in metastatic than in primary tumors. Therefore we assessed by immunohistochemical analysis, tissue microarrays containing tissue from primary and associated metastatic breast cancers for expression of CD24, CD44, E-cadherin and vimentin to evaluate candidate cancer-initiating cell populations in breast cancer subtypes and metastatic lesions. The occurrence of CD24-/44+ and CD24+/44- cells did not differ in primary vs matched lymph node or distant and locoregional metastatic lesions; E-cadherin expression was decreased in primary vs lymph node metastases (P=0.018) but not decreased in distant and locoregional metastases relative to primary tumor, whereas vimentin, was more frequently expressed in lymph node and distant and locoregional metastases (P=0.013, P=0.004) than in matched primary cancers. Thus, the frequency of CD24-/44+ cells does not differ in metastases relative to the primary breast cancer but differs by tumor stage and subtype.

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.

Piwil2, a member of AGO/PIWI family of proteins, has been reported to be expressed in precancerous stem cells (pCSCs), tumor cell lines and various types of human cancers. However, the significance of piwil2 expression in breast cancer has not been investigated. In this study, archival formalin-fixed, paraffin-embedded breast cancer specimens at various developmental stages were prepared as tissue microarrays (TMAs) and examined for the expressions of piwil2, estrogen receptor (ER), progesterone receptor (PR) and a cell proliferation marker Ki67 by immunohistochemical (IHC) staining and human epidermal growth factor receptor 2 (HER2) by fluorescence in situ hybridization (FISH). The correlation of piwil2 expression with ER, PR and Ki67 were analyzed statistically. The piwil2 was detected in all of breast cancer TMA cores. In contrast, ER, PR, HER2, and Ki67 were detected only in 66.1%, 54.5%, 36.0%, and 47% of the TMA cores, respectively. Piwil2 was expressed in cytoplasm (Cyt), nucleus (N) or both cytoplasm and nucleus (C-N). The N pattern was less observed in breast precancers, whereas all three patterns were observed in invasive and metastatic cancers. While the Cyt pattern was significantly correlated with ER expression (p = 0.002); N pattern was significantly correlated with Ki67 expression (p =0.001). ER and Ki67 expressions were reduced and increased, respectively, with the expression patterns being shifted from Cyt --> C-N --> N. In conclusion, piwil2 is expressed in various stages of breast cancers and has the potential to be used a novel biomarker.