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1.  Genomic and Expression Analysis of Microdissected Inflammatory Breast Cancer 
Purpose
Inflammatory breast cancer (IBC) is a unique clinical entity characterized by rapid onset of erythema and swelling of the breast often without an obvious breast mass. Many studies have examined and compared gene expression between IBC and non-IBC (nIBC), repeatedly finding clusters associated with receptor subtype, but no consistent gene signature associated with IBC has been validated. Here we compared microdissected IBC tumor cells to microdissected nIBC tumor cells matched based on estrogen and HER-2/neu receptor status.
Methods
Gene expression analysis and comparative genomic hybridization were performed. An IBC gene set and genomic set were identified using a training set and validated on the remaining data. The IBC gene set was further tested using data from IBC consortium samples and publically available data.
Results
Receptor driven clusters were identified in IBC; however no IBC-specific gene signature was identified. Fifteen genes were correlated between increased genomic copy number and gene overexpression data. An expression-guided gene set upregulated in the IBC training set clustered the validation set into two clusters independent of receptor subtype but segregated only 75% of samples in each group into IBC or nIBC. In a larger consortium cohort and in published data the gene set failed to optimally enrich for IBC samples. However, this gene set had a high negative predictive value for excluding the diagnosis of IBC in publically available data (100%). An IBC enriched genomic data set accurately identified 10/16 cases in the validation data set.
Conclusions
Even with microdissection, no IBC-specific gene signature distinguishes IBC from nIBC. Using microdissected data, a validated gene set was identified that is associated with IBC tumor cells. IBC comparative genomic hybridization data are presented, but a validated genomic data set that identifies IBC is not demonstrated.
doi:10.1007/s10549-013-2501-6
PMCID: PMC3677826  PMID: 23568481
Inflammatory breast cancer; CGH; array; gene signature
2.  Establishment and validation of circulating tumor cell-based prognostic nomograms in first-line metastatic breast cancer patients 
Purpose
Circulating tumor cells (CTC) represent a new outcome-associated biomarker independently from known prognostic factors in metastatic breast cancer (MBC). The objective here was to develop and validate nomograms that combined baseline CTC counts and the other prognostic factors to assess the outcome of individual patients starting first-line treatment for MBC.
Experimental Design
We used a training set of 236 MBC patients starting a first-line treatment from the MD Anderson Cancer Center to establish nomograms that calculated the predicted probability of survival at different time points: 1, 2, and 5 years for overall survival (OS) and 6 months and 1 and 2 years for progression-free survival (PFS). The covariates computed in the model were: age, disease subtype, visceral metastases, performance status, and CTC counts by CellSearch. Nomograms were independently validated with 210 MBC patients from the Institut Curie who underwent first-line chemotherapy. The discriminatory ability and accuracy of the models were assessed using Harrell’s c-statistic and calibration plots at different time points in both training and validation datasets.
Results
Median follow-up was of 23 and 29 months in the MD Anderson and Institut Curie cohorts, respectively. Nomograms demonstrated good C-statistics: 0.74 for OS and 0.65 for PFS and discriminated OS prediction at 1, 2, and 5 years, and PFS prediction at 6 months and 1 and 2 years.
Conclusions
Nomograms, which relied on CTC counts as a continuous covariate, easily facilitated the use of a web-based tool for estimating survival, supporting treatment-decisions and clinical trial stratification in first-line MBC.
doi:10.1158/1078-0432.CCR-12-3137
PMCID: PMC3662240  PMID: 23340302
circulating tumor cells; first-line; metastatic breast cancer; nomogram; survival
3.  A Phase I Study of Weekly Everolimus (RAD001) in Combination with Docetaxel in Patients with Metastatic Breast Cancer 
Cancer  2011;118(9):2378-2384.
Background
Inhibition of mTOR with everolimus may result improve efficacy of taxanes. Everolimus and docetaxel are both metabolized by CYP3A4, which could result in a pharmacokinetic (PK) interaction.
Patients and Methods
15 patients with metastatic breast cancer were treated with docetaxel (doses of 40-75 mg/m2 IV on day 1 of a 21 day cycle) in combination with everolimus (doses ranging from 20-50 mg po on days 1 and 8 of a 21 day cycle) in a phase I trial using the continuous reassessment method (CRM) to determine maximum tolerated dose (MTD). The first two patients developed DLT (neutropenic infection), prompting a mandatory dose reduction and PK evaluation of both everolimus and docetaxel for patients enrolled in subsequent dosing cohorts.
Results
15 patients were treated. Dose limiting toxicity included grade 3 mucositis (n=1), prolonged grade 4 neutropenia (n=1), and grade 3 infection/febrile neutropenia (n=3). Day 8 of everolimus was commonly held for neutropenia despite a dose reduction in docetaxel to 40mg/m2. Eleven patients underwent complete PK evaluation for everolimus and 9 patients underwent complete PK evaluation for both everolimus and docetaxel. Widely variable changes in clearance were seen for both drugs and the study was terminated due to lack of efficacy and concerns regarding toxicity seen with the combination.
Conclusion
Weekly everolimus in combination with Q 3-week docetaxel was associated with excessive neutropenia and variable clearance of both drugs making combination therapy unpredictable, even at low doses of both drugs.
doi:10.1002/cncr.26571
PMCID: PMC3893000  PMID: 22006179
Phase I; everolimus; docetaxel
4.  Prognostic value of HER2-positive circulating tumor cells in patients with metastatic breast cancer 
International journal of clinical oncology  2011;17(2):10.1007/s10147-011-0260-0.
Backgrounds
The presence of ≥5 circulating tumor cells (CTCs) in 7.5 ml blood is a poor prognostic marker in metastatic breast cancer (MBC). However, the role of human epidermal growth factor receptor 2 (HER2) status in CTCs is not known.
Methods
We prospectively assessed the prognostic value of this parameter for patients with MBC who started a new line of systemic therapy. The CTC count (≥5 or <5) and the HER2 status in CTCs at the initiation of the therapy and 3–4 weeks later (first follow-up) were determined.
Results
The median follow-up time of the 52 enrolled patients was 655.0 days (18–1,275 days). HER2-positive CTCs were present in 14 of the 52 patients (26.9%) during the study period. Eight of 33 patients (24.2%) with HER2-negative primary tumors had HER2-positive CTCs during the study period. At first follow-up, patients with HER2-positive CTCs had significantly shorter progression-free (n = 6; P = 0.001) and overall (P = 0.013) survival than did patients without HER2-positive CTCs (n = 43) in log-rank analysis. In multivariate analysis, HER2-positive CTCs at first follow-up (P = 0.029) and the number of therapies patients received before this study (P = 0.006) were independent prognostic factors in terms of progression-free survival. The number of therapies (P = 0.001) and a count of ≥5 CTCs (P = 0.043) at baseline were independent prognostic factors in terms of overall survival.
Conclusions
We showed that HER2 status in CTCs may be a prognostic factor for MBC. Well-powered prospective studies are necessary to determine the potential role of HER2-targeted therapies for patients with HER2-positive CTCs and HER2-negative primary tumors.
doi:10.1007/s10147-011-0260-0
PMCID: PMC3860324  PMID: 21671160
Circulating tumor cell; Breast neoplasm; HER2; Metastasis
5.  Serum HER2 levels determined by two methods in patients with metastatic breast cancer 
International journal of clinical oncology  2011;17(1):10.1007/s10147-011-0253-z.
Background
The role and the optimal measurement method of serum HER2 levels are not defined in patients with metastatic breast cancer (MBC). We prospectively assessed the prognostic value of serum HER2 levels in MBC using two methods, enzyme immunoassay (EIA) and chemiluminescence immunoassay (CLIA).
Methods
We collected blood samples from patients with MBC at baseline and at subsequent 3- to 4-week intervals up to 12 weeks. Samples were divided, and serum HER2 levels were determined using EIA and CLIA. We also determined whether serum HER2 levels had decreased by ≥20% at first follow-up. These results were evaluated against overall survival, progression-free survival, and tumor response.
Results
We obtained 196 samples from 52 patients. In 59 samples from patients who received trastuzumab, serum HER2 positivity rates were significantly lower for EIA (n = 22) than for CLIA (n = 33, P = 0.042); in 137 samples from patients who did not receive trastuzumab, there was no significant difference in rates of serum HER2 positivity for CLIA (n = 83) and EIA (n = 80). Serum HER2 level at baseline, the level at first follow-up, and a decrease of ≥20% between baseline and first follow-up were not associated with overall survival, progression-free survival, and tumor response.
Conclusions
Chemiluminescence immunoassay was a more sensitive method than EIA for measuring serum HER2 levels in patients who received trastuzumab. However, because serum HER2 levels did not correlate with patient outcome, we do not currently recommend measuring serum HER2 levels by either method for prognostic evaluation in patients with MBC.
doi:10.1007/s10147-011-0253-z
PMCID: PMC3860357  PMID: 21607830
Breast neoplasm; HER2; Metastasis; Serum HER2; Trastuzumab
6.  Epithelial-Mesenchymal Transition and Stem Cell Markers in Patients with HER2-Positive Metastatic Breast Cancer 
Molecular cancer therapeutics  2012;11(11):2526-2534.
Currently, there is extensive information about circulating tumor cells (CTCs) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition inducing transcription factors (EMT-TFs) and cancer stem cell features in HER2+ metastatic breast cancer (MBC) patients. Epithelial cells were enriched from peripheral blood mononuclear cells (PBMCs) using antibody-coated anti-CD326 antibody (CD326+) magnetic beads, and the residual CD326− PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326−CD45−). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). Additionally, PBMCs were analyzed using multi-parameter flow cytometry for ALDH activity and cancer stem cells (CSCs) that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326−CD45− cell fraction of 60.7% of patients. The CD326−CD45− fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH+/CD133+ cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P=0.038). Our data indicate that HER2+ MBC patients have EMT-CTCs. Moreover, an enrichment of cancer stem cells was found in CD326−CD45− cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in HER2+ MBC patients treated with trastuzumab-based therapy.
doi:10.1158/1535-7163.MCT-12-0460
PMCID: PMC3500676  PMID: 22973057
circulating tumor cells; epithelial to mesenchymal transition; stem cells; HER2; CD133; metastatic breast cancer
7.  Presence of anaplastic lymphoma kinase in inflammatory breast cancer 
SpringerPlus  2013;2:497.
Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKβ in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 μM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+ IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.
Electronic supplementary material
The online version of this article (doi:10.1186/2193-1801-2-497) contains supplementary material, which is available to authorized users.
doi:10.1186/2193-1801-2-497
PMCID: PMC3791224  PMID: 24102046
Inflammatory breast cancer; Anaplastic lymphoma kinase; Reverse phase protein arrays; Crizotinib
8.  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.
doi:10.1186/1756-9966-32-70
PMCID: PMC3850122  PMID: 24294976
Inflammatory breast cancer; EZH2; Cancer stem cell; Tumor spheroid formation
9.  Expression of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Primary Breast Cancer: The Effect of Neoadjuvant Therapy 
Epithelial cancer cells are likely to undergo epithelial mesenchymal transition (EMT) prior to entering the peripheral circulation. By undergoing EMT, circulating tumor cells (CTCs) lose epithelial markers and may escape detection by conventional methods. Therefore, we conducted a pilot study to investigate mRNA transcripts of EMT-inducing transcription factors (TFs) in tumor cells from the peripheral blood (PB) of primary breast cancer (PBC) patients.
Peripheral blood mononuclear cells were isolated from 52 stages I–III PBC patients and 30 healthy donors (HD) and sequentially depleted of EpCAM+ cells and CD45+ leukocytes, henceforth referred to as CD45−. The expression levels of EMT-inducing TFs (TWIST1, SNAIL1, SLUG, ZEB1, and FOXC2) in the CD45− cells were determined using qRT-PCR. The highest level of expression by the CD45− cell fraction of HD was used as “cut off” to determine if samples from PBC patients overexpressed any EMT-inducing TFs. In total, 15.4% of PBC patients overexpressed at least one of the EMT-inducing TF transcripts. Overexpression of any EMT-inducing TF transcripts was more likely to be detected in PBC patients who received neoadjuvant therapies (NAT) than patients who received no NAT (P = 0.003). Concurrently, CTCs were detected in 7 out of 38 (18.4%) patients by CellSearch® and 15 out of 42 (35.7%) patients by AdnaTest™. There was no association between the presence of CTCs measured by CellSearch® or AdnaTest™.
In summary, our results demonstrate that CTCs with EMT phenotype may occur in the peripheral circulation of PBC patients and NAT is unable to eliminate CTCs undergoing EMT.
doi:10.1002/ijc.26037
PMCID: PMC3169728  PMID: 21387303
circulating tumor cells; epithelial-mesenchymal transition; primary breast cancer; neoadjuvant therapy
10.  Recurrent granulomatous mastitis mimicking inflammatory breast cancer 
BMJ Case Reports  2011;2011:bcr0720103156.
Granulomatous mastitis (GM) is an uncommon benign breast lesion. Diagnosis is a matter of exclusion from other inflammatory, infectious and granulomatous aetiologies. Here, we presented an atypical GM case, which had clinical and radiologic features overlapping with inflammatory breast cancer (IBC). The disease had multiple recurrences. The patient is a 40-year-old Caucasian woman with a sudden onset of left breast swelling accompanied by diffuse skin redness, especially of the subareolar region and malodorous yellow nipple discharge from the left nipple. The disease progressed on antibiotic treatment and recurred after local resection. A similar lesion developed even after bilateral mastectomy. GM may show clinical/radiologic features suggestive of IBC. Multiple recurrences can be occasionally encountered. GM after recurrence could be much more alarming clinically. Pathology confirmation is the key for accurate diagnosis and a multidisciplinary approach is important to rule out IBC.
doi:10.1136/bcr.07.2010.3156
PMCID: PMC3062046  PMID: 22715267
11.  A randomized phase II study of lapatinib + pazopanib versus lapatinib in patients with HER2+ inflammatory breast cancer 
This multi-center Phase II study evaluated lapatinib, pazopanib, and the combination in patients with relapsed HER2+ inflammatory breast cancer. In Cohort 1, 76 patients were randomized 1:1 to receive lapatinib 1,500 mg + placebo or lapatinib 1,500 mg + pazopanib 800 mg (double-blind) once daily until disease progression, unacceptable toxicity, or death. Due to high-grade diarrhea observed with this dose combination in another study (VEG20007), Cohort 1 was closed. The protocol was amended such that an additional 88 patients (Cohort 2) were randomized in a 5:5:2 ratio to receive daily monotherapy lapatinib 1,500 mg, lapatinib 1,000 mg + pazopanib 400 mg, or monotherapy pazopanib 800 mg, respectively. The primary endpoint was overall response rate (ORR). Secondary endpoints included duration of response, progression-free survival (PFS), overall survival, and safety. In Cohort 1, ORR for the lapatinib (n = 38) and combination (n = 38) arms was 29 and 45 %, respectively; median PFS was 16.1 and 14.3 weeks, respectively. Grade ≥3 adverse events (AEs) were more frequent in the combination arm (71 %) than in the lapatinib arm (24 %). Dose reductions and interruptions due to AEs were also more frequent in the combination arm (45 and 53 %, respectively) than in the lapatinib monotherapy arm (0 and 11 %, respectively). In Cohort 2, ORR for patients treated with lapatinib (n = 36), lapatinib + pazopanib (n = 38), and pazopanib (n = 13) was 47, 58, and 31 %, respectively; median PFS was 16.0, 16.0, and 11.4 weeks, respectively. In the lapatinib, combination, and pazopanib therapy arms, grade ≥3 AEs were reported for 17, 50, and 46 % of patients, respectively, and the incidence of discontinuations due to AEs was 0, 24, and 23 %, respectively. The lapatinib–pazopanib combination was associated with a numerically higher ORR but no increase in PFS compared to lapatinib alone. The combination also had increased toxicity resulting in more dose reductions, modifications, and treatment delays. Activity with single-agent lapatinib was confirmed in this population.
doi:10.1007/s10549-012-2369-x
PMCID: PMC3539065  PMID: 23239151
Lapatinib; Pazopanib; Inflammatory breast cancer; HER2-positive breast cancer
12.  Triple-Negative Subtype Predicts Poor Overall Survival and High Locoregional Relapse in Inflammatory Breast Cancer 
The Oncologist  2011;16(12):1675-1683.
The value of hormone receptor and human epidermal growth factor receptor 2 expression for predicting overall survival, distant relapse, and locoregional relapse was examined in patients with inflammatory breast cancer. Triple-negative disease was associated with worse outcomes, indicating the need for developing new locoregional and systemic treatment strategies for patients with this aggressive subtype.
Background.
Numerous studies have demonstrated that expression of estrogen/progesterone receptor (ER/PR) and human epidermal growth factor receptor (HER)-2 is important for predicting overall survival (OS), distant relapse (DR), and locoregional relapse (LRR) in early and advanced breast cancer patients. However, these findings have not been confirmed for inflammatory breast cancer (IBC), which has different biological features than non-IBC.
Methods.
We retrospectively analyzed the records of 316 women who presented to MD Anderson Cancer Center in 1989–2008 with newly diagnosed IBC without distant metastases. Most patients received neoadjuvant chemotherapy, mastectomy, and postmastectomy radiation. Patients were grouped according to receptor status: ER+ (ER+/PR+ and HER-2−; n = 105), ER+HER-2+ (ER+/PR+ and HER-2+; n = 37), HER-2+ (ER−/PR− and HER-2+; n = 83), or triple-negative (TN) (ER−PR−HER-2−; n = 91). Kaplan–Meier and Cox proportional hazards methods were used to assess LRR, DR, and OS rates and their associations with prognostic factors.
Results.
The median age was 50 years (range, 24–83 years). The median follow-up time and median OS time for all patients were both 33 months. The 5-year actuarial OS rates were 58.7% for the entire cohort, 69.7% for ER+ patients, 73.5% for ER+HER-2+ patients, 54.0% for HER=2+ patients, and 42.7% for TN patients (p < .0001); 5-year LRR rates were 20.3%, 8.0%, 12.6%, 22.6%, and 38.6%, respectively, for the four subgroups (p < .0001); and 5-year DR rates were 45.5%, 28.8%, 50.1%, 52.1%, and 56.7%, respectively (p < .001). OS and LRR rates were worse for TN patients than for any other subgroup (p < .0001–.03).
Conclusions.
TN disease is associated with worse OS, DR, and LRR outcomes in IBC patients, indicating the need for developing new locoregional and systemic treatment strategies for patients with this aggressive subtype.
doi:10.1634/theoncologist.2011-0196
PMCID: PMC3248766  PMID: 22147002
Inflammatory breast cancer; Estrogen receptor; Progesterone receptor; HER-2; Molecular subtypes
13.  Circulating tumor cells (CTCs) in breast cancer: a diagnostic tool for prognosis and molecular analysis 
Metastatic breast cancer (MBC) is characterized by a combination of tumor growth, proliferation and metastatic progression and is typically managed with palliative intent. The benefit of standard systemic therapies is relatively limited and the disease is considered incurable suggesting the need to investigate the biological drivers of the various phases of the metastatic process in order to improve the selection of molecularly driven therapies. The detection, enumeration and molecular analysis of circulating tumor cells (CTCs) provide an intriguing opportunity to advance this knowledge. CTCs enumerated by the Food and Drugs Administration-cleared CellSearch® system are an independent prognostic factor of progression-free survival (PFS) and overall survival (OS) in MBC patients. Several published papers demonstrated the poor prognosis for MBC patients that presented basal CTC count ≥5 in 7.5 mL of blood. Therefore, the enumeration of CTCs during treatment for MBC provides a tool with the ability to predict progression of disease earlier than standard timing of anatomical assessment using conventional radiological tests. During the metastatic process cancer cells exhibit morphological and phenotypic plasticity undergoing epithelial-mesenchymal transition (EMT). This important phenomenon is associated with down regulation of epithelial marker (e.g., EpCAM) with potential limitations in the applicability of current CTCs enrichment methods. Such observations translated in a number of investigations aimed at improving our capabilities to enumerate and perform molecular characterization of CTCs. Theoretically, the phenotypic analysis of CTCs can represent a “liquid” biopsy of breast tumor that is able to identify a new potential target against the metastatic disease and advanced the development and monitoring of personalized therapies.
doi:10.3978/j.issn.1000-9604.2012.11.03
PMCID: PMC3551332  PMID: 23359451
Circulating tumor cells (CTCs); metastatic breast cancer (MBC); epithelial-mesenchymal transition (EMT); cancer stem cells
14.  Considerations in the development of circulating tumor cell technology for clinical use 
This manuscript summarizes current thinking on the value and promise of evolving circulating tumor cell (CTC) technologies for cancer patient diagnosis, prognosis, and response to therapy, as well as accelerating oncologic drug development. Moving forward requires the application of the classic steps in biomarker development–analytical and clinical validation and clinical qualification for specific contexts of use. To that end, this review describes methods for interactive comparisons of proprietary new technologies, clinical trial designs, a clinical validation qualification strategy, and an approach for effectively carrying out this work through a public-private partnership that includes test developers, drug developers, clinical trialists, the US Food & Drug Administration (FDA) and the US National Cancer Institute (NCI).
doi:10.1186/1479-5876-10-138
PMCID: PMC3478228  PMID: 22747748
Circulating tumor cells; Prognostic biomarker; Predictive biomarker; Analytical validation; Clinical validation; Biomarker qualification; Oncologic drug development
15.  Primary breast cancer patients with high risk clinicopathologic features have high percentages of bone marrow epithelial cells with ALDH activity and CD44+CD24lo cancer stem cell phenotype 
Background
Cancer stem cells (CSCs) are purported to be epithelial tumor cells expressing CD44+CD24lo that exhibit aldehyde dehydrogenase activity (Aldefluor+). We hypothesized that if CSCs are responsible for tumor dissemination, disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore, we assessed the presence of Aldefluor+ epithelial (CD326+CD45dim) cells for the presence of the CD44+CD24lo phenotype in BM of patients with primary breast cancer (PBC).
Methods
BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analyzed for Aldefluor+ epithelial cells with or without CD44+CD24lo expression by flow cytometry. BM aspirates from 3 healthy donors (HD) were subjected to identical processing and analyses and served as controls.
Results
Patients with triple-receptor-negative (TN) tumors had a significantly higher median percentage of CD44+CD24lo CSC within Aldefluor+ epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumors or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44+CD24lo CSC within Aldefluor+ epithelial cell population above the highest level of HD. Furthermore, patients who received NACT were more likely to have percentages of Aldefluor+ epithelial cells greater than the highest level of HD (P=0.004).
Conclusion
The percentage of CD44+CD24lo CSC in the BM is higher in PBC patients with high risk tumor features. The selection or enrichment of Aldefluor+ epithelial cells by NACT may represent an opportunity to target these cells with novel therapies.
doi:10.1016/j.ejca.2011.01.011
PMCID: PMC3116032  PMID: 21334874
16.  Advancements in the Treatment of Metastatic Breast Cancer (MBC): The Role of Ixabepilone 
Journal of Oncology  2012;2012:703858.
Successful management of breast cancer in the metastatic setting is often confounded by resistance to chemotherapeutics, in particular anthracyclines and taxanes. The limited number of effective treatment options for patients with more aggressive biological subtypes, such as triple-negative metastatic breast cancer, is especially concerning. As such, a therapy clinically proven to be effective in this subtype would be of great value. Ixabepilone, a novel synthetic lactam analog of epothilone B, demonstrated better clinical outcomes in metastatic disease, particularly in triple-negative breast cancer. Most recently, studies have shown the activity of ixabepilone in the neoadjuvant setting, suggesting a role for this drug in primary disease. Notably, treating in the neoadjuvant setting might allow clinicians to explore the predictive value of biomarkers and response to treatment, as pharmacogenomic approaches to therapy continue to evolve. In this article, we review the efficacy and safety data of ixabepilone as a monotherapy and as a component of combination therapy for metastatic and primary breast cancer.
doi:10.1155/2012/703858
PMCID: PMC3356906  PMID: 22645612
17.  Mesenchymal stem cells expressing GD2 and CD271 correlate with breast cancer-initiating cells in bone marrow 
Cancer Biology & Therapy  2011;11(9):812-815.
Purpose
The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients.
Results
The percentages of BCIC (Aldefluor+CD326+CD44+CD24−) correlated with the percentages of BM-MSC, either CD45−GD2+CD200+CD271+ (Kedall's τ = 0.684, p = 0.004) or CD45−GD2+CD271+ in the bone marrow (Kedall's τ = 0.464, p = 0.042).
Experimental Design
Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45−) with a “stem-like” phenotype (CD44+CD24low/−, ALDH activity). BM-MSC was defined as CD34−CD45− cells that co-expressed GD2, CD271 and/or CD200 within CD326-depleted BM-MNC.
Conclusions
There was a positive correlation between mesenchymal stem cells expressing GD2 and CD271 and breast cancer-initiating cells in BM of patients with primary breast cancer.
doi:10.4161/cbt.11.9.15178
PMCID: PMC3230296  PMID: 21358274
mesenchymal stem cells; bone marrow; microenvironment; cancer-initiating cells; cancer stem cells
18.  Trastuzumab in Primary Inflammatory Breast Cancer (IBC): High Pathological Response Rates and Improved Outcome 
The breast journal  2010;10.1111/j.1524-4741.2010.00953.x.
Inflammatory breast cancer (IBC) represents a rare but aggressive and lethal form of locally advanced breast cancer (LABC) and frequently with HER-2 neu overexpressed or amplified. We retrospectively identified 16 newly diagnosed HER-2/neu-positive IBC patients who were treated with preoperative trastuzumab. We determined the pathological complete response rate (pCR) when trastuzumab was added to preoperative chemotherapy in patients with HER2/neupositive IBC. Furthermore, we assessed the expression of CXCR4 in metastatic recurrence sites. Ten patients (62.5%) achieved a pCR. Six patients (37.5%) achieved a partial response. Median follow-up of all patients was 24.2 months. Four (25%) patients have experienced a progression, of which three were in the brain. Two-year progression-free survival was 59.4% (95% CI 35–100). High expression of CXCR4 was detected in the brain metastases. We conclude that in spite of high pCR rates among women with HER-2/neu-positive IBC treated with neoadjuvant trastuzumab-based regimens the outcome remains dismal and brain recurrences are frequent. CXCR4 may represent a novel therapeutic target.
doi:10.1111/j.1524-4741.2010.00953.x
PMCID: PMC3097300  PMID: 20626396
CXCR4; inflammatory breast cancer; trastuzumab
19.  Prognostic Value of EMT-Circulating Tumor Cells in Metastatic Breast Cancer Patients Undergoing High-Dose Chemotherapy with Autologous Hematopoietic Stem Cell Transplantation 
Journal of Cancer  2012;3:369-380.
Background: Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer (MBC) patients treated by conventional dose chemotherapy. The aim of this study was to determine the role of CTCs and CTCs undergoing epithelial-mesenchymal transition (EMT) in metastatic breast cancer. We used the platform of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation (AHSCT) to study the CTCs and CTCs with EMT.
Patients and methods: CTCs were enumerated in 21 MBC patients before apheresis and 1 month after AHSCT. CD34-depleted apheresis products were analyzed for CD326+ epithelial and Aldefluor+ cancer stem cells (CSC) by flow cytometry and were depleted of CD45+ cells and assessed for EMT-inducing transcription factors (EMT-TF) by quantitative RT-PCR.
Results: Patients with ≥ 5 CTCs/7.5 mL of peripheral blood 1 month after AHSCT had shorter progression-free survival (PFS) (P=0.02) and overall survival (OS) (P=0.02). Patients with apheresis products containing high percentages of CD326+ epithelial cells or overexpressing EMT-TF had shorter PFS. In multivariate analysis, low percentage of CD326+ epithelial cells and response to HDCT with AHSCT were associated with longer PFS, whereas lower CTCs after AHSCT was associated with longer OS. High CTCs, 1 month after AHSCT correlated with shorter PFS and OS in MBC patients undergoing HDCT and AHSCT, while CTCs with EMT and CSCs phenotype in apheresis products are associated with relapse.
Conclusion: Our data suggest that CTC and CTCs with EMT are prognostic in MBC patients undergoing HDCT followed by AHSCT.
doi:10.7150/jca.5111
PMCID: PMC3471078  PMID: 23074378
metastatic breast cancer; circulating tumor cells; epithelial-mesenchymal transition; high-dose chemotherapy; autologous hematopoietic stem cell transplantation.
20.  GPR30 and estrogen receptor expression: New insights into hormone dependence of inflammatory breast cancer 
Background
GPR30 is a novel G protein-coupled estrogen receptor (ER) associated with metastases in breast cancer (BC) and poor survival in endometrial and ovarian tumors. The association of GPR30 expression with inflammatory breast cancer (IBC), an aggressive and commonly hormone-independent form of BC, has not been studied.
Methods
GPR30, ER, progesterone receptor (PR), epidermal growth factor receptor (EGFR) and HER-2 expression were assessed by immunohistochemistry (and FISH for HER-2) in 88 primary IBCs. GPR30 expression was correlated with patient overall survival (OS), disease free survival (DFS), pathologic variables, and other biomarkers.
Results
GPR30 expression was found in 69% of IBC cases. ER, PR, HER-2 and EGFR were found in 43%, 35%, 39%, and 34% of IBC cases, respectively. GPR30 expression correlated inversely with ER expression (P=0.02). Co-expression of ER and GPR30 was found in 24% of IBC samples; 19% expressed only ER and 46% expressed only GPR30. Univariate analysis showed no association between GPR30 expression and OS or DFS. However, co-expression of ER and GPR30 was associated with improved OS (P<0.03) and marginally with DFS (P<0.06); the absence of both ER and GPR30 was associated with worse OS and DFS (P=0.03 for both). Multivariate analysis identified ER as an independent prognostic factor of OS (P=0.008) and DFS (P=0.02).
Conclusions
The majority of IBC tumors are GPR30-positive, suggesting that estrogen signaling may be active in ER-negative IBC patients. These findings suggest potential new therapeutic targets for IBC such as novel endocrine agents or direct modulation of GPR30.
doi:10.1007/s10549-009-0631-7
PMCID: PMC2904403  PMID: 19902352
Inflammatory breast cancer; GPR30; Hormone receptors; growth factor receptors; overall survival; disease-free survival
21.  Dyadic Coping in Metastatic Breast Cancer 
Objective
Couples facing metastatic breast cancer (MBC) must learn to cope with stressors that can affect both partners' quality of life as well as the quality of their relationship. Common dyadic coping involves taking a “we” approach, whereby partners work together to maintain their relationship while jointly managing their shared stress. This study prospectively evaluated whether common dyadic coping was associated with less cancer-related distress and greater dyadic adjustment for female MBC patients and their male partners.
Design
Couples (N = 191) completed surveys at the start of treatment for MBC (baseline), and 3 and 6 months later.
Main Outcome Measures
Cancer-related distress was assessed with the Impact of Events Scale; dyadic adjustment was assessed using the short-form of the Dyadic Adjustment Scale.
Results
Multilevel models using the couple as the unit of analysis showed that the effects of common positive dyadic coping on cancer-related distress significantly differed for patients and their partners. Whereas partners experienced slightly lower levels of distress, patients experienced slightly higher levels of distress. Although patients and partners who used more common negative dyadic coping experienced significantly greater distress at all times, the association was stronger for patients. Finally, using more common positive dyadic coping and less common negative dyadic coping was mutually beneficial for patients and partners in terms of greater dyadic adjustment.
Conclusion
Our findings underscore the importance of couples working together to manage the stress associated with MBC. Future research may benefit from greater focus on the interactions between patients and their partners to address ways that couples can adaptively cope together.
doi:10.1037/a0018165
PMCID: PMC3138118  PMID: 20230090
metastases; breast cancer; couples; dyadic coping; distress; dyadic adjustment
22.  Circulating tumor cells as prognostic and predictive markers in metastatic breast cancer patients receiving first-line systemic treatment 
Introduction
Circulating tumor cells (CTCs) represent an independent predictor of outcome in patients with metastatic breast cancer (MBC). We assessed the prognostic impact of CTCs according to different first-line systemic treatments, and explored their potential predictive value in MBC patients.
Methods
We retrospectively evaluated 235 newly diagnosed MBC patients, treated at the University of Texas MD Anderson Cancer Center. All patients had a baseline CTC assessment performed with CellSearch®. Progression-free survival and overall survival were compared with the log-rank test between groups, according to CTC count (< 5 vs. ≥ 5) and type of systemic therapy. We further explored the predictive value of baseline CTCs in patients receiving different treatments.
Results
At a median follow-up of 18 months, the CTC count was confirmed to be a robust prognostic marker in the overall population (median progression-free survival 12.0 and 7.0 months for patients with CTC < 5 and ≥ 5, respectively; P < 0.001). Conversely, in patients with human epidermal growth factor receptor-2-overexpressed/amplified tumors receiving trastuzumab or lapatinib, the baseline CTC count was not prognostic (median progression-free survival 14.5 months for patients with CTC < 5 and 16.1 months for those with CTC ≥ 5; P = 0.947). Furthermore, in patients with human epidermal growth factor receptor-2 normal tumors, a baseline CTC count ≥ 5 identified subjects who derived benefit from more aggressive treatments, including combination chemotherapy and chemotherapy plus bevacizumab.
Conclusions
This analysis suggests that the prognostic information provided by CTC count may be useful in patient stratifications and therapeutic selection, particularly in the group with positive CTCs, in which various therapeutic choices may procure differential palliative benefit.
doi:10.1186/bcr2907
PMCID: PMC3218956  PMID: 21699723
23.  Differential Regulation of The Aggressive Phenotype of Inflammatory Breast Cancer Cells By Prostanoid Receptors EP3 and EP4 
Cancer  2010;116(11 Suppl):2806-2814.
Background
Although inflammatory breast cancer (IBC) is recognized as the most lethal variant of locally advanced breast cancer, there are few molecular signatures of IBC that have been identified that can be used as targets to develop therapeutics to effectively inhibit the aggressive phenotype displayed by IBC tumors.
Methods
Real time PCR, western blotting, modified Boyden chamber invasion assays, vasculogenic mimicry assays, and gelatin zymography were used in the present studies. Agonists and antagonists of EP3 and EP4 and EP4 short hairpin RNA knockdown approaches were used as tools to assess the role of prostanoid receptors EP3 and EP4 in regulation of specific biological activities of IBC breast cancer cells.
Results
The present studies revealed that IBC breast cancer cell lines, SUM149 and SUM190, express high levels of Cox-2 mRNA and protein, produce abundant levels of PGE2 and produce both EP3 and EP4 receptor proteins. Studies using the EP4 antagonist GW627368X and shRNA molecular knockdown approaches revealed a role for EP4 in regulating invasion of IBC cells. EP3 but not EP4 regulates the ability of SUM149 cells to undergo vasculogenic mimicry (VM), which is the ability to form capillary-like structures, a characteristic exhibited by very aggressive tumor types. Inhibition of VM by sulprostone was associated with an inhibition of matrix metalloprotease-2 (MMP-2) enzyme activity.
Conclusions
The prostanoid receptors EP3 and EP4 differentially regulate activities exhibited by IBC cells that have been associated with the aggressive phenotype of this lethal variant of breast cancer. While EP4 regulates invasion, EP3 regulates VM and the associated increased MMP-2 enzyme activity.
doi:10.1002/cncr.25167
PMCID: PMC2889924  PMID: 20503412
inflammatory breast cancer; cyclooxygenase-2; prostanoid receptor; EP3; EP4; PGE2; invasion; vasculogenic mimicry; MMP-2
24.  Circulating tumor cells in breast cancer: A tool whose time has come of age 
BMC Medicine  2011;9:43.
Circulating tumor cells (CTCs) are isolated tumor cells disseminated from the site of disease in metastatic and/or primary cancers, including breast cancer, that can be identified and measured in the peripheral blood of patients. As recent technical advances have rendered it easier to reproducibly and repeatedly sample this population of cells with a high degree of accuracy, these cells represent an attractive surrogate marker of the site of disease.
Currently, CTCs are being integrated into clinical trial design as a surrogate for phenotypic and genotypic markers in correlation with development of molecularly targeted therapies. As CTCs play a crucial role in tumor dissemination, translational research is implicating CTCs in several biological processes, including epithelial to mesenchymal transition. In this mini-review, we review CTCs in metastatic breast cancer, and discuss their clinical utility for assessing prognosis and monitoring response to therapy. We will also introduce their utility in pharmacodynamic monitoring for rational selection of molecularly targeted therapies and briefly address how they can help elucidate the biology of cancer metastasis.
doi:10.1186/1741-7015-9-43
PMCID: PMC3107794  PMID: 21510857
25.  Differential radiosensitizing effect of valproic acid in differentiation versus self-renewal promoting culture conditions 
Purpose
It has been shown that valproic acid (VA) enhances proliferation and self-renewal of normal hematopoietic stem cells and that breast cancer stem/progenitor cells can be resistant to radiation. Based on these data, we hypothesized that VA would fail to radiosenstize breast cancer stem/progenitor cells grown to 3D mammospheres.
Materials and Methods
We used the MCF7 breast cancer cell line grown under stem cell promoting culture conditions (3D, mammosphere) and standard non-stem cell monolayer culture conditions (2D) to examine the effect of pretreatment with VA on radiation sensitivity in clonogenic survival assays and on the expression of embryonic stem cell transcription factors.
Results
3D cultured MCF-7 cells express higher levels of Oct4, Nanog and Sox2. 3D passage enriched self-renewal and increased radioresistance in 3D mammosphere formation assays. VA radiosensitized adherent cells but radioprotected 3D cells in single fraction clonogenic assays. Moreover, fractionated radiation sensitized VA-treated adherent MCF7 cells, but did not have a significant effect in VA-treated single cells grown to mammospheres.
Conclusion
We conclude that VA may preferentially radiosensitize differentiated cells over those expressing stem cell surrogates, and that stem cell promoting culture is a useful tool for in vitro evaluation of novel cancer therapeutic agents and radiosensitizers.
doi:10.1016/j.ijrobp.2009.09.052
PMCID: PMC2892870  PMID: 20159363
Valproic Acid; Radiation; Breast Cancer Stem/Progenitors cells; Mammospheres; 3D culture

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