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.
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.
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.
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.
We previously reported survival trends among patients with inflammatory breast cancer (IBC) over a 30-year-period before 2005. Here we evaluated survival outcomes for women with IBC diagnosed before or after October 2006, in the era of HER2-directed therapy and after opening a dedicated multidisciplinary IBC clinic.
We retrospectively identified and reviewed 260 patients with newly diagnosed IBC without distant metastasis, 168 treated before October 2006 and 92 treated afterward. Most patients received anthracycline and taxane-based neoadjuvant chemotherapy, mastectomy, and postmastectomy radiation. Survival outcomes were compared between the two groups.
Median follow-up time was 29 months for the entire cohort (39 mo and 24 mo for patients treated before and after October 2006). Patients treated more recently were more likely to have received neoadjuvant HER2-directed therapy for HER2-positive tumors (100% vs. 54%, P<0.001). No differences were found in receipt of hormone therapy. Three-year overall survival (OS) rates were 63% for those treated before and 82% for those treated after October 2006 (log-rank P=0.02). Univariate Cox analysis demonstrated better OS among patients treated after October 2006 than among those treated beforehand (hazard ratio [HR] 0.5, 95% confidence Interval [CI] 0.34-0.94); a trend toward improved survival was noted in the multivariate analysis (HR=0.47, 95% CI 0.19-1.16, P=0.10). Significant factors in the multivariate model included HER2-directed therapy (HR=0.38, 95% CI 0.17-0.84, P=0.02) and estrogen receptor positivity (HR=0.32, 95% CI 0.14-0.74, P=0.01).
Survival improved in the context of the IBC clinic and prompt initiation of neoadjuvant Her-2 directed therapeutics.
Inflammatory breast cancer; survival; multidisciplinary clinic; targeted therapy
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.
Breast cancer (BC); circulating tumor cells (CTCs); liquid biopsy; precision medicine
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.
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.
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.
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.
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.
Farnesyl transferase inhibitor; Tipifarnib; Ras; Breast cancer; Neoadjuvant chemotherapy; Inflammatory breast cancer
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.
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.
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).
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.
In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with RPKM values (Reads Per Kilobase per Million mapped reads1) for ERBB2 (14.4, 400 and 300 for SUM149, SUM 190 and SKBR3 respectively and for EGFR 60.1, not detected and 1.4 for the same 3 cell lines. We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g. 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs. total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used the following bioinformatics sites, GeneGo, PathwayCommons and NCI receptor signaling networks to identify pathways which contained the four main oncogenes, had good coverage in the transcriptomic and proteomic data sets as well as significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling, caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings: branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 sub-pathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.
We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 ‘missing’ proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of ‘missing’ proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV and single nucleotide variant (SNV) databases and the construction of websites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer associated genes we have focused the clustering of cancer associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of co-expression through coordinated regulation.
Chromosome-Centric Human Proteome Project; Chromosome 17 Parts List; ERBB2; Oncogene
The goal of this study was to determine whether gene expression differences exist between inflammatory breast cancers (IBC) and stage-matched non-IBC patiens stratified by hormone receptor and HER2 status.
We used Affymetrix GeneChips to analyze 82 tumor samples (25 T4d, 25 patients and 57, T4a-c patients) of newly diagnosed breast cancers. Genes that were differentially expressed between the IBC and non-IBC specimens were identified using the t-test, and differential expression of gene sets was assessed using gene set analysis. Three distinct clinical subtypes of IBC and non-IBC were compared: ER-positive/HER2-normal, HER2-amplified, and ER-negative/HER2-normal.
When we compared expression data from all IBC with all non-IBC, we found no significant differences after adjusting for multiple testing. When IBC and non-IBC tumors were compared by clinical subtype, however, significant differences emerged. Complement and immune system-related pathways were overexpressed in ER-positive/HER2-normal IBC. Protein translation and mTOR signaling were overexpressed in HER2-amplified IBC. Apoptosis-, neural-, and lipid metabolism-related pathways were overexpressed in ER-negative/HER2-normal IBC compared with non-IBC of the same receptor phenotype. In this stage-matched case-control study, the survival curves of patients with IBC and non-IBC were similar for all three clinical subtypes.
IBC tumors can be divided into molecular and clinical subtypes similar to those of non-IBC. Clinical subtypes of IBC show molecular differences compared with similar subtypes of non-IBC.
Inflammatory breast cancer; receptor subtypes; cDNA microarray; Gene set analysis
Epidermal growth factor receptor (EGFR) overexpression has been associated with prognostic and predictive value in inflammatory breast cancer (IBC). Epidermal growth factor receptor 2 (HER2) overexpression is observed at a higher rate in IBC compared with noninflammatory breast cancer. Current clinically available anti-HER2 therapies are effective only in patients with HER2 amplified breast cancer, including IBC. AZD8931 is a novel small-molecule equipotent inhibitor of EGFR, HER2, and HER3 signaling. In this study, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel using preclinical models of EGFR-overexpressed and HER2 non-amplified IBC cells.
Two IBC cell lines SUM149 and FC-IBC-02 derived from pleural effusion of an IBC patient were used in this study. Cell growth and apoptotic cell death were examined in vitro. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. AZD8931 was given by daily oral gavage at doses of 25 mg/kg, 5 days/week for 4 weeks. Paclitaxel was subcutaneously injected twice weekly.
AZD8931 significantly suppressed cell growth of IBC cells and induced apoptosis of human IBC cells in vitro. Significantly, we showed that AZD8931 monotherapy inhibited xenograft growth and the combination of paclitaxel + AZD8931 was demonstrably more effective than paclitaxel or AZD8931 alone treatment at delaying tumor growth in vivo in orthotopic IBC models.
AZD8931 single agent and in combination with paclitaxel demonstrated signal inhibition and antitumor activity in EGFR-overexpressed and HER2 non-amplified IBC models. These results suggest that AZD8931 may provide a novel therapeutic strategy for the treatment of IBC patients with HER2 non-amplified tumors.
Inflammatory breast cancer; EGFR; HER2; HER3; AZD8931; Paclitaxel
Inflammatory breast cancer (IBC) is the most aggressive and deadly form of breast cancer. Disease-specific research and conferences have been organized since 2008 with the intent to bring together experts in various disciplines. This report focus on the Third International IBC Conference held in Philadelphia on December 2012.
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.
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.
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.
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.
Inflammatory breast cancer; CGH; array; gene signature
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.
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.
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.
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.
circulating tumor cells; first-line; metastatic breast cancer; nomogram; survival
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.
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.
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.
Phase I; everolimus; docetaxel
Purpose: We hypothesized that aldehyde dehydrogenase 1 (ALDH1) staining in breast cancer tumor cells might be a simple surrogate for the presence of circulating tumor cells (CTCs) or disseminated tumor cells (DTCs).
Experimental Design: Whole tissue primary tumor sections from 121 patients enrolled in a clinical trial assessing CTCs and DTCs at the time of surgery were stained for ALDH1 and scored by a dedicated breast pathologist blinded to outcome. Clinical data was extracted and staining was correlated to clinical variables and outcome by Fisher's exact test, the Log rank test and Cox proportional hazards regression analysis respectively. P < 0.05 was considered significant.
Results: ALDH1 staining in tumor cells was present in 12% of cases (15/121). In univariate analysis, ALDH1 tumor staining predicted worse overall survival (71% vs. 91% at 5 years P = 0.0074) and was an independent predictor on multivariable analysis of worse overall survival, (HR 4.93) after adjusting for stage, ER, grade, LVI, age and neoadjuvant chemotherapy (P = 0.04).
ALDH1 was significantly associated with estrogen receptor (ER) negative (P value = 0.029) primary tumors but not the presence of CTCs or DTCs by multivariate logistic regression. Positive ALDH staining in non-tumor cells of any pattern or morphology was common but did not correlate with CTCs or DTCs, other clinical variables, or outcome.
Conclusion: ALDH1 tumor staining was associated with ER -negative breast cancer and was an independent predictor of OS. However, it did not correlate to putative cancer stem cell surrogates CTCs and/or DTCs.
ALDH1; breast cancer; cancer stem cells; circulating tumor cells; disseminated tumor cells; micrometastatic disease.
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.
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.
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.
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.
Circulating tumor cell; Breast neoplasm; HER2; Metastasis
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).
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.
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.
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.
Breast neoplasm; HER2; Metastasis; Serum HER2; Trastuzumab
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.
circulating tumor cells; epithelial to mesenchymal transition; stem cells; HER2; CD133; metastatic breast cancer
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.
Inflammatory breast cancer; Anaplastic lymphoma kinase; Reverse phase protein arrays; Crizotinib
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.
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.
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.
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.
Inflammatory breast cancer; EZH2; Cancer stem cell; Tumor spheroid formation
Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-013-2600-4) contains supplementary material, which is available to authorized users.
CD44; NOTCH3; MYC; ATAD2; ALK; FAK1/PTK2
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.
circulating tumor cells; epithelial-mesenchymal transition; primary breast cancer; neoadjuvant therapy
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.
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.
Lapatinib; Pazopanib; Inflammatory breast cancer; HER2-positive breast cancer
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.
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.
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.
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).
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.
Inflammatory breast cancer; Estrogen receptor; Progesterone receptor; HER-2; Molecular subtypes