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.
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.
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
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
Matrix metalloproteinases (MMPs) are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. The aim of this study was to assess correlation between CTCs and tumor MMP1 in BC.
Study included 149 primary BC patients treated by surgery from March 2012 to March 2013. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSepTM selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of healthy donors (n = 60) were considered as CTC positive. Expression of MMP1 in surgical specimens was evaluated by immunohistochemistry.
CTCs were detected in 24.2% patients. CTCs exhibiting only epithelial markers were present in 8.7% patients, whereas CTCs with epithelial-mesenchymal transition (EMT) markers (CTC_EMT) were observed in 13.4% of patients and CTCs co-expressing both markers were detected in 2.0% patients. Patients with CTC_EMT in peripheral blood had significantly increased expression of MMP1 in tumor cells (p = 0.02) and tumor associated stroma (p = 0.05) than those of patients without CTC_EMT. In multivariate analysis, CTC_EMT and tumor grade were independently associated with MMP1 expression in cancer cells, while CTC_EMT and Ki67 were independently associated with MMP1 expression in cancer associated stroma.
Our data suggest link between MMP1 and CTCs with EMT phenotype and support role of MMPs and EMT in tumor dissemination.
Circulating tumor cells; Matrix-metaloproteinase 1; Primary breast cancer
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
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
Altered serum microRNA (miRNA) levels may be correlated with a dysregulated expression pattern in parental tumor tissue and reflect the clinical evolution of disease. The overexpression of miR-21, miR-10b, and miR-19a is associated with the acquisition of malignant characteristics (increased tumor cell proliferation, migration, invasion, dissemination, and metastasis); thus, we determined their utility as serum biomarkers for aggressive breast cancer (HER2-overexpressed or -amplified [HER2+] and inflammatory breast cancer [IBC]).
In this prospective study, we measured miR-21, miR-10b, and miR-19a levels using quantitative reverse transcriptase-polymerase chain reaction in the serum of 113 breast cancer patients and determined their association with clinicopathologic factors and clinical outcome. Thirty healthy donors with no history of cancer were enrolled as controls.
Patients with non-metastatic HER2+ breast cancer had higher serum miR-21 median levels than patients with non-metastatic HER2− disease (p = 0.044); whereas patients with metastatic HER2+ breast cancer had higher serum miR-10b median levels than patients with metastatic HER2− disease (p = 0.0004). There were no significant differences in serum miR-19a median levels between HER2+ and HER2− groups, regardless of the presence of metastases. High serum miR-19a levels were associated with IBC (p = 0.039). Patients with metastatic IBC had significantly higher serum miR-19a median levels than patients with metastatic non-IBC (p = 0.019). Finally, high serum miR-19a levels were associated with longer progression-free survival time (10.3 vs. 3.2 months; p = 0.022) and longer overall survival time (median not reached vs. 11.2 months; p = 0.003) in patients with metastatic HER2+ IBC.
High levels of miR-21 and miR-10b were present in the serum of patients with non-metastatic and metastatic HER2+ breast cancer, respectively. High levels of serum miR-19a may represent a biomarker for IBC that is predictive for favorable clinical outcome in patients with metastatic HER2+ IBC.
Because of its high rate of metastasis, inflammatory breast cancer (IBC) has a poor prognosis compared with non-inflammatory types of breast cancer (non-IBC). In a recent study, Lehmann and colleagues identified seven subtypes of triple-negative breast cancer (TNBC). We hypothesized that the distribution of TNBC subtypes differs between TN-IBC and TN-non-IBC. We determined the subtypes and compared clinical outcomes by subtype in TN-IBC and TN-non-IBC patients.
We determined TNBC subtypes in a TNBC cohort from the World IBC Consortium for which IBC status was known (39 cases of TN-IBC; 49 cases of TN-non-IBC). We then determined the associations between TNBC subtypes and IBC status and compared clinical outcomes between TNBC subtypes.
We found the seven subtypes exist in both TN-IBC and TN-non-IBC. We found no association between TNBC subtype and IBC status (P = 0.47). TNBC subtype did not predict recurrence-free survival. IBC status was not a significant predictor of recurrence-free or overall survival in the TNBC cohort.
Our data show that, like TN-non-IBC, TN-IBC is a heterogeneous disease. Although clinical characteristics differ significantly between IBC and non-IBC, no unique IBC-specific TNBC subtypes were identified by mRNA gene-expression profiles of the tumor. Studies are needed to identify the subtle molecular or microenvironmental differences that contribute to the differing clinical behaviors between TN-IBC and TN-non-IBC.
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).
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.
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).
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.
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.
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).
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.
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.
mesenchymal stem cells; bone marrow; microenvironment; cancer-initiating cells; cancer stem cells
To determine the effects of thalidomide and placebo on anorexia-cachexia and its related symptoms, body composition, resting metabolic rate, and serum cytokines and their receptors in patients with advanced cancer.
Included in the study were patients with advanced cancer with weight loss greater than 5% in 6 months and who reported anorexia, fatigue, and one of the following: anxiety, depression, or sleep disturbances. Patients on chemotherapy within 2 weeks prior or during the study were excluded from the study. Patients were randomly assigned to either 100 mg thalidomide or placebo once a day for 14 days. The Edmonton Symptom Assessment Scale (ESAS), Functional Assessment of Anorexia/Cachexia Therapy (FAACT), Functional Assessment of Cancer Illness Therapy (FACIT-F), Hospital Anxiety Depression Scale (HADS) Pittsburgh Sleep Quality Index (PSQI) were utilized, and in addition body composition, Resting Energy Expenditure (REE), and serum cytokine levels were assessed.
Of the 31 patients entered in the study, 15 were assigned to the thalidomide group and 16 to the placebo group. However only 21/31 patients were able to complete the study. Compared with their baseline values, both the thalidomide and the placebo groups showed significant reduction in cytokines. Tumor necrosis factor (TNF)-α (p=0.04) and its receptors TNFR1 (p=0.04), TNFR2 (p=0.04), and interleukin (IL)-8 (p=0.04) were statistically significant in the thalidomide group. In the placebo group, TNF-α (p=0.008), TNFR1 (p=0.005), TNFR2 (p=0.005), IL-RA (p=0.005), IL-6 (p=0.005), and IL-8 (p=0.005) were statistically significant. However, improvement in these symptoms and cytokine levels were not significantly different in the thalidomide group compared with the placebo group. None of the patients withdrew from the study because of toxicity of either thalidomide or placebo.
Based on the poor accrual rate and attrition observed in this study, it is important that future research on thalidomide as a treatment for cancer-related anorexia-cachexia symptoms (ACS) in patients with advanced cancer use less stringent entry criteria and less exhaustive outcome measures.
Although preliminary reports suggest that ALK gene amplification may occur in inflammatory breast cancer (IBC), data are limited. We performed a comprehensive investigation of the status of ALK gene in IBC.
We used core biopsy (CB) samples from 30 IBC patients for immunohistochemistry (IHC), 25 of these samples for fluorescence in situ hybridization (FISH) of ALK gene rearrangement, 8 for chromosome 2 aneusomy, and 20 microdissected frozen CBs for array comparative genomic hybridization (CGH) and mRNA analysis.
All 30 samples were negative for ALK protein expression by IHC. FISH analysis showed no EML4-ALK gene rearrangement in any samples, although 16 of the 25 samples (64%) contained 3 to 4 extra copies of the ALK gene, and chromosome 2 aneusomy was found in 7 of 8 samples that had extra copies of the ALK gene. Only 3 of the 20 samples showed evidence of mild ALK gene amplification by array CGH. mRNA analysis revealed that mRNA expression of ALK was not significantly higher in these samples compared with samples that showed no evidence of ALK gene amplification in CGH analysis, nor was mRNA expression of ALK significantly different in tumor compared with 5 normal breast samples (P > 0.05, t test).
Our comprehensive evaluation suggests that ALK gene rearrangement did not occur in the IBC patients studied. The significance of our finding of mildly increased copy numbers of the ALK gene resulting from chromosome 2 aneusomy rather than mild amplification of the ALK gene requires further investigation.
WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.
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.
metastatic breast cancer; circulating tumor cells; epithelial-mesenchymal transition; high-dose chemotherapy; autologous hematopoietic stem cell transplantation.
Youth infected with HIV at birth often have sleep disturbances, neurocognitive deficits, and abnormal psychosocial function which are associated with and possibly resulted from elevated blood cytokine levels that may lead to a decreased quality of life. To identify molecular pathways that might be associated with these disorders, we evaluated 38 HIV-infected and 35 uninfected subjects over 18-months for intracellular cytokine levels, sleep patterns and duration of sleep, and neurodevelopmental abilities. HIV infection was significantly associated with alterations of intracellular pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12), sleep factors (total time asleep and daytime sleep patterns), and neurocognitive factors (parent and patient reported problems with socio-emotional, behavioral, and executive functions; working memory-mental fatigue; verbal memory; and sustained concentration and vigilance. By better defining the relationships between HIV infection, sleep disturbances, and poor psychosocial behavior and neurocognition, it may be possible to provide targeted pharmacologic and procedural interventions to improve these debilitating conditions.
pediatric HIV infection; intracellular cytokines; sleep behavior; neurodevelopment; neurocognition; path analysis
This article reviews the current status of multidisciplinary care for patients with inflammatory breast cancer. Future avenues of research to advance the care of patients with this disease are also presented.
We review the current status of multidisciplinary care for patients with inflammatory breast cancer (IBC) and discuss what further research is needed to advance the care of patients with this disease.
We performed a comprehensive review of the English-language literature on IBC through computerized literature searches.
Significant advances in imaging, including digital mammography, high-resolution ultrasonography with Doppler capabilities, magnetic resonance imaging, and positron emission tomography–computed tomography, have improved the diagnosis and staging of IBC. There are currently no established molecular criteria for distinguishing IBC from noninflammatory breast cancer. Such criteria would be helpful for the diagnosis and development of novel targeted therapies. Combinations of neoadjuvant systemic chemotherapy, surgery, and radiation therapy have led to an improved prognosis; however, the overall 5-year survival rate for patients with IBC remains very low (∼30%). Sentinel lymph node biopsy and skin-sparing mastectomy are not recommended for patients with IBC.
Optimal management of IBC requires close coordination among medical, surgical, and radiation oncologists, as well as radiologists and pathologists. There is a need to identify molecular changes that define the pathogenesis of IBC to enable eradication of IBC with the use of IBC-specific targeted therapies.
Inflammatory breast cancer; Systemic therapy; Targeted therapy
To investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery.
Patients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998–2000. Real-time RT-PCR quantification of a DTC multimarker panel consisting of cytokeratin 19, mammaglobin A and TWIST1 mRNA was performed in BM samples obtained from 154 patients three weeks (BM2) and/or six months after surgery (BM3). The results were compared to previously published data from pre-operative BM analyses for the same patients.
DTCs were identified in post-operative BM samples (BM2 and/or BM3) from 23 (15%) of the 154 patients investigated. During a median follow-up of 98 months, 10 (44%) of these patients experienced systemic relapse as compared to 16 (12%) of 131 DTC-negative patients. Kaplan-Meier estimates of systemic recurrence-free- and breast-cancer specific survival demonstrated significantly shorter survival for patients with persistent DTCs in BM after surgery (p≤0.001). By multivariate Cox regression analyses, persistent DTCs after surgery was an independent predictor of both systemic recurrence-free- (HR = 5.4, p < 0.001) and breast-cancer specific survival (HR = 5.3, p < 0.001). Furthermore, the prognostic value of DTCs in BM was similar for pre- and post surgery samples. However, patients with DTCs both before and after surgery (BM1 and BM2/3) had a particularly poor prognosis (systemic recurrence-free survival: HR = 7.2, p < 0.0001 and breast-cancer specific survival: HR = 8.0, p < 0.0001).
Detection of persistent DTCs in BM samples obtained after surgery identified non-metastatic breast cancer patients at high risk for systemic relapse, and with reduced breast-cancer specific survival. Furthermore, patients with positive DTC status both before and after surgery had a particularly poor prognosis.
Breast cancer; Minimal residual disease; Multimarker real-time PCR; Bone marrow; DTC; prognosis
Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.
Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.
Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).
DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).
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.
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.
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.
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.
Purpose: Sodium stibogluconate (SSG), a small molecule inhibitor of protein tyrosine phosphatases, combined with IFN-alpha-2b (IFN-α) inhibited solid tumor cell line growth in vitro. We conducted a phase I clinical trial with SSG plus IFN-α in advanced cancer patients to assess tolerance, maximum tolerated dose (MTD) and immune system effects.
Experimental Design: SSG was administered intravenously alone for five days of week 1, cycle 1 (21 days per cycle) and together with IFN-α 2b s (3 million units sc TIW) in week 2, and after a rest during week 3, on a 2-week on/1-week off cycle. SSG dose levels were 400, 600, 900, 1125, and 1350 mg/m2.
Results: Twenty-four patients were studied. Common toxicities included asymptomatic elevated serum lipase, thrombocytopenia, fatigue, fever, chills and anemia. The dose-limiting toxicities (DLT) were hypokalemia, thrombocytopenia, fatigue, pancreatitis and skin rash. The MTD was 900 mg/m2 SSG and IFN-α, 3 million units TIW. At this dose, patients had a significantly lower number of regulatory T cells (TR Cells) (p = 0.012), myeloid dendritic cells (mDC) (p = 0.028); higher percentage of natural killer (NK) cells that synthesized perforin (p = 0.046) and of plasmacytoid dendritic cells (pDC) that secreted IFN-α (p = 0.018) in response to activation through toll-like receptor (TLR) 7 and TLR 8 by CL097, the highly water-soluble derivative of the imidazoquinoline compound R848.
Conclusions: SSG in combination with IFN-α 2b was well tolerated and augmented cellular immune parameters.
sodium stibogluconate; phase 1; interferon alpha; immunity; cancer
Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.
We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.
We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.
MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.
Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced by serial xenograft passages through transplantation. Herein we fully characterize cancer stem cell-like features in 293T human embryonic kidney cells.
293T cells can be readily cultured and passaged as spheres in serum-free stem cell promoting culture conditions. Cells cultured in vitro as three-dimensional spheres (3D) were shown to contain higher ALDH1 and CD44+/CD24- population compared to monolayer cells. These cells were also resistant to radiation and upregulate stem cell survival signaling including β-catenin, Notch1 and Survivin in response to radiation. Moreover, 3D spheres generated from the 293T cells have increased expression of mesenchymal genes including vimentin, n-cadherin, zeb1, snail and slug as well as pro-metastatic genes RhoC, Tenascin C and MTA1. In addition, microRNAs implicated in self-renewal and metastases were markedly reduced in 3D spheres.
293T cells exhibit a cancer stem cell-like phenotype when cultured as 3D spheres and represent an important research tool for studying the molecular and biological mechanisms of cancer stem cells and for testing and developing novel targets for cancer therapy.