A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors

Publisher's version (útgefin grein).Human breast cancer is believed to arise in luminal progenitors within the normal breast. A subset of these are double positive (DP) for basal and luminal keratins and localizes to a putative stem cell zone within ducts. We here present a new protocol based on a combination of CD146 with CD117 and CD326 which provides an up to thirty fold enrichment of the DP cells. We show by expression profiling, colony formation, and morphogenesis that CD146high/CD117high/CD326high DP cells belong to a luminal progenitor compartment. While these DP cells are located quite uniformly in ducts, with age a variant type of DP (vDP) cells, which is mainly CD146-negative, accumulates in lobules. Intriguingly, in specimens with BRCA1 mutations known to predispose for cancer, higher frequencies of lobular vDP cells are observed. We propose that vDP cells are strong candidates for tracing the cellular origin of breast cancer.We thank Lena Kristensen, Tove Marianne Lund and Anita Sharma Friismose for expert technical assistance. Benedikte Thuesen and Trine Foged Henriksen, Capio CFR Hospitaler are acknowledged for providing breast biopsy material. The Core Facility for Integrated Microscopy (University of Copenhagen) is acknowledged for confocal microscope accessibility. This work was supported by Novo Nordisk Fonden and Danish Research Council grant 10-092798 (to DanStem), Toyota-Fonden Denmark and Anita og Tage Therkelsens Fond (to R.V.), Familien Erichsens Mindefond and Vera og Carl Johan Michaelsens Legat (to J.K.), Harboefonden, Else og Mogens Wedell-wedellborgs Fond and Danish Cancer Society Grant R146-A9257 (to L.R.-J.).Peer Reviewe

Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients

We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules

Immunoexpression analysis and prognostic value of BLCAP in breast cancer

Bladder Cancer Associated Protein (BLCAP, formerly Bc10), was identified by our laboratory as being down-regulated in bladder cancer with progression. BLCAP is ubiquitously expressed in different tissues, and several studies have found differential expression of BLCAP in various cancer types, such as cervical and renal cancer, as well as human tongue carcinoma and osteosarcoma. Here we report the first study of the expression patterns of BLCAP in breast tissue. We analyzed by immunohistochemistry tissue sections of normal and malignant specimens collected from 123 clinical high-risk breast cancer patients within the Danish Center for Translational Breast Cancer Research (DCTB) prospective study dataset. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. We observed weak immunoreactivity for BLCAP in mammary epithelial cells, almost exclusively localizing to the cytoplasm and found that levels of expression of BLCAP were generally higher in malignant cells as compared to normal cells. Quantitative IHC analysis of BLCAP expression in breast tissues confirmed this differential BLCAP expression in tumor cells, and we could establish, in a 62-patient sample matched cohort, that immunostaining intensity for BLCAP was increased in tumors relative to normal tissue, in more than 45% of the cases examined, indicating that BLCAP may be of value as a marker for breast cancer. We also analyzed BLCAP expression and prognostic value using a set of tissue microarrays comprising an independent cohort of 2,197 breast cancer patients for which we had follow-up clinical information