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
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
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.
Circulating tumor cells (CTCs); metastatic breast cancer (MBC); epithelial-mesenchymal transition (EMT); cancer stem cells
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).
Circulating tumor cells; Prognostic biomarker; Predictive biomarker; Analytical validation; Clinical validation; Biomarker qualification; Oncologic drug development
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.
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.
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
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.
CXCR4; inflammatory breast cancer; trastuzumab
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.
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.
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.
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).
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.
Inflammatory breast cancer; GPR30; Hormone receptors; growth factor receptors; overall survival; disease-free survival
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.
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.
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.
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.
metastases; breast cancer; couples; dyadic coping; distress; dyadic adjustment
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.
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.
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.
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.
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.
inflammatory breast cancer; cyclooxygenase-2; prostanoid receptor; EP3; EP4; PGE2; invasion; vasculogenic mimicry; MMP-2
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.
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.
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.
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.
Valproic Acid; Radiation; Breast Cancer Stem/Progenitors cells; Mammospheres; 3D culture
The aims of this study were to determine outcomes for patients with inflammatory breast cancer (IBC) treated with multimodality therapy, to identify factors associated with locoregional recurrence, and to determine which patients may benefit from radiation dose escalation.
Methods and Materials
We retrospectively reviewed 256 consecutive patients with nonmetastatic IBC treated at our institution between 1977 and 2004.
The 192 patients who were able to complete the planned course of chemotherapy, mastectomy, and postmastectomy radiation had significantly better outcomes than the 64 patients who did not. The respective 5-year outcome rates were: locoregional control (84% vs. 51%), distant metastasis–free survival (47% vs. 20%), and overall survival (51% vs. 24%) (p < 0.0001 for all comparisons). Univariate factors significantly associated with locoregional control in the patients who completed plan treatment were response to neoadjuvant chemotherapy, surgical margin status, number of involved lymph nodes, and use of taxanes. Increasing the total chest-wall dose of postmastectomy radiation from 60 Gy to 66 Gy significantly improved locoregional control for patients who experienced less than a partial response to chemotherapy, patients with positive, close, or unknown margins, and patients <45 years of age.
Patients with IBC who are able to complete treatment with chemotherapy, mastectomy, and postmastectomy radiation have a high probability of locoregional control. Escalation of postmastectomy radiation dose to 66 Gy appears to benefit patients with disease that responds poorly to chemotherapy, those with positive, close, or unknown margin status, and those <45 years of age.
Inflammatory breast cancer; Radiation dose; Locoregional therapy
Inflammatory breast cancer (IBC) is a rare but aggressive type of advanced breast cancer. Epidermal growth factor receptor (EGFR) expression is an independent poor prognostic factor in IBC. The purpose of this study was to determine the impact on IBC tumorigenicity and metastasis of blocking the EGFR pathway.
IBC cell lines, which express high level of EGFR, were treated with EGFR siRNA and with the EGFR tyrosine kinase inhibitor erlotinib. The role of EGFR in IBC cell proliferation, motility, invasiveness, and change of the expression levels of epithelial-mesenchymal transition (EMT) markers were examined. The role of mitogen-activated protein kinase (ERK) 1/2 in erlotinib activity was also studied. The activity of erlotinib in tumor growth and metastasis was examined in an orthotopic xenograft model of IBC.
Erlotinib inhibited proliferation and anchorage-independent growth of IBC cells, and this inhibition was ERK dependent. Erlotinib inhibited cell motility and invasiveness and reversed the mesenchymal phenotype of IBC cells to epithelial phenotype in three-dimensional culture. Erlotinib dramatically inhibited IBC tumor growth in a xenograft model. Interestingly, erlotinib inhibited spontaneous lung metastasis, even at a low dose that had no significant impact on primary tumor growth. These erlotinib-treated tumors were converted to epithelial phenotype from mesenchymal phenotype.
The EGFR pathway is involved in tumor growth and metastasis of IBC. Targeting EGFR through the ERK pathway may represent an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in EGFR-expressing IBC.
inflammatory breast cancer; tyrosine kinase inhibitor; EGFR; erlotinib; metastasis
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.
Early detection of cellular events is important to predict the outcome of the patients. This study was aimed to use 99mTc EC-annexin V to image tumor cells undergoing apoptosis.
In ten patients with breast cancer, scintigraphic images and dosimetric estimates were obtained after administering 99mTc EC-annexin V.
Nine of the 10 cases showed detectable 99mTc EC-annexin V uptake in tumor. Higher values of T/N ratios are associated with patient after treatment.
Apoptosiscan be quantified using 99mTc EC-annexin V.