Cells In Breast Cancer Patients Research Articles

Abstract P1-15-02: Gene expression profiling of doxorubicin cardiotoxicity in peripheral blood cells of breast cancer patients

Abstract Background: Doxorubicin (DOX), a widely used anti-cancer drug for treatment of breast cancer is known for its cardiotoxicity. DOX cardiotoxicity is cumulative-dose-dependent and begins with the first dose of chemotherapy. To date, no biomarker for early presymptomatic detection of DOX cardiotoxicity has been validated. Our previous data indicated that peripheral blood mononuclear cells (PBMCs) can be used as a surrogate tissue for identification of biomarkers for DOX cardiotoxicity. The aim of this study was to analyze PBMC gene expression induced by a single dose of DOX-based chemotherapy in breast cancer patients and correlated the data with DOX-induced cardiotoxicity. Materials and Methods. Blood samples of 33 women treated for breast cancer with DOX-based chemotherapy were collected before the start and after the first cycle of chemotherapy. Total RNA was isolated from PBMC and whole-genome gene expression was performed using Illumina HumanHT-12 v4 Expression BeadChip array. Gene expression data were log2 – transformed and gene transcripts with average log2-intensities > 7 were considered to be expressed. The group-specific means were analyzed via repeated-measures with ANOVA for expression changes after DOX. Genes with p-value<0.05 were considered differentially expressed. Cardiac function was assessed before and after the completion of chemotherapy by echocardiogram and/or multigated acquisition scan. An absolute decrease of left ventricle ejection fraction >10% or <55% was considered abnormal. Differentially expressed genes (DEG) of patients who developed abnormal LVEF decrease were compared with DEG of patients who did not. Results. A single dose of DOX-based chemotherapy resulted in 235 DEG in PBMC (P<0.05, FDR<0.05), mapped to cell death, oxygen transport and iron ion binding. Further analysis identified 87 DEG in the PBMC of eight (n=8) women who developed abnormal decline in LVEF from the baseline in comparison with women who did not (n=25). Most of the 87 DEG encode proteins secreted by activated neutrophils, such as alpha-defensins, arginase, cathepsin G, elastase, haptoglobin. The functional analysis of the 87 DEG showed enrichment for inflammatory response, immune response, cell death and peptidase activity. Discussion. The results from this study indicated that elevated neutrophil-associated transcripts in the early stages of DOX-based chemotherapy were independent of the neutrophil count. These data suggest an association between the neutrophils activation after a single dose of DOX-based chemotherapy and later impairment of cardiac function. The early PBMC transcriptome signature can be used in the future development of biomarkers for DOX-associated cardiotoxicity. Citation Format: Todorova VK, Siegel ER, Makhoul I, Marquette M, Wei JY, Klimberg VS. Gene expression profiling of doxorubicin cardiotoxicity in peripheral blood cells of breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-15-02.

Circulating Tumor Cells in Breast Cancer Patients.

Breast cancer is the most commonly diagnosed cancer among women, resulting in an estimated 40,000 deaths in 2014.1 Metastasis, a complex, multi-step process, remains the primary cause of death for these patients. Although the mechanisms involved in metastasis have not been fully elucidated, considerable evidence suggests that metastatic spread is mediated by rare cells within the heterogeneous primary tumor that acquire the ability to invade into the bloodstream. In the bloodstream, they can travel to distant sites, sometimes remaining undetected and in a quiescent state for an extended period of time before they establish distant metastases in the bone, lung, liver, or brain. These occult micrometastatic cells (circulating tumor cells, CTCs) are rare, yet their prognostic significance has been demonstrated in both metastatic and non-metastatic breast cancer patients. Because repeated tumor tissue collection is typically not feasible and peripheral blood draws are minimally invasive, serial CTC enumeration might provide "real-time liquid biopsy" snapshots that could be used to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. In addition, characterizing CTCs might aid in the development of novel, personalized therapies aimed at eliminating micrometastases. This review describes current CTC isolation, detection, and characterization strategies in operable breast cancer.

Circulating tumor cells in breast cancer patients.

Circulating tumor cells (CTCs) represent apotential non-invasive substitute for real-time tissue biopsy. Moreover, detection of CTCs in breast cancer patients has been reported as astrong prognostic factor. Biomarkers on CTCs have been analysed and correlated to tissue biopsies from breast cancer patients. Discordance in expression of potential biomarkers between primary tumor, metastatic sites and CTCs has been observed. Potential analytical confounding factors include lack of analytical consistency, varying sensitivities and specificities of used assays and differences in analytical ranges among various reported studies. Besides, clonal evolution within primary tumor (and metastatic sites) that leads to intratumor heterogeneity must be accounted for. Nevertheless, several on-going trials are exploring CTCs detection and biomarker profiling in view of personalising cancer treatment based on these real-time results. In this work, we will review CTCs in breast cancer patients and focus on identification of novel prognostic biomarkers.

Influence of Radiotherapy on Frequency of Sister Chromatid Exchange, Micronuclei and Binuclear Cells in Breast Cancer Patients

The aim of this study was to evaluate any cytogenetic change in sister chromatid exchange (SCE), micronuclei (MN) and binuclear cell (BNC) of peripheral blood lymphocytes in breast cancer patients treated with postoperative radiation therapy (RT). Frequency of the SCE, MN and BNC were examined in 22 breast cancer patients received RT and 10 healthy individuals. All parameters were measured before (RT-a), at the completion of (RT-b) and three months after the completion of (RT-c) RT. A significant difference emerged in SCE (p=0.008) and MN (p=0.004) between RT-a and control groups. There was not any statistically significant difference in the SCE frequencies between RT-b and RT-c measurements. The frequencies of MN were also significantly higher in RT-b (p=0.005) and RT-c (p=0.005) than in control group. MN levels decreased to pre-RT levels three months after completion of treatment. Increasing MN and SCE frequencies following radiotherapy is an expected situation. Decrease in MN frequency at 3-month after the completion of RT suggests that expected repair continues. Persistent SCE at the same period suggests that recovery in SCE has not completed yet and a longer period of time is needed.

Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.

BackgroundTrastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab.Patients and methodsTwenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients’ peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by 51Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab.ResultsWe found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006).ConclusionsAlthough this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0680-0) contains supplementary material, which is available to authorized users.

Paired-like Homeodomain Transcription factor 2 expression by breast cancer bone marrow disseminated tumor cells is associated with early recurrent disease development.

The presence of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients is prognostic for early relapse. In the present study, we analyzed the gene expression profiles from BM cells of breast cancer patients to identify molecular signatures associated with DTCs and their relevance to metastatic outcome. We analyzed BM from 30 patients with stage II/III breast cancer by gene expression profiling and correlated expression with metastatic disease development. A candidate gene, PITX2, was analyzed for expression and phenotype in breast cancer cell lines. PITX2 was knocked down in the MDAMB231 cell lines for gene expression analysis and cell invasiveness. Expression of various signaling pathway molecules was confirmed by RT–PCR. We found that the expression of Paired-like Homeobox Transcription factor-2 (PITX2) is absent in the BM of normal healthy volunteers and, when detected in the BM of breast cancer patients, is significantly correlated with early metastatic disease development (p = 0.0062). Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells. Three genes—NKD1, LEF1, and DKK4—were significantly downregulated in response to PITX2 suppression. Expression of PITX2 in BM of early-stage breast cancer patients is associated with risk for early disease recurrence. Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-015-3576-z) contains supplementary material, which is available to authorized users.

Peripheral blood NK cells expressing HLA-G, IL-10 and TGF-β in healthy donors and breast cancer patients

Human natural killer (NK) cells are not only professional cytotoxic cells integrated into effector branch of innate immunity, but they are also regulatory cells, managing different immune processes. Immunoregulatory NK cells, expressing HLA-G and IL-10, have been generated in vitro from human hematopoietic progenitors and found in vivo among decidual NK cells of pregnant women. Human peripheral blood NK cells have been shown to acquire suppressive properties after HLA-G uptake during trogocytosis. Moreover, it has been shown that circulating NK cells contain a trace amount of cells producing TGF-β and IL-10, which exert a suppressive influence upon innate and adaptive immunity. In this study, we report on a minor subset of peripheral blood HLA-G+ NK cells possessing suppressive activity toward effector functions of NK cells. Further we demonstrate an increased number of circulating HLA-G+, IL-10+, and TGF-β+ NK cells in breast cancer patients which might impair efficiency of anti-tumor immunity.

Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients.

IntroductionThe truncated form of human epidermal growth factor receptor 2 (p95HER2) lacks the HER2 extracellular domain and has been associated with poor prognosis and resistance to trastuzumab. In the present study, the expression of p95HER2 was investigated on circulating tumor cells (CTCs) from breast cancer patients.MethodsTriple-staining immunofluorescent experiments were performed on peripheral blood mononuclear cells’ (PBMCs) cytospins obtained from patients with early (n = 24) and metastatic (n = 37) breast cancer. Cells were stained with the pancytokeratin (A45-B/B3) antibody coupled with antibodies against the extracellular (ECD) and the intracellular (ICD) domains of HER2. Slides were analyzed with either confocal laser scanning microscopy or with the Ariol system.ResultsHER2-positive CTCs were identified in 55.6 % of early and 65.2 % of metastatic CTC-positive breast cancer patients. p95HER2-positive CTCs were identified in 11.1 % of early and 39.1 % of metastatic breast cancer patients (p = 0.047). In 14 patients with metastatic HER2-positive breast cancer, CTCs were also analyzed before and after first-line trastuzumab therapy. Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %. Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03).Conclusionsp95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients. Their incidence is increased in the metastatic setting and their presence is associated with poor survival. Longitudinal studies during anti-HER2 treatment are required to determine the clinical relevance of p95HER2-expressing CTCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0624-x) contains supplementary material, which is available to authorized users.

The Hedgehog signalling pathway mediates drug response of MCF-7 mammosphere cells in breast cancer patients.

BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

Increased P-35, EBI3 Transcripts and Other Treg Markers in Peripheral Blood Mononuclear Cells of Breast Cancer Patients with Different clinical Stages.

Currently, cancer as a major problem around the world threatens human health and has a high incidence in developing countries. Many reports have indicated that patients suffering from cancer demonstrate decreased antitumor immune responses as well as a high prevalence of T regulatory population. It has been reported that Foxp3(+)Tregs exert suppression by cell contact-dependent mechanisms which are mediated by soluble factors such as immunosuppressive cytokines like IL-10, TGF-β and IL-35. Consequently there is a great need to identify prognostic and diagnostic biomarkers of regulatory T cells for vaccine and drug development. In this study IL-10, TGF-β, IL-35 and Foxp3 mRNA gene expression has been measured in peripheral blood of 40 breast cancer patients and 40 normal age-matched women using quantitative real-time PCR (qRT-PCR) method with Master Mix reaction containing SYBER Green. GAPDH gene was used as housekeeping gene. Our data demonstrated a significant up-regulation of IL-10, TGF-β, P35, EBI3 and Foxp3 gene expression in patients' peripheral blood compared to normal healthy controls (p<0.05). The data suggests that the immune system is suppressed in breast cancer patients, which may be due to elevated Treg cells population. These results may be useful for diagnostic or therapeutic purposes. However it may require more investigations.

Abstract PD1-4: Somatic leukemogenic mutations associated with infiltrating white blood cells in breast cancer patients

Abstract Background: In the last few decades, theoretical models of cancer growth and progression have long focused on the aberrations of cancer cells alone, such as the abnormal mitotic and invasive characteristics of cancer cells. More recent research across multiple solid tumors suggests a critical interplay between solid tumors and immune regulating cells. Mounting evidence suggests that the immune system can tip the scales of cancer progression, eliciting either an anti-tumor or pro-tumor immune response depending upon varying stimulating and inhibitory factors. Here, we are the first to demonstrate novel mutations including leukemogenic mutations among tumor infiltrating lymphocytes in breast cancer patients. Methods: We obtained 17 primary breast cancer samples from patients who presented for either a lumpectomy or mastectomy as part of an IRB approved biospecimen protocol. Of the 17 patient samples, 13 had triple negative breast cancer, 2 had ER+, HER2+ disease, and 2 had ER+, HER2- disease. In the 17 samples, we used fluorescent activated cell sorting to separate CD45-positive hematopoietic cells from CD45-negative epithelial cells. We then performed exome sequencing of tumor-infiltrating hematopoietic cells to investigate for the presence of pathogenic mutations in tumor-associated leukocytes. In this first step, we identified candidate mutations in known cancer genes, including BCOR, NOTCH2, TET2, NF1, EZH2, and JAK1. As a validation step, we then performed capture-based sequencing of tumor-infiltrating leukocytes in 20 breast cancer samples matched to each patient’s germline DNA sample (buccal swab). In 10 of the 20 patients, we identified and validated somatic mutations. Of note, 6 of these patients harbored mutations known to be associated with leukemia, including DNTM3A, TET2, and BCOR. Most of these mutations were present in at least 5-20% of reads. This suggests that these mutations were present in enriched subclones and were not rare alleles occurring in a minority of hematopoietic stem cells. Lastly, we performed 454 deep sequencing analysis of microdissected tumor DNA samples and confirmed the absence of these mutations in breast cancer cells. Conclusion: Our data demonstrate somatic mutations in tumor infiltrating leukocytes in breast tumors which were not identified in matched germline or tumor DNA samples. Notably, some of these mutations have been implicated in the pathogenesis of lymphoid and myeloid malignancies. This observation suggests a unique relationship between cancer cells and mutant infiltrating leukocytes. We are now investigating the functional interaction between cancer cells and hematopoietic cells. Our findings reframe our understanding of carcinogenesis and offer novel opportunities for cancer detection and treatment. Citation Format: Elizabeth A Comen, Maria Kleppe, Hannah Wen, Britta Weigelt, Lennart Bastian, Brian Blum, Franck T Rapaport, Matt Keller, Nicolas Socci, Agnes Viale, Daoqi You, Robert Benezra, Edi Brogi, Jorge Reis-Filho, Michael Berger, Ross Levine, Larry Norton. Somatic leukemogenic mutations associated with infiltrating white blood cells in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-4.

Abstract P4-15-20: Insulin-like growth factor receptor-1 (IGF-1R) expression is highly correlated with HER2 amplification on circulating epithelial tumor cells (CETCs) in breast cancer - this may be the reason for resistance to trastuzumab

Abstract Background: HER2 status has been the most extensively studied biomarker in circulating epithelial tumor cells of breast cancer patients. HER2 status can change during recurrence of disease and HER2 overexpression is a mandatory requirement to administer anti-HER2-directed drugs. Nevertheless, 70% of patients with HER2-positive breast cancers develop intrinsic or secondary resistance to trastuzumab. This resistance has been associated with the activation of an alternative signalling pathway such as the insulin-like growth factor (IGF) pathway. Therefore we investigated the expression of IGF-IR on the CETCs in addition to HER2 amplification in breast cancer patients to identify patients who might benefit from a combined targeted therapy against HER2 and IGF-IR. Methods: CETCs were determined from blood of 30 breast cancer patients. The number of vital CETCs and the expression of IGF-IR were investigated using the maintrac® approach. Fluorescence in situ hybridisation was used for analysis of HER2 amplification in CETCs. Results: CETCs could be detected in all breast cancer patients. The number of CETCs ranged from 4 to 163 in 100 µl of cell suspension. IGF-IR expression on the surface of CETCs was detected in all patients. Setting a cut-off 30% positive cells as HER2 positive in 75% of patients HER2 positive CETCs were observed irrespective of the status in the primary tumor. In contrast, only 6% of patients changed their HER2 status from positive tissue to negative CETCs. A statistically high correlation was found between the percentage of IGF-IR positive and HER2 positive CETCs. A statistically significant association was found between IGF-IR expression or HER2 amplification and ER/PR receptor status. The higher frequency of HER2 amplified circulating tumor cells might be due to the difference in preparation steps between tissue and blood borne cells and one of the reasons for trastuzumab resistance and for the response of some HER2 negative patients to trastuzumab. Re-evaluation of HER2 status in CETCs could be a valid strategy with potential clinical applications. Conclusion: Our results demonstrate a parallel expression of IGF-IR and HER2 amplification in CETCs. IGF-IR may be involved in the development of resistance to trastuzumab and may be an important potential therapeutic target in breast cancers with HER2 positive circulating tumor cells. Combining targeting of IGF-IR and HER2 may be a rational approach to improve response to trastuzumab in the sub-group of CETCs that express both, HER2 and IGF-IR. Citation Format: Dorothea Zimon, Monika Pizon, Ulrich Pachmann, Katharina Pachmann. Insulin-like growth factor receptor-1 (IGF-1R) expression is highly correlated with HER2 amplification on circulating epithelial tumor cells (CETCs) in breast cancer - this may be the reason for resistance to trastuzumab [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-20.

Clinical implications of circulating tumor cells of breast cancer patients: role of epithelial-mesenchymal plasticity.

There is increasing interest in circulating tumor cells (CTCs) due to their purported role in breast cancer metastasis, and their potential as a “liquid biopsy” tool in breast cancer diagnosis and management. There are, however, questions with regards to the reliability and consistency of CTC detection and to the relationship between CTCs and prognosis, which is limiting their clinical utility. There is increasing acceptance that the ability of CTCs to alter from an epithelial to mesenchymal phenotype plays an important role in determining the metastatic potential of these cells. This review examines the phenotypic and genetic variation, which has been reported within CTC populations. Importantly, we discuss how the detection and characterization of CTCs provides additional and often differing information from that obtained from the primary tumor, and how this may be utilized in determining prognosis and treatment options. It has been shown for example that hormone receptor status often differs between the primary tumor and CTCs, which may help to explain failure of endocrine treatment. We examine how CTC status may introduce alternative treatment options and also how they may be used to monitor treatment. Finally, we discuss the most interesting current clinical trials involving CTC analysis and note further research that is required before the breast cancer “liquid biopsy” can be realized.

OSTEOPROTEGERIN AS A PREDICTIVE MARKER OF THE COURSE OF BREAST CANCER

Despit e th e considerable progress in research of breast cancer (BC), taking into consideration the division into molecular subtypes, the mechanics of metastasis development in BC patients are not definitely investigated, especially at favorable prognosis. Research of new informative diagnostic and prognostic markers represents the modern problems of BC pathology in a new way. Thi s paper is devoted to study of osteoprotegerin (OPG) expression levels in tumor cells in BC patients. The study covered 83 female patients with regional BC (T2–4N0–3M0), treated from 2003 to 2010. The inclusion criteria were histologically proved diagnosis of invasive BC, age over 18 y. o., ECOG 0 or 1. In order to study OPG levels, we carried out immune histochemical test, which was carried out according to standard protocol on the sections of biopsy material. The OPG antibody from GeneTex, used in the study, was rabbit polyclonal, dilution 1:1000, incubation time – 30 minutes. Vizualization system was Real EnVision, anti-rabbit (by DAKO, Denmark). Immunohistochemical staining was studied by counting of number of positive cells in reference to total number of malignant cells. Expression over 50 % of cells was regarded as high, less than 50 % – as low. Average value of OPG expression level was 49 %, median – 50 %. Minimal value – 0 %, maximum value – 90 %. The patients were divided into 2 groups according their expression levels: Group 1 – OPG (high) expression (more than median), Group 2 – OPG (low) expression (less than median). OPG (high) Group was made by 47 patients, OPG (low) Group – by 36. When analyzing clinical and pathological characteristics of BC patients, taking into consideration OPG expression, there were noticed no reliable differences with respect to availability o r absence o f affected regional lymph nodes, category T and Ki-67 indice. The group with positive estogen-progesteron receptors showed reliably more often the high values of OPG than the group with negative receptors (p < 0,05). When analyzing the duration till the progression and overall survival, a difference between the survival and time to progression (rise of metastases) in patients with low OPG levels and the same exponents in patients with high OPG levels was established.

Obesity, proinflammatory mediators, adipose tissue progenitors, and breast cancer.

There is emerging evidence that obesity is associated with an increase in the incidence, severity, and mortality from different types of cancer, including postmenopausal breast cancer. Here, we discuss the role of white adipose tissue (WAT) cells and of related soluble factors in the local and metastatic growth of this neoplastic disease. Moreover, we discuss the recent increase in the use of WAT-derived progenitor cells in breast cancer patients to enhance the quality of breast reconstruction and the related risks. In several murine models, WAT cells and progenitors were found to have cooperative roles in promoting local breast cancer. Moreover, they were found to contribute to adipocytes and pericytes supporting the cancer vasculature, and stimulated the metastatic progression of breast cancer. There are some clinically retrospective data showing a significant increase in the frequency of intraepithelial neoplasia in patients who received a lipofilling procedure for breast reconstruction compared with controls. Preclinical models and clinical studies are urgently needed to investigate how to inhibit the tumor-promoting activity of WAT cells and progenitors. The risks associated with the use of WAT cells for breast reconstructions should be better investigated retrospectively and prospectively.

High GINS2 transcript level predicts poor prognosis and correlates with high histological grade and endocrine therapy resistance through mammary cancer stem cells in breast cancer patients

GINS2, a subunit of the GINS complex, is overexpressed in lung adenocarcinoma and metastatic breast tumor; however, its prognostic power and possible molecular mechanisms in breast cancer (BC) remain unclear. In this study, we aimed to explore the function of GINS2 in BC. The association between GINS2 transcript level and the clinical outcome of BC patients were estimated using Kaplan-Meier plots, multivariate cox regression analysis, forest plots, and receiver operating characteristics curves. Gene set enrichment analysis (GSEA) was performed to explore the mechanisms underlying the effects of the GINS2 transcript. High GINS2 transcript level was correlated with poor relapse free survival (log-rank P ≤ 0.001 in six cohorts; forest plot: total n = 1,420, total RR = 1.72, 95% CI 1.45-2.03; multivariate cox regression analysis: n = 906, HR 2.36, 95% CI 1.88-2.97), and distant metastasis free survival (log-rank P < 0.01 in 3 cohorts; forest plot: total n = 691, total RR 1.91, 95% CI 1.36-2.67; multivariate cox regression analysis: n = 442, HR 2.43, 95% CI 1.70-3.47). BC patients with higher GINS2 transcript levels showed poorer tamoxifen efficacy in a dose-dependent manner. GINS2 expression was significantly downregulated under mutated p53-depleted condition in MDA-468 and MDA-MB-231 cells, upregulated in mammary cancer stem cells (MaCSCs) (P = 0.003), and correlated with upregulated genes in mammary stem cells (GSEA: P < 0.01). Our study, for the first time, demonstrates that GINS2 is an independent prognostic marker and is associated with lung metastasis, histological grade, and endocrine therapy resistance in BC patients, which may attribute to mutant p53 and MaCSCs.

Abstract 3030: Correlation of circulating tumor cells (CTCs) expressing stemness and EMT phenotypes with immunosuppressive cells in metastatic breast cancer patients

Abstract Background: The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of breast cancer (BC) patients is an unfavourable prognostic factor; however further characterization of these cells is required to understand their biological role. The presence of stemness and epithelial-to-mesenchymal transition (EMT) markers in CTCs could be an important marker of enhanced metastatic capacity. On the other hand, myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) play an essential role in tumor-immune evasion, by suppressing the normal function of both CD4+and CD8+ T cells. Increased levels of MDSCs and Tregs have been correlated with advanced disease and poor prognosis. Recent findings in preclinical models and primary tumours indicate that immune cells can induce EMT and stemness characteristics of cancer cells; however this has not been demonstrated in circulation. The purpose of this study was to evaluate the correlation of CTCs expressing stemness and EMT phenotypes with immunosuppressive cells in BC patients. Patients and Methods: PB was obtained from 38 metastatic BC patients prior to treatment. Triple immunofluorescence was performed on PBMCs cytospin preparations using antibodies against putative markers for epithelial cells (anti-pancytokeratin, A45-B/B3), stemness (anti-ALDH1) and EMT (anti-TWIST) phenotypes. Quantification of ALDH1 expression levels and characterization of TWIST subcellular localization was performed using the ARIOL system. The frequency of different immune cell subpopulations was also evaluated in PB using flow cytometry and was correlated to the presence of distinct CTC phenotypes. Results: Cytokeratin+ CTCs were detected in 16/38 (42%) patients. Patients with detectable CTCs had a higher percentage of CD4+ Tregs compared to those without CTCs (p=0.022). Moreover, elevated levels of CD4+ Tregs were associated with detectable stem cell-like CTCs (ALDH1high) (p=0.023), while in patients with non-stem CTCs (ALDH1low/-) an increased frequency of MDSCs [CD15+CD14-HLA-DR-Lin-CD33+CD11b+] occurred (p=0.020). The detection of EMT-like CTCs was not associated with the incidence of MDSCs and Tregs. Conclusions: The detection of CTCs using ARIOL system was associated with a higher incidence of CD4+ Tregs and MDSCs in metastatic BC patients. However, different immunosuppressive cell subpopulations were correlated with phenotypically different CTCs according to EMT and stemness characteristics. These findings indicate that the immune system may be differentially involved in regulating CTCs with different biological behaviour or reversely, CTCs with different characteristics might induce specific subpopulations of immunosuppressive cells. Citation Format: Despoina Aggouraki, Maria Papadaki, Eleni Kyriaki Vetsika, Anna Koutoulaki, Athanasios Kotsakis, Galatea Kallergi, Sofia Agelaki, Dimitris Mavroudis, Vassilis Georgoulias. Correlation of circulating tumor cells (CTCs) expressing stemness and EMT phenotypes with immunosuppressive cells in metastatic breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2014-3030

Abstract 4018: Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients

Abstract Introduction: Circulating Tumor Cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. Triple-negative breast cancers have a more aggressive clinical outcome than other forms of breast cancer. The aim of our work was to develop a quantitative real-time multiplex RT-qPCR assay for studying the expression of ER, PR, HER-2 and EGFR in CTC of breast cancer patients. Materials and Methods: A quadruplex quantitative real time RT-qPCR assay for ER, PR, HER-2 and EGFR was developed based on the de novo in-silico design of eight primers and four dual hybridization probes. All primers and probes were designed to avoid cross-reactions and primer dimer formation and to match the assay conditions, such as amplicon sizes, and melting temperatures. The specificity of all primer and hybridization probe sequences was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). Each probe set included a 3′-fluorescein (F) donor probe and a 5′- LC acceptor probe that was different for each gene set: ER at 610nm, PR at 640nm, HER-2 at 670nm, EGFR at 705nm. A color compensation test was performed by using pure dye spectra so that spectral overlap between dyes was corrected. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. The assay was designed, so that only 1 μL of cDNA is used and the total reaction volume is 10 μL. Results: The assay sensitivity was evaluated by performing spiking experiments. For this reason known numbers of MCF7 and SKBR-3 cells, as well as external quantification calibrators ranging from 105 copies/μL to 10 copies/μL, were used. The specificity was evaluated by carefully designed cross reaction studies, performed for each gene target in the presence of all other targets. Our assay, under optimized conditions has shown a very high specificity for each analyte. Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative multiplex real-time RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR. The assay is currently validated in CTC isolated from a large number of breast cancer patients. Citation Format: Areti D. Strati, Evi S. Lianidou. Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2014-4018

Abstract 4076: Characterizing tumor-specific memory stem like T cells in blood and bone marrow of breast cancer patients

Abstract The identification of memory T cells with stem cell-like properties has been reported recently in both humans and mice. Such stem cell-like memory T cells (Tscm) upon adoptive transfer into a humanized mouse model had superior self-renewal, proliferation and mediated potent anti tumor immunity. However, very little is known about their specificities, physiological function and contribution to anti-tumor immune responses in cancer patients. We investigated the presence of Tscm cells in the peripheral blood (PB) and bone marrow (BM) of breast cancer patients by multicolor flow cytometry analyses with a panel of human T cell markers to distinguish naïve (Tn), central memory (Tcm), effector memory (Tem) and Tscm (CCR7+, CD45RO-, CD127+, CD45RA+, CD95+, CXCR3+, IL2Rβ+) population. Tscm cells were detected in both CD4+ and CD8+ T cells in the PB and BM of breast cancer patients. Interestingly, we observed a significant increase (3-fold) in the number of Tscm cells among the CD4+ and CD8+ T cells in breast cancer patients as compared to healthy donors. This was significantly higher among the CD8+ T cells as compared to CD4+ T cells. We further evaluated the expression of exhaustion markers (PD-1 &amp; LAG3) and found that majority of Tscm cells (&amp;gt;95%) did not express PD-1 &amp; LAG3 and comprised the least exhausted T cell compartment as compared to Tn, Tem and Tcm. To investigate the contribution of different subsets of memory T cells to tumor-specific T cells in breast cancer patients, we used a panel of breast cancer associated MHC class-I pentamers against human epidermal growth factor receptor 2 (HER2348-356 , HER2435-443), Mucin-1 (MUC112-20) and Carcinoembryonic antigen (CEA571-579). Cumulative analyses of tumor-specific pentamer+ CD8+ T cells in PB and BM showed that 20-25% of total tumor-specific T cells were of Tscm phenotype. We observed higher percentages of tumor-specific pentamer+CD8+ T cells among the total Tscm cells in BM as compared to PB, however this difference was not significant. We performed ex vivo re-stimulation assays using autologous dendritic cells loaded with several breast cancer-specific peptides with PB and BM of breast cancer patients to further assess the frequency and function of tumor-specific Tscm cells. Cumulative analyses showed that 20-23% of tumor-specific IFN-γ+ CD8+ and 26-32% IL-2+ CD4+ had a Tscm phenotype. In summary, we report the presence of Tscm cells in the PB and BM of breast cancer patients and a significant increase in their number as compared to healthy donors. Importantly, we could show that Tscm contribute to the population of functional and tumor reactive CD8+ and CD4+ T cells in PB and BM of breast cancer patients. Our further aim is to investigate the therapeutic potential of Tscm cells in adoptive T cell experiments with humanized mouse model of breast cancer. Citation Format: Mudita Pincha, Paranchai Boonsawat, Christoph Domschke, Philipp Beckhove. Characterizing tumor-specific memory stem like T cells in blood and bone marrow of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2014-4076