RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix™ technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach.In this study, we established a workflow including the micro-fluidic Parsortix™ technology for the molecular detection of CTC related transcripts. Background levels of EpCAM, PPIC, TUSC3, and MAL2 were efficiently removed due to an up to 106-fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix™-enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%).The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.

Objective To analyze the characteristics of invasive lobular carcinoma (ILC) compared with invasive ductal carcinoma (IDC) and to investigate the impact of histology on axillary lymph node (ALN) involvement in luminal A subtype tumors. Methods We retrospectively analyzed patients diagnosed with ILC or IDC from 2012 to 2016 who underwent surgery. Patients constituted 493 primary early breast cancer cases (82 ILC; 411 IDC). Results Compared with IDC, ILC tumors were significantly more likely to be grade 2, estrogen receptor- (ER) positive (+), have a lower proliferation rate (Ki67 <14%), and a higher pathological T stage (pT2–4). The luminal A subtype was significantly more common in ILC compared with IDC. In a multivariate regression model, grade 2, ER+, progesterone receptor-positive, pT2, and pT3 were significantly associated with ILC. Additionally, with the luminal A subtype, ALN involvement (pathological node stage (pN)1–3) was significantly more frequent with ILC versus IDC. Conclusions Our data suggest that grade 2, positive hormone receptor status, and higher pathological T stage are associated with ILC. With the luminal A subtype, ALN involvement was more frequent with ILC versus IDC.

BRCA-1 mutation-associated triple-negative breast cancer (TNBC) has been hypothesized to exhibit a phenotype that is distinct from non-mutation carriers. We have analyzed immunohistochemically detected cytokeratins 5 and 14, epidermal growth factor receptor (EGFR), claudin (CLDN) 3, 4, and 7, and E-cadherin in 57 TNBC (32 BRCA1 and 8 BRCA2 tumors, 17 WT tumors). Positive staining of CLDN3 and negative EGFR expression in TNBC are associated with a BRCA1 mutation. EGFR and CLDN3 expression was able to predict the presence of BRCA1 mutation (area under curve 0.802, p < 0.001). This could help in guiding the decision for BRCA testing.