Da Cruz Paula Arnaud, Leitão Catarina, Marques Oriana, Rosa Ana Margarida, Santos Ana Helena, Rêma Alexandra, de Fátima Faria Maria, Rocha Ana, Costa José Luís, Lima Margarida, Lopes Carlos
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.
Department of Pathology, Portuguese Oncology Institute (IPO), Porto, Portugal.
Virchows Arch. 2017 Mar;470(3):311-322. doi: 10.1007/s00428-017-2068-4. Epub 2017 Jan 23.
Breast cancer epithelial cells with the CD44/CD24 phenotype 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.
具有CD44/CD24表型的乳腺癌上皮细胞具有肿瘤起始细胞和上皮-间质转化（EMT）能力。大规模平行测序可能是深入了解这些细胞分子特征的一种有趣方法。我们对通过流式细胞术从43例活检和6例乳房切除术样本中分离出的CD44/CD24/细胞角蛋白（Ck）/CD45细胞进行了特征分析，这些样本包含不同的良性和恶性乳腺病变。使用Ion Torrent Ampliseq癌症热点面板v2（CHPv2）来鉴定从分离出的CD44/CD24/Ck/CD45细胞中提取的DNA中的体细胞突变。对相应的福尔马林固定、石蜡包埋（FFPE）块的切片进行E-钙黏蛋白和波形蛋白免疫组织化学检测。从非恶性病变到恶性病变，CD44/CD24/Ck/CD45细胞的百分比显著增加，并且与波形蛋白表达的显著增加相关。非恶性病变仅存在单核苷酸多态性（SNP）。在原位导管癌（DCIS）分离出的CD44/CD24/Ck/CD45细胞中发现了肿瘤抑制基因p53（TP53）、Notch同源物1（NOTCH1）、磷酸酶和张力蛋白同源物（PTEN）以及v-akt小鼠胸腺瘤病毒癌基因同源物1（AKT1）的突变。在浸润性导管癌（IDC）中还鉴定出了集落刺激因子1受体（CSF1R）、原癌基因ret（RET）和TP53基因的其他突变。即使使用从少量细胞中提取的少量DNA，大规模平行测序技术在这类应用中也显示出极其有效。现在需要使用更大的队列进行更多研究，以设计出适合这种表型的突变谱。
