Analysis of EpCAM positive cells isolated from sentinel lymph nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles

Abstract

IntroductionThe presence of tumor cells in the axillary lymph nodes is the most important prognostic factor in early stage breast cancer. However, the optimal method for sentinel lymph node (SLN) examination is still sought and currently many different protocols are employed. To examine two approaches for tumor cell detection we performed, in sequence, immunomagnetic enrichment and RT-PCR analysis on SLN samples from early stage breast cancer patients. This allowed us to compare findings based on the expression of cell surface proteins with those based on detection of intracellular transcripts.MethodsEnrichment of EpCAM and Mucin 1 expressing cells from fresh SLN samples was achieved using magnetic beads coated with the appropriate antibodies. All resulting cell fractions were analyzed by RT-PCR using four chosen breast epithelial markers (hMAM, AGR2, SBEM, TFF1). Gene expression was further analyzed using RT-PCR arrays and markers for epithelial to mesenchymal transition (EMT).ResultsBoth EpCAM and Mucin 1 enriched for the epithelial-marker expressing cells. However, EpCAM-IMS identified epithelial cells in 71 SLNs, whereas only 35 samples were positive with RT-PCR targeting breast epithelial transcripts. Further analysis of EpCAM positive but RT-PCR negative cell fractions showed that they had increased expression of MMPs, repressors of E-cadherin, SPARC and vimentin, all transcripts associated with the process of epithelial to mesenchymal transition.ConclusionsThe EpCAM IMS-assay detected tumor cells with epithelial and mesenchymal-like characteristics, thus proving to be a more robust marker than pure epithelial derived biomarkers. This finding has clinical implications, as most methods for SLN analysis today rely on the detection of epithelial transcripts or proteins.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells

“…Neither in DG75 CBF1 wt nor in DG75 CBF1 ko cells statistically significant changes (p ≤ 0.05) of cellular transcript abundance in response to estrogen treatment were observed, proving that target gene activation is strictly dependent on ER/EBNA2 (S1A Fig). In addition, estrogen responsive target genes described in the literature did not change expression levels proving that the estrogen receptor response is not functional in DG75 B cells (S1B Fig) [23–27]. It is important to note that EBNA2 not only activates a set of direct target genes but thereby initiates a cascade of secondary events, which are included in our target gene lists and in total reflect EBNA2 functions.…”

Immediate Postoperative Inflammation Is an Important Prognostic Factor in Breast Cancer

“…There have been several studies showing that this inflammation was associated with cancer progression. Many authors have shown that inflammation can enhance tumor cell proliferation, invasion, angiogenesis, and eventual metastasis [17,18,19,20,21,22,23,24]. …”

“…Surgery-induced inflammation could induce tumor growth, modify the immune response, and even exacerbate tumor dissemination [17,18,19,20,21,22,23,24]. Therefore, we hypothesized that the inflammation induced by surgery could impact the clinical outcome in breast cancer.…”

The estrogen-regulated anterior gradient 2 (AGR2) protein in breast cancer: a potential drug target and biomarker

“…Detection of metastatic cells in the lymph node or circulating tumor cells (CTCs) in the peripheral blood is strongly associated with poor clinical outcome in breast cancer [41-43]. AGR2 may be a fitting target for detection of metastatic cells as it has been found to promote metastasis in functional experiments and to be expressed in metastatic breast cancer cells in humans.…”