title: Molecular characterization of CD44+/CD24-/Ck+/CD45-cells in benign and malignant breast lesions.
abstract: Breast cancer epithelial cells with the CD44+/CD24-/lowphenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24-/cytokeratin(Ck)+/CD45-cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24-/Ck+/CD45-cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24-/Ck+/CD45-cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24-/Ck+/CD45-cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.
authors.affiliation: Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Jorge Viterbo Ferreira Street, n° 288, 4050-313, Porto, Portugal. arnaudcpaula@hotmail.com.|Department of Pathology, Portuguese Oncology Institute (IPO), Porto, Portugal. arnaudcpaula@hotmail.com.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Da Cruz Paula
authors.affiliation: Institute of Research and Health Innovation, University of Porto, Porto, Portugal.|IBMC - Institute of Molecular and Cell Biology, Porto, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Leitão
authors.affiliation: Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Jorge Viterbo Ferreira Street, n° 288, 4050-313, Porto, Portugal.|Institute of Research and Health Innovation, University of Porto, Porto, Portugal.|Unit for Multidisciplinary Biomedical Research (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal.|Basic and Clinical Research on Iron Biology, Institute of Molecular and Cell Biology (IBMC)/i3s, Porto, Portugal.
authors.forename: Oriana
authors.initials: O
authors.lastname: Marques
authors.affiliation: Institute of Research and Health Innovation, University of Porto, Porto, Portugal.|Basic and Clinical Research on Iron Biology, Institute of Molecular and Cell Biology (IBMC)/i3s, Porto, Portugal.
authors.forename: Ana Margarida
authors.initials: AM
authors.lastname: Rosa
authors.affiliation: IBMC - Institute of Molecular and Cell Biology, Porto, Portugal.|Laboratory of Cytometry, Department of Hematology, Santo António Hospital (HSA), Porto Hospital Centre (CHP), Porto, Portugal.
authors.forename: Ana Helena
authors.initials: AH
authors.lastname: Santos
authors.affiliation: Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Jorge Viterbo Ferreira Street, n° 288, 4050-313, Porto, Portugal.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Rêma
authors.affiliation: Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Jorge Viterbo Ferreira Street, n° 288, 4050-313, Porto, Portugal.
authors.forename: Maria
authors.initials: M
authors.lastname: de Fátima Faria
authors.affiliation: IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.|Medical Faculty of the University of Porto, Porto, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Rocha
authors.affiliation: Institute of Research and Health Innovation, University of Porto, Porto, Portugal.|IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.|Medical Faculty of the University of Porto, Porto, Portugal.
authors.forename: José Luís
authors.initials: JL
authors.lastname: Costa
authors.affiliation: IBMC - Institute of Molecular and Cell Biology, Porto, Portugal.|Laboratory of Cytometry, Department of Hematology, Santo António Hospital (HSA), Porto Hospital Centre (CHP), Porto, Portugal.
authors.forename: Margarida
authors.initials: M
authors.lastname: Lima
authors.affiliation: Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Jorge Viterbo Ferreira Street, n° 288, 4050-313, Porto, Portugal. calopes@icbas.up.pt.|Department of Pathology, Portuguese Oncology Institute (IPO), Porto, Portugal. calopes@icbas.up.pt.
authors.forename: Carlos
authors.initials: C
authors.lastname: Lopes
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D001941:Breast Diseases / Q000235:genetics / Q000401:mortality / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics / Q000401:mortality / Q000473:pathology*; D015820:Cadherins / Q000032:analysis / Q000096:biosynthesis; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000401:mortality / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000235:genetics / Q000401:mortality / Q000473:pathology; D004252:DNA Mutational Analysis; D005260:Female; D005434:Flow Cytometry; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000096:biosynthesis; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000096:biosynthesis; D014411:Neoplastic Stem Cells / Q000473:pathology*; D010641:Phenotype; D000090063:Proto-Oncogene Mas
chemical_list: D014408:Biomarkers, Tumor; C497483:CD44 protein, human; D015820:Cadherins; D018960:Hyaluronan Receptors; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
keywords: Breast cancer; Breast cancer stem cell markers; CD24; CD44; Ion Torrent AmpliSeq Cancer Hotspot panel v2; Next-generation sequencing