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1.  Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer 
The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial–mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.
doi:10.3390/ijms17101665
PMCID: PMC5085698  PMID: 27706044
metastatic breast cancer (MBC); circulating tumor associated cells; circulating tumor cells (CTCs); circulating tumor cell clusters (CTC clusters); epithelial–mesenchymal transition (EMT); cancer associated macrophage-like cells (CAMLs); size-exclusion technology
2.  Real-time HER2 status detected on circulating tumor cells predicts different outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer patients 
BMC Cancer  2016;16:526.
Background
This study was initiated to investigate the difference in HER2 status between tumor tissue and circulating tumor cells (CTCs), as well as the predictive value of CTC HER2 status for predicting the outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer (MBC) patients.
Methods
HER2 expression on CTCs was detected using a CellSearch system within 7 days before a new line of anti-HER2 therapy was begun. According to the criterion proposed in our previous report, patients were defined as CTC HER2-positive or -negative. After close follow-up, the correlation between CTC HER2 status and the outcome of the treatment was evaluated by statistical analysis.
Results
CTCs were detected in 57.4 % (58/101) of the patients. Notably, 62.1 % (36/58) of these patients had an inconsistent HER2 status between their tissue and CTCs. The discordant rate may correlate with the time interval between histological and CTC HER2 testing and is more likely to occur in the subgroup of patients with an interval of > 1 year than in those with an interval < 1 year (70.7 % vs. 41.2 %, P = 0.043). For PFS, positive HER2 status on CTCs was shown to be a valuable predictor, both in univariate (HR = 0.321, 95%CI, 0.156–0.62, P = 0.0011) and multivariate (HR = 0.383, 95%CI, 0.166–0.831, P = 0.019) Cox regression analysis. Meanwhile, Kaplan-Meier survival curves revealed that the median PFS of CTC HER2-positive patients was significantly longer than CTC HER2-negative ones (8.5 vs. 3.5 months, P < 0.001).
Conclusions
HER2 status on CTCs was different from that of tumor tissues and predicted a different outcome of the patients’ anti-HER2 therapy. This difference may be correlated with the time interval between tissue and CTC HER2 testing, indicating the necessity of real-time HER2 analysis for histologically HER2-positive MBC patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2578-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-016-2578-5
PMCID: PMC4960713  PMID: 27456503
Circulating Tumor Cells (CTCs); Human Epidermal Growth Factor Receptor 2 (HER2); Metastatic Breast Cancer (MBC); Anti-HER2 therapy; Real-time HER2 status
3.  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 < 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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-016-0706-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-016-0706-4
PMCID: PMC4855427  PMID: 27142282
Circulating tumor cells; Mitotic index of CTCs; Blood based biopsy; Breast cancer cell motility
4.  Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers 
Oncotarget  2016;7(18):26724-26738.
Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient's disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access.
doi:10.18632/oncotarget.8494
PMCID: PMC5042010  PMID: 27049831
liquid biopsy; CTC; cfDNA; metastatic breast cancer; next generation sequence
5.  International Expert Consensus on Primary Systemic Therapy in the Management of Early Breast Cancer: Highlights of the Fifth Symposium on Primary Systemic Therapy in the Management of Operable Breast Cancer, Cremona, Italy (2013) 
Expert consensus-based recommendations regarding key issues in the use of primary (or neoadjuvant) systemic treatment (PST) in patients with early breast cancer are a valuable resource for practising oncologists. PST remains a valuable therapeutic approach for the assessment of biological antitumor activity and clinical efficacy of new treatments in clinical trials. Neoadjuvant trials provide endpoints, such as pathological complete response (pCR) to treatment, that potentially translate into meaningful improvements in overall survival and disease-free survival. Neoadjuvant trials need fewer patients and are less expensive than adjuvant trial, and the endpoint of pCR is achieved in months, rather than years. For these reasons, the neoadjuvant setting is ideal for testing emerging targeted therapies in early breast cancer. Although pCR is an early clinical endpoint, its role as a surrogate for long-term outcomes is the key issue. New and better predictors of treatment efficacy are needed to improve treatment and outcomes. After PST, accurate management of post-treatment residual disease is mandatory. The surgery of the sentinel lymph-node could be an acceptable option to spare the axillary dissection in case of clinical negativity (N0) of the axilla at the diagnosis and/or after PST. No data exists yet to support the modulation of the extent of locoregional radiation therapy on the basis of the response attained after PST although trials are underway.
doi:10.1093/jncimonographs/lgv023
PMCID: PMC5009414  PMID: 26063896
6.  The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells 
Background
Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC.
Methods
Cell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models.
Results
CEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines.
Conclusions
CEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-016-0694-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-016-0694-4
PMCID: PMC4806466  PMID: 27009091
CEP-37440; FAK1; ALK; IBC; Inflammatory breast cancer; Triple-negative breast cancer; TNBC
7.  Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner 
Background
Inflammatory breast cancer (IBC) is a very aggressive and lethal subtype of breast cancer that accounts for about 4 % of all breast cancers diagnosed in the United States. Despite the efforts of several investigators to identify the molecular factors driving the aggressive phenotype of IBC, a great deal is still unknown about the molecular underpinnings of the disease. In the present study, we investigated the role of interferon-induced transmembrane protein 1 (IFITM1), a well-known interferon-stimulated gene (ISG), in promoting the aggressiveness of SUM149 IBC cells.
Methods
Western blot and real-time polymerase chain reaction analyses were performed to assess the protein and messenger RNA (mRNA) levels of IFITM1 and other ISGs in three IBC cell lines: SUM149, MDA-IBC-3, and SUM190. IFITM1 expression and cellular localization were assessed by using immunofluorescence, while the tumorigenic potential was assessed by performing cell migration, invasion, and colony formation assays. Small interfering RNA and short hairpin RNA knockdowns, enzyme-linked immunosorbent assays, and luciferase assays were performed to determine the functional significance of IFITM1 and signal transducers and activators of transcription 1 and 2 (STAT1/2) in SUM149 cells.
Results
We found that IFITM1 was constitutively overexpressed at the mRNA and protein levels in triple-negative SUM149 IBC cells, but that it was not expressed in SUM190 and MDA-IBC-3 IBC cells, and that suppression of IFITM1 or blockade of the IFNα signaling pathway significantly reduced the aggressive phenotype of SUM149 cells. Additionally, we found that knockdown of STAT2 abolished IFITM1 expression and IFITM1 promoter activity in SUM149 cells and that loss of STAT2 significantly inhibited the ability of SUM149 cells to proliferate, migrate, invade, and form 2-D colonies. Notably, we found that STAT2-mediated activation of IFITM1 was particularly dependent on the chromatin remodeler brahma-related gene 1 (BRG1), which was significantly elevated in SUM149 cells compared with SUM190 and MDA-IBC-3 cells.
Conclusions
These findings indicate that overexpression of IFITM1 enhances the aggressive phenotype of triple-negative SUM149 IBC cells and that this effect is dependent on STAT2/BRG1 interaction. Further studies are necessary to explore the potential of IFITM1 as a novel therapeutic target and prognostic marker for some subtypes of IBCs.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-016-0683-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-016-0683-7
PMCID: PMC4761146  PMID: 26897526
Inflammatory breast cancer; IFITM1; JAK/STAT signaling; Interferon-stimulated genes; BRG1
8.  Inflammatory Breast Cancer Management in the National Comprehensive Cancer Network (NCCN): The Disease, The Recurrence Pattern, and The Outcome 
Clinical breast cancer  2014;15(1):1-7.
Background
Inflammatory breast cancer (IBC) is an uncommon clinicopathologic entity characterized by rapid progression and aggressive behavior. We used the NCCN Outcomes Database to characterize recurrence patterns and outcomes.
Methods
Patients with newly diagnosed IBC treated between 1999 and 2009 at 12 NCCN institutions were identified and baseline characteristics obtained. Patients had multimodality therapy if they received two of three treatments: surgery, perioperative (neoadjuvant or adjuvant) chemotherapy, or perioperative radiation. First site of recurrence/metastatic diagnosis was identified. Overall survival was calculated based on stage at diagnosis and receipt of multimodality therapy.
Results
We identified 673 patients, of which 195 (29%) had metastatic disease at presentation. Median follow-up was 29 months. Of stage III patients, 82% received >1 treatment modality. Among 203 stage III patients who recurred, the most frequent sites of first recurrence were bone (28%), central nervous system (CNS), lung, and liver (all 21%). HER2 positive and triple negative subtypes had higher rates of CNS recurrence (p=0.001). Median survival was 66 months (95% CI 54-107) for stage III and 26 months (95% CI 22-33) for stage IV. Among 82% of stage III patients receiving multimodality therapy, median survival was 107 months (95% CI 71-Not Reached).
Conclusions
This large, retrospective, multi-institutional study confirms the aggressive clinical features, unique recurrence patterns and adverse prognosis of IBC. The high rate of CNS recurrence among high-risk subtypes, despite the inflammatory nature of the breast cancer, suggests that new strategies are needed for earlier detection or prevention of brain metastases to improve long-term prognosis.
doi:10.1016/j.clbc.2014.05.005
PMCID: PMC4422394  PMID: 25034439
Inflammatory breast cancer; metastatic breast cancer; brain metastases; triple negative breast cancer; multimodality therapy
9.  Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples 
PLoS ONE  2016;11(1):e0147400.
Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.
doi:10.1371/journal.pone.0147400
PMCID: PMC4726586  PMID: 26808060
10.  CCR5 receptor antagonists block metastasis to bone of v-Src-oncogene-transformed metastatic prostate cancer cell lines 
Cancer research  2014;74(23):7103-7114.
Src family kinases (SFKs) integrate signal transduction for multiple receptors, regulating cellular proliferation invasion and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. In order to determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cells (PEC) lines were grown in vivo vs. tissue cultures. The whole body, bone and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors.
doi:10.1158/0008-5472.CAN-14-0612
PMCID: PMC4294544  PMID: 25452256
Prostate Cancer; bone metastasis; v-Src; brain metastasis; CCR5
11.  A Preclinical Model of Inflammatory Breast Cancer to Study the Involvement of CXCR4 and ACKR3 in the Metastatic Process 
Translational Oncology  2015;8(5):358-367.
Inflammatory breast cancer (IBC) is an aggressive and invasive tumor, accounting for 2.5% of all breast cancer cases, and characterized by rapid progression, regional and distant metastases, younger age of onset, and lower overall survival. Presently, there are no effective therapies against IBC and a paucity of model systems. Our aim was to develop a clinically relevant IBC model that would allow investigations on the role of chemokine receptors in IBC metastasis. Primary cultures of tumor cells were isolated from pleural exudates of an IBC patient and grown as spheres or monolayers. We developed a human xenograft model where patient-derived IBC cells, stably transduced with lentiviral vectors expressing fluorescent and bioluminescent markers, were inoculated directly into the left ventricle of mice. Our in vivo data show that these IBC cells (FC-IBC02A) are able to seed and proliferate into various organs, including brain, lungs, lymph nodes, and bone, closely replicating the metastatic spread observed in IBC patients. Moreover, cells were able to generate tumors when grafted in the mammary fat pad of mice. RT-PCR and microscopy studies revealed expression of both CXCR4 and ACKR3 receptors in FC-IBC02A cells. Furthermore, CXCL12 (the endogenous chemokine ligand of these receptors) induced transendothelial migration of these cells and stimulated signaling pathways involved in cell survival and migration - an effect reduced by CXCR4 or ACKR3 antagonists. This new model can be used to develop chemokine-based pharmacological approaches against the IBC metastatic process. This work also provides the first evidence of ACKR3 expression in IBC cells.
doi:10.1016/j.tranon.2015.07.002
PMCID: PMC4631055  PMID: 26500026
12.  Molecular subtyping improves diagnostic stratification of patients with primary breast cancer into prognostically defined risk groups 
Combined use of MammaPrint and a molecular subtyping profile (BluePrint) identifies disease subgroups with marked differences in long-term outcome and response to neo-adjuvant therapy. The aim of this study was to evaluate the prognostic value of molecular subtyping using MammaPrint and BluePrint in women with early-stage breast cancer (BC) treated at US institutions following National Comprehensive Cancer Network standard guidelines. Tumor samples were collected from stage 1-2B consecutively diagnosed BC patients (n = 373) who underwent lumpectomy or mastectomy with an axillary staging procedure between 1992 and 2010 at two institutes (NorthShore University HealthSystem and Fox Chase Cancer Center) in the United States of America, with a median follow-up time of 9.5 years. MammaPrint low-risk patients had a 10-year DMFS of 96 % (95 %CI 92.8–99.4), while MammaPrint high-risk patients had a 10-year DMFS of 87 % (95 %CI 81.9–92.1) with a hazard ratio of 3.62 (95 %CI 1.38–9.50) (p = 0.005). Uni- and multivariate analyses included age, tumor size, grade, ER, and Her2; in multivariate analysis, MammaPrint reached near-significance (HR 3.01; p 0.08). When comparing BluePrint molecular subtyping with clinical stratification, the prognosis (10-year DMFS) was significantly different in 10-year DMFS between the different molecular subtypes (p < 0.001). This retrospective study with 10-year follow-up data provides valuable insight into prognosis of patients with primary BC comparing clinical with molecular subtyping. The BluePrint molecular stratification assay identifies patients with significantly different outcomes compared with standard clinical molecular stratification.
doi:10.1007/s10549-015-3587-9
PMCID: PMC4621695  PMID: 26424167
Breast cancer; Molecular subtyping; Molecular assay; Prognosis
13.  Arterial Blood, Rather Than Venous Blood, is a Better Source for Circulating Melanoma Cells 
EBioMedicine  2015;2(11):1821-1826.
Background
CTCs provide prognostic information and their application is under investigation in multiple tumor types. Of the multiple variables inherent in any such process, none is more important to outcome than the appropriateness of the sample source. To address this question, we investigated CTCs in paired peripheral venous and arterial blood specimens obtained from stage IV uveal melanoma patients.
Methods
Blood specimens were obtained from both common femoral arteries and antecubital veins in 17 uveal melanoma patients with multiple hepatic metastases for CTC measurements.
Finding
CTCs were detectable with greater frequency (100%) and in larger numbers (median 5, range 1 to 168) in all arterial blood specimens than in venous samples (52.9%; median 1, range 0 to 8). Patients with hepatic as well as extra-hepatic metastasis showed higher number of arterial CTCs, compared to patients with liver-only metastasis (p = 0.003). There was no significant association between the number of arterial CTCs and the tumor burden within the liver in patients who had liver-only metastases.
Interpretation
Our data indicate that arterial blood specimens might be a better source of circulating uveal melanoma cells. Although less conveniently processed, perhaps arterial blood should be evaluated as sample source for measurement of CTCs.
Highlights
•CTCs were detectable in 100% of arterial blood obtained from metastatic uveal melanoma patients, while only 53% of venous blood was positive for CTCs.
CTCs have been investigated to provide prognostic information in multiple tumor types. Of the multiple variables, none is more important than the appropriateness of the sample source. Blood specimens were obtained from both femoral arteries and antecubital veins in 17 uveal melanoma patients with multiple hepatic metastases. CTCs were detectable with greater frequency (100%) and in larger numbers in all arterial blood specimens than in venous samples (52.9%). Our data indicate that arterial blood specimens might be a better source of circulating uveal melanoma cells. Although less convenient, arterial blood should be evaluated as sample source for measurement of CTCs.
doi:10.1016/j.ebiom.2015.09.019
PMCID: PMC4740300  PMID: 26870807
Ab, antibody; AKTi, AKT inhibitor; BCNU, bischlorethylnitrosourea; DEBDOX, drug-eluting beads with doxorubicin; EDTA, ethylenediaminetetraacetic acid; HMW-MAA, high molecular weight melanoma associated antigen; Ipi, ipilimumab; LN, lymph node; MEKi, MEK inhibitor; METi, MET inhibitor;; TACE, transarterial chemoembolization; VPA, valproic acid; XRT, radiation therapy; Uveal melanoma; Circulating tumor cells; Hepatic metastasis; Arterial venous; Peripheral venous; CTC count
14.  Pre-Clinical studies of Notch Signaling Inhibitor RO4929097 in Inflammatory Breast Cancer Cells 
Basal breast cancer, common among patients presenting with inflammatory breast cancer, has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling that promotes the breast cancer stem cell phenotype. Herein we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 inflammatory breast cancer (IBC) cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1 and HeyL and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1uM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8, cytokines known to mediate the efficacy of notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest additional targeting agents may be required to selectively target IBC stem cells through notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.
doi:10.1007/s10549-012-2075-8
PMCID: PMC4545650  PMID: 22547109
Notch; Cancer stem cells; R04929097; Inflammatory breast cancer; Radiation
15.  Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells 
PLoS ONE  2015;10(7):e0132710.
Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.
doi:10.1371/journal.pone.0132710
PMCID: PMC4514595  PMID: 26207636
16.  Prognostic Significance of HER-2 Status in Women With Inflammatory Breast Cancer 
Cancer  2008;112(9):1905-1911.
BACKGROUND
Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer with poorly understood prognostic variables. The purpose of this study was to define the prognostic impact of HER-2 status on survival outcomes of patients with IBC.
METHODS
In all, 179 patients with IBC, diagnosed between 1989 and 2005, with known HER-2 status, and treated with an anthracycline-based chemotherapy regimen without trastuzumab, were included in the analysis. Patients with HER-2-positive disease who received trastuzumab at the time of disease recurrence were included. Survival outcomes were estimated by the Kaplan-Meier product limit method and compared across groups using the log-rank statistic. A Cox proportional hazards model was fitted to determine the association of survival outcomes with HER-2 status after adjusting for patient and tumor characteristics.
RESULTS
A total of 111 patients (62%) had HER-2-negative disease and 68 (38%) had HER-2-positive disease. The median follow-up among all patients was 35 months. At the time of the analysis, 62 patients (55.9%) with HER-2-negative disease and 42 patients (61.8%) with HER-2-positive disease had a recurrence. Thirty-one patients (73.8%) with HER-2-positive disease who had a disease recurrence went on to receive trastuzumab. On univariate analysis, no statistically significant difference was observed for either recurrence-free survival (P = .75) or overall survival (P = .24) between patients who had HER-2-positive disease and those who had HER-2-negative disease. In a multivariate model, HER-2 status did not appear to significantly affect recurrence-free survival (hazards ratio [HR] of 0.75; 95% confidence interval [95% CI], 0.46–1.22 [P = .241]). In the multivariate model, patients with HER-2-positive disease had a decreased hazard of death (HR of 0.56; 95% CI, 0.34–0.93 [P = .024]) compared with patients with HER-2-negative disease.
CONCLUSIONS
HER-2 status, in the absence of trastuzumab, did not appear to significantly affect recurrence-free survival. After adjusting for other characteristics, the addition of trastuzumab in the metastatic setting significantly improved survival in the HER-2-positive group above and beyond that of the HER-2-negative group. This gives us further insight into the biology of this aggressive disease and underlines the major effect of targeted intervention.
doi:10.1002/cncr.23350
PMCID: PMC4437796  PMID: 18300243
inflammatory breast cancer; HER-2 status; survival outcomes; targeted intervention
17.  Molecular characterization and targeted therapeutic approaches in breast cancer 
Despite the wide improvements in breast cancer (BC) detection and adjuvant treatment, BC is still responsible for approximately 40,000 deaths annually in the United States. Novel biomarkers are fundamental to assist clinicians in BC detection, risk stratification, disease subtyping, prediction of treatment response, and surveillance, allowing a more tailored approach to therapy in both primary and metastatic settings. In primary BC, the development of molecular profiling techniques has added prognostic and predictive information to conventional biomarkers - estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Moreover, the application of next-generation sequencing and reverse-phase protein microarray methods in the metastatic setting holds the promise to further advance toward a personalized management of cancer. The improvement in our understanding on BC biology associated with the study of the genomic aberrations characterizing the most common molecular subtypes allows us to explore new targets for drug development. Finally, the integration of cancer stem cell-targeted therapies and immune therapies in future combination regimens increases our chances to successfully treat a larger proportion of women with more aggressive and resistant metastatic disease. This article reviews the current state of novel biological markers for BC, the evidence to demonstrate their clinical validity and utility, and the implication for therapeutic targeting.
doi:10.1186/s13058-015-0560-9
PMCID: PMC4407294  PMID: 25902832
18.  Emergence of constitutively active estrogen receptor-α mutations in pretreated advanced estrogen receptor positive breast cancer 
Purpose
We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone dependent breast cancer and to delineate the functional roles of the most commonly detected alterations.
Experimental Design
We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER positive (ER+/HER2–) and, as controls, 115 ER negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes.
Results
Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% (9/76, 95% CI 6%-21%) in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25, 95% CI 7%-41%). These mutations were not detected in primary or treatment naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments.
Conclusions
In this study we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer.
doi:10.1158/1078-0432.CCR-13-2332
PMCID: PMC3998833  PMID: 24398047
breast cancer; estrogen receptor; ESR1 mutations; endocrine resistance
19.  Phase I/II Study of G3139 (Bcl-2 Antisense Oligonucleotide) in Combination with Doxorubicin and Docetaxel in Breast Cancer 
Purpose
Preclinical data showed enhancement of breast cancer cell death when G3139 was combined with anthracyclines and taxanes. We evaluated the efficacy and safety of a Bcl-2 antisense oligonucleotide, G3139, in combination with doxorubicin (A) and docetaxel (T) in patients with locally advanced breast cancer (LABC).
Experimental Design
Following a brief phase I to determine the phase II dose, patients with locally advanced breast cancer received G3139 administered by continuous i.v. infusion for 5 to 7 days with bolus A (50mg/m2) and T (75 mg/m2) administered on either day 3 or 6 of therapy with G3139. Cycles were repeated every 21 days × 6 in the neoadjuvant setting. Serial plasma samples were obtained for pharmacokinetic analysis. Tissue samples were obtained before and after therapy for pharmacodynamic analysis of Bcl-2 expression.
Results
Thirty patients (median age, 49 years; range, 24–71years) received 160 cycles. During the phase I portion of the trial, the dose of G3139 was escalated from 3 to 7 mg/kg/d (i.v. for 5 days) in combination with AT. During the phase II portion of the trial, several doses and schedules of G3139 were evaluated. There were no pathologic complete responses. Pharmacodynamic studies showed limited Bcl-2 down-regulation in the primary tumors.
Conclusions
G3139 in combination with doxorubicin and docetaxel is well tolerated. No pathologic complete response was seen and pharmacodynamic studies showed very little down-regulation of Bcl-2 in primary tumors, perhaps related to issues with insufficient drug delivery to the intact tumor.
doi:10.1158/1078-0432.CCR-08-1104
PMCID: PMC4375958  PMID: 19047121
20.  Factors Predicting Additional Disease in the Axilla in Patients With Positive Sentinel Lymph Nodes After Neoadjuvant Chemotherapy 
Cancer  2008;112(12):2646-2654.
BACKGROUND
The utility of sentinel lymph node (SNL) biopsy (SLNB) as a predictor of axillary lymph node status is similar in patients who receive neoadjuvant chemotherapy and patients who undergo surgery first. The authors of this study hypothesized that patients with positive SLNs after neoadjuvant therapy would have unique clinicopathologic factors that would be predictive of additional positive non-SLNs distinct from patients who underwent surgery first.
METHODS
One hundred four patients were identified who received neoadjuvant chemotherapy, had a positive SLN, and underwent axillary dissection between 1997 and 2005. At the time of presentation, 66 patients had clinically negative lymph nodes by ultrasonography, and 38 patients had positive lymph nodes confirmed by fine-needle aspiration. Eighteen factors were assessed for their ability to predict positive non-SLNs using chi-square and logistic regression analysis with a bootstrapped, backwards elimination procedure. The resulting nomogram was tested by using a patient cohort from another institution.
RESULTS
Patients with clinically negative lymph nodes at presentation were less likely than patients with positive lymph nodes to have positive non-SLNs (47% vs 71%; P = .017). On multivariate analysis, lymphovascular invasion, the method for detecting SLN metastasis, multicentricity, positive axillary lymph nodes at presentation, and pathologic tumor size retained grouped significance with a bootstrap-adjusted area under the curve (AUC) of 0.762. The resulting nomogram was validated in the external patient cohort (AUC, 0.78).
CONCLUSIONS
A significant proportion of patients with positive SLNs after neoadjuvant chemotherapy had no positive non-SLNs. The use of a nomogram based on 5 predictive variables that were identified in this study may be useful for predicting the risk of positive non-SLNs in patients who have positive SLNs after chemotherapy.
doi:10.1002/cncr.23481
PMCID: PMC4365777  PMID: 18442039
neoadjuvant therapy; nomogram; breast cancer; sentinel lymph node biopsy
21.  Circulating tumor cells in newly diagnosed inflammatory breast cancer 
Introduction
Circulating tumor cells (CTCs) are an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. The prognostic value of a CTC count in newly diagnosed IBC has not been established. The aim of this study was to assess the prognostic value of a baseline CTC count in patients with newly diagnosed IBC.
Methods
This retrospective study included 147 patients with newly diagnosed IBC (77 with locally advanced and 70 with metastatic IBC) treated with neoadjuvant therapy or first-line chemotherapy during the period from January 2004 through December 2012 at The University of Texas MD Anderson Cancer Center. CTCs were detected and enumerated by using the CellSearch system before patients were started with chemotherapy.
Results
The proportion of patients with ≥1 CTC was lower among patients with stage III than among patients with metastatic IBC (54.5% versus 84.3%; P = 0.0002); the proportion of patients with ≥5 CTCs was also lower for stage III than for metastatic IBC (19.5% versus 47.1%; P = 0.0004). Patients with fewer than five CTCs had significantly better progression-free survival (PFS) (hazard ratio (HR) = 0.60; P = 0.02) and overall survival (HR = 0.59; P = 0.03) than patients with five or more CTCs. Among patients with stage III IBC, there was a nonsignificant difference in PFS (HR = 0.66; 95% confidence interval (CI), 0.31 to 1.39; P = 0.29) and OS (HR = 0.54; 95% CI, 0.24 to 1.26; P = 0.48) in patients with no CTCs compared with patients with one or more CTCs. In multivariate analysis, CTC was prognostic for PFS and OS independent of clinical stage.
Conclusions
CTCs can be detected in a large proportion of patients with newly diagnosed IBC and are a strong predictor of worse prognosis in patients with newly diagnosed IBC.
doi:10.1186/s13058-014-0507-6
PMCID: PMC4318180  PMID: 25572591
22.  CTC enumeration and characterization: moving toward personalized medicine 
The primary cause of tumor-related death in breast cancer (BC) is still represented by distant metastasization. The dissemination of tumor cells from the primary tumor to distant sites through bloodstream cannot be early detected by standard imaging methods. The enumeration of circulating tumor cells (CTCs) represents an effective prognostic and predictive biomarker, which is able to monitor efficacy of adjuvant therapies, detect early development of (micro)metastases and at last, assess therapeutic responses of advanced disease earlier than traditional imaging methods. Moreover, since repeated tissue biopsies are invasive, costly and not always feasible, the assessment of tumor characteristics on CTCs, by a peripheral blood sample as a ‘liquid biopsy’, represents an attractive opportunity. The implementation of molecular and genomic characterization of CTCs could contribute to improve the treatment selection and thus, to move toward more personalized treatments. This review describes the current state of the art on CTC detection strategies, the evidence to demonstrate their clinical validity, and their potential impact for both future clinical trial design and, decision-making process in our daily practice.
doi:10.3978/j.issn.2305-5839.2014.09.06
PMCID: PMC4245508  PMID: 25489582
Breast cancer (BC); circulating tumor cells (CTCs); liquid biopsy; precision medicine
23.  TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients 
Introduction
Circulating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.
Methods
CTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs.
Results
From 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele.
Conclusions
We are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.
doi:10.1186/s13058-014-0445-3
PMCID: PMC4303125  PMID: 25307991
24.  Phase I–II study of the farnesyl transferase inhibitor tipifarnib plus sequential weekly paclitaxel and doxorubicin–cyclophosphamide in HER2/neu-negative inflammatory carcinoma and non-inflammatory estrogen receptor-positive breast carcinoma 
Tipifarnib (T) is a farnesyl transferase inhibitor (FTI) that enhances the antineoplastic effects of cytotoxic therapy in vitro, has activity in metastatic breast cancer, and enhances the pathologic complete response (pCR) rate to neoadjuvant doxorubicin–cyclophosphamide (AC) chemotherapy. We, therefore, performed a phase I–II trial of T plus neoadjuvant sequential weekly paclitaxel and 2-week AC chemotherapy in locally advanced breast cancer. Eligible patients with HER2-negative clinical stage IIB–IIIC breast cancer received 12 weekly doses of paclitaxel (80 mg/m2) followed by AC (60/600 mg/m2 every 2 weeks and filgrastim), plus T (100 or 200 mg PO on days 1–3 of each P dose, and 200 mg PO on days 2–7 of each AC cycle). The trial was powered to detect an improvement in breast pCR rate from 15 to 35 % (α = 0.10, β = 0.10) in two strata, including ER and/or PR-positive, non-inflammatory (stratum A) and inflammatory carcinoma (stratum B). Of the 60 patients accrued, there were no dose-limiting toxicities among the first six patients treated at the first T dose level (100 mg BID; N = 3) or second T dose level (200 mg BID; N = 3) plus paclitaxel. Breast pCR occurred in 6/33 patients (18 %, 95 % confidence intervals (CI) 7–36 %) and 1/22 patients (4 %, 95 % CI 0–8 %) in stratum B. Combination of the FTI T with weekly paclitaxel–AC is unlikely to be associated with a breast pCR rate of 35 % or higher in patients with locally advanced HER2/neu-negative inflammatory or non-inflammatory ER- and/or PR-positive breast carcinoma.
doi:10.1007/s10549-013-2704-x
PMCID: PMC3999640  PMID: 24068539
Farnesyl transferase inhibitor; Tipifarnib; Ras; Breast cancer; Neoadjuvant chemotherapy; Inflammatory breast cancer
25.  Circulating tumor cells as early predictors of metastatic spread in breast cancer patients with limited metastatic dissemination 
Introduction
Traditional factors currently used for prognostic stratification do not always adequately predict treatment response and disease evolution in advanced breast cancer patients. Therefore, the use of blood-based markers, such as circulating tumor cells (CTCs), represents a promising complementary strategy for disease monitoring. In this retrospective study, we explored the role of CTC counts as predictors of disease evolution in breast cancer patients with limited metastatic dissemination.
Methods
A total of 492 advanced breast cancer patients who had a CTC count assessed by CellSearch prior to starting a new line of systemic therapy were eligible for this analysis. Using the threshold of 5 CTCs/7.5 ml of blood, pretreatment CTC counts were correlated in the overall population with metastatic site distribution, evaluated at baseline and at the time of treatment failure, using Fisher’s exact test. Time to visceral progression and time to the development of new metastatic lesions and sites were estimated in patients with nonvisceral metastases and with single-site metastatic disease, respectively, by the Kaplan-Meier method. Survival times were compared between groups according to pretreatment CTC count by logrank test.
Results
In the overall population, a pretreatment level ≥5 CTCs/7.5 ml was associated with an increased baseline number of metastatic sites compared with <5 CTCs/7.5 ml (P = 0.0077). At the time of treatment failure, patients with ≥5 CTCs/7.5 ml more frequently developed new metastatic lesions and sites compared with those with <5 CTCs/7.5 ml (development of new lesions: P = 0.0002; development of new sites: P = 0.0031). Among patients with disease originally confined to nonvisceral sites, ≥5 CTCs/7.5 ml was associated with remarkably shorter time to visceral metastases (P = 0.0021) and overall survival (P = 0.0006) compared with <5 CTCs/7.5 ml. In patients with single-site metastatic disease, ≥5 CTCs/7.5 ml was associated with a significant reduction of the time to development of new metastatic sites (P = 0.0051) and new lesions (P = 0.0002) and with worse overall survival (P = 0.0101).
Conclusion
Our results suggest that baseline CTC counts can be used as an early predictor of metastatic potential in breast cancer patients with limited metastatic dissemination.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0440-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0440-8
PMCID: PMC4303121  PMID: 25223629

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