BackgroundCirculating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. Epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue.MethodsThis study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. Expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score.ResultsCTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT.ConclusionsIn this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features.

No abstract

BackgroundDermatofibrosarcoma protuberans (DFSP) is a relatively common soft-tissue tumor. A more aggressive appearing fibrosarcoma may arise in DFSP, changing its biological behavior. CD34 and apolipoprotein-D are highly expressed in DFSP, but their prognostic significance is uncertain.MethodsDFSP and fibrosarcomatous-DFSP (FS-DFSP) patients referred to our institute between 1982 and 2009 were identified. Fibrosarcomatous changes, expression of CD34 and apolipoprotein-D were evaluated.Results40 patients, (median age 43 years, 55% males) were identified. Tumor was located in the limbs in 60%, in the trunk in 40%. Thirty-seven patients had localized and 3 had metastatic disease. Thirteen (32%) patients were FS-DFSP. All but one underwent surgery with adequate surgical margins in 72%. 7 FS-DFSP received also radiotherapy (RT). Chemotherapy was administered to 3 patients with FS-DFSP. With a median follow-up of 49 months, the 5-OS was 90%. Local recurrence rate was 23%: 42% FS-DFSP, 15% DFSP. Metastases developed in three FS-DFSP patients. The 5-year EFS was 70% in localized patients. Histology (DFSP 75% vs. FS-DFSP 52%, p = 0.002), surgical margins (adequate 74% vs. inadequate 55%, p = 0.02), site (limb 47% vs. trunk 100%), CD34 expression (CD34 positive: 70% vs. CD34 negative: 33%, p = 0.05), and apolipoprotein-D expression (Apo-D positive: 73% vs. Apo-D negative: 33%, p = 0.02) influenced the 5-year EFS, whereas sex, use of RT or number of previous surgical treatments did not.ConclusionsPatients with DFSP have a high survival probability. Site, adequate surgical margins, presence of the fibrosarcomatous component, lack of CD34 expression and apolipoprotein-D influence outcome.

Circulating tumor cells (CTCs) play a pivotal role in tumor dissemination and progression, and are considered to be a critical part of the metastatic cascade. The aim of the present research article was to examine breast cancer-specific mutations in primary breast cancer (PBC) using targeted resequencing. A total of 78 patients with PBC were enrolled into this translational study. Reverse transcription-quantitative PCR assay for the expression of epithelial markers (CK19) or epithelial-to-mesenchymal transition (EMT)-related genes (TWIST1, SNAIL1, SLUG and ZEB1) was applied for identification of CTCs prior to surgery. Total DNA was isolated from fresh frozen primary tumors. Sequencing was performed by Agilent SureSelect target enrichment and Illumina paired-end sequencing on the MiSeq platform. The most commonly affected genes were TP53 (mutated in 21 tumors; 26.9%), followed by PIK3CA (mutated in 16 tumors; 20.5%) and BRCA1/2 (mutated in 7 tumors, BRCA1 n=2 and BRCA2 n=5; 9.0%). In our cohort, a significantly higher proportion of patients with epithelial CTCs harbored mutations in the BRCA1/2 genes in the tumor tissue. There were no mutations in specific genes associated with CTCs with the EMT phenotype. To the best of our knowledge, this study is the first to report a correlation between the presence of epithelial CTCs in the peripheral blood and mutations of the BRCA1/2 genes in primary tumor tissue.

ANXA2 stromal expression might play a key role in aggressive tumor phenotype associated with increased EMT CTCs release, however, other factors beyond ANXA2 are responsible for coagulation activation mediated by CTCs in breast cancer patients.

Background: CTCs represent a heterogeneous population of cells with different phenotypes and biological values. Epithelial to mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased resistance to chemotherapy that may be under detected by currently approved assays. The aim of this study was to characterize CTCs based on the expression of epithelial and mesenchymal markers in primary breast cancer (BC) and to correlate them with patients'/tumor characteristics. Methods: This prospective translational study included 422 patients with primary BC enrolled from March 2012 to February 2015. Blood for CTC detection was drawn before surgery (422 patients), before 1st cycle (95 patients) and before 2nd cycle (53 patients) of adjuvant therapy. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using RossetteSep kit (StemCell Technologies) negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of 60 healthy donors. Results: Totally, CTCs were detected in 116/422 (27.5%) patients before surgery, in 21/95 (22.1%) patients after surgery and before 1st cycle and in 19/53 (35.8%) of patients before 2nd cycle of adjuvant therapy. Before surgery, CTCs exhibited only epithelial markers in 38 (9.0%) patients, only EMT markers in 68 (16.1%) of patients, while in 10 (2.4%) patients CTCs with both epithelial and EMT markers were detected. Epithelial CTCs were more often detected before surgery compared to after surgery (11.4% vs. 2.1%; p = 0.003), while mesenchymal CTCs were more often detected after the 1st cycle of chemotherapy as opposed to detection before surgery (30.2% vs. 18.2%; p = 0.05). Patients with N2-3 disease had more often detectable CTCs compared to patients with N0-1 disease (41.4% vs. 24.9%, p = 0.01) and this was mainly driven by mesenchymal CTCs (31.0% for N2-3 vs. 16.0% for N0-1; p = 0.007). Similarly, patients that lacked p53 expression (wild type TP53) in primary tumor had more often CTCs with EMT phenotype opposite to patients with p53 expression (p = 0.02). Presence of epithelial CTCs was significantly associated with lower absolute lymphocyte (p = 0.02) and neutrophil (p = 0.02) counts in peripheral blood. Conclusions: Our results support the concept of CTCs phenotypic heterogeneity in breast cancer patients. These results support the role of EMT in cancer pathogenesis and suggest that CTCs with EMT phenotype are involved in tumor dissemination while their increase after chemotherapy might be a mechanism of treatment resistance. Moreover, these data suggest inverse relationship between immune cells and epithelial CTCs which stress the role of immune cells in tumor dissemination. Citation Format: Mego M, Jurisova S, Karaba M, Minarik G, Benca J, Sedlackova T, Manasova D, Malejcikova M, Sieberova G, Macuch J, Gronesova P, Sufliarsky J, Pindak D, Cristofanilli M, Reuben JN, Mardiak J. Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer. [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 P2-02-04.

Background: Circulating tumor cells (CTCs) are prognostic in breast cancer (BC) patients. CTCs represent a heterogeneous population of cells with different phenotypes and biological value. Epithelial-mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased chemotherapy resistance and may be responsible for undetection of CTC by conventional methods in a fraction of patients with early or advanced breast cancer. The aim of this study was to characterize CTCs based on the expression of epithelial and mesenchymal markers in non-metastatic breast cancer patients before surgery. Methods: This prospective ongoing translational study consists of 36 patients with non-metastatic breast cancer. CTCs were detected before surgery, before 1st cycle and before 2nd cycle of adjuvant therapy. Herein, we present preliminary data related to CTCs detection before surgery. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using RossetteSep kit (StemCell Technologies) negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45−) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, FOXC2) and epithelial (CK19, EpCAM) gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of 30 healthy donors (HD). Results: Median age was 62 year (range: 40–80 years). TWIST1, SNAIL1, SLUG, FOXC2, CK19 and EpCAM were overexpressed in CD45- PBMC in 2.8%, 2.8%, 11.1%, 13.9%, 19.4% and 2.8% respectively. Totally, CTCs were detected in 36.2% of patients. CTCs with only epithelial markers were present in peripheral blood of 5.6% patients; CTC with EMT phenotype were present in 13.9% of patients, while in 16.7% of patients CTCs exhibited both epithelial and mesenchymal markers. Overexpression of different EMT-related transcription factors in peripheral blood samples were mutually exclusive. There was no association between the presence of CTCs and analyzed clinico-pathological variables. Conclusions: CTCs with EMT phenotypes are more prevalent compared to CTCs with epithelial phenotype in early breast cancer patients before surgery, and EMT genes may be involved in the dissemination of CTCs. These data support the concept of CTCs heterogeneity in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-12.

Background: CTCs play a major role in tumor dissemination and progression, and represent one of the key components of the metastatic cascade. The aim of this study was to identify signaling pathways associated with presence of CTCs in primary breast cancer (PBC) patients using a comprehensive genomics approach. Methods: This translational study included 78 patients with PBC. CTCs were detected before surgery by quantitative RT-PCR assay for expression of epithelial (EP; CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1). Total DNA and RNA were extracted, in parallel, from fresh frozen primary tumor and the microRNA and mRNA expression profiles were obtained using Human microRNA Microarray v21.0 and SurePrint G3 Human Gene Expression v3 (Agilent Technologies). Next generation sequencing (NGS) was performed by Illumina Multiplex Sequencing using MiSeq Sequencing Reagent Kit V3. Results:Mutations in BRCA1/2 genes in tumor tissue were more common in patients with epithelial CTCs (CTC_EP) compared to patients without epithelial CTCs in peripheral blood (23.5% vs. 0%, p = 0.02), while there were no mutations in specific genes associated with CTC with EMT phenotype (CTC_EMT).Further, we identified 90 genes and 7 miRs that were expressed at significantly different levels in tumors with presence of CTC_EP and 199 genes and 13 miRs specifically associated with CTC_EMT, compared to tumors with non-detectable CTCs. We also identified 39 overlapping genes and 7 miRs, that were expressed at significantly different levels in tumors with CTC_EP and/or CTC_EMT compared to tumors with non-detectable CTCs. Overlapping genes and miRs with highest different levels in expression were ATAD3A, TMEM201, DCPS, DOCK9-AS2, TRAF2 and miR-5195-3p, miR-188-5p, miR-6780a-5p, miR-6757-5p. Signalling pathways associated with these genomic alterations belong to several critically functional groups, such as immune response, signal transduction, cell proliferation, cell cycle progression, or apoptosis were significantly differentially based on CTCs status. Conclusions: We identified for the first time various genomic alterations in primary tumor tissue of PBC associated with different CTCs subpopulations in peripheral blood. We hypothesize that these genomic alterations could play a role in tumor dissemination and progression and might lead to identification of new therapeutic targets. Citation Format: Mego M, Tokar T, Minarik G, Hajduk M, Karaba M, Benca J, Sedlackova T, Repiska G, Krasnicanova L, Macuch J, Sieberova G, Pindak D, Cristofanilli M, Reuben JM, Jurisica I, Mardiak J. Comprehensive analysis of genomic alterations in tumor tissue associated with presence of various subpopulations of circulating tumor cells (CTCs) in primary breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-05.