부산대학교 도서관

Keywords: CDKN1B; p27; mutation; copy number variation, CNV; breast cancer; young breast cancer patients; liquid biopsy; ovarian cancer; head and neck squamous cell carcinoma Abstract The CDKN1B gene, encoding for the CDK inhibitor p27.sup.kip1, is mutated in defined human cancer subtypes, including breast, prostate carcinomas and small intestine neuroendocrine tumors. Lessons learned from small intestine neuroendocrine tumors suggest that CDKN1B mutations could be subclonal, raising the question of whether a deeper sequencing approach could lead to the identification of higher numbers of patients with mutations. Here, we addressed this question and analyzed human cancer biopsies from breast (n=396), ovarian (n=110) and head and neck squamous carcinoma (n=202) patients, using an ultra-deep sequencing approach. Notwithstanding this effort, the mutation rate of CDKN1B remained substantially aligned with values from the literature, showing that essentially only hormone receptor-positive breast cancer displayed CDKN1B mutations in a relevant number of cases (3%). However, the analysis of copy number variation showed that another fraction of luminal breast cancer displayed loss (8%) or gain (6%) of the CDKN1B gene, further reinforcing the idea that the function of p27.sup.kip1 is important in this type of tumor. Intriguingly, an enrichment for CDKN1B alterations was found in samples from premenopausal luminal breast cancer patients (n=227, 4%) and in circulating cell-free DNA from metastatic luminal breast cancer patients (n=59, 8.5%), suggesting that CDKN1B alterations could correlate with tumor aggressiveness and/or occur later during disease progression. Notably, many of the identified somatic mutations resulted in p27.sup.kip1 protein truncation, leading to loss of most of the protein or of its C-terminal domain. Using a gene-editing approach in a luminal breast cancer cell line, MCF-7, we observed that the expression of p27.sup.kip1 truncating mutants that lose the C-terminal domains failed to rescue most of the phenotypes induced by CDKN1B gene knockout, indicating that the functions retained by the C-terminal portion are critical for its role as an oncosuppressor, at least in luminal breast cancer. [c] 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. Article Note: Equal contributions B Belletti and G Baldassarre share last co-authorship. No conflicts of interest were declared. CAPTION(S): Supplementary materials and methods Figure S1. p27 protein expression correlates with CDKN1B copy number in breast cancer samples Figure S2. p27 mutations in liquid biopsies of metastatic breast cancer patients correlates with worse progression-free survival Figure S3. CDKN1B truncation mutants display altered protein stability compared with the WT form Figure S4. Loss of CDKN1B and expression of C-terminal deletion mutants alter S-phase entry and cytonuclear localization in LBC cells Figure S5. Silencing of p27 induces increased anchorage independent growth in LBC cells Figure S6. Loss of CDKN1B alters the growth of LBC cells in 3D-Matrigel Table S1. Clinicopathological features of young ( Table S2. Clinicopathological features of breast cancer patients analyzed by liquid biopsy (n=62) Table S3. Clinicopathological features of ovarian cancer patients (n=110) Table S4. Clinicopathological features of head and neck cancer patients (n=202) Table S5. Mutations of CDKN1B detected by multi-gene panel and not confirmed by targeted sequencing in breast cancer patients Byline: Davide Viotto, Francesca Russo, Ilaria Anania, Ilenia Segatto, Gian Luca Rampioni Vinciguerra, Alessandra Dall'Acqua, Riccardo Bomben, Tiziana Perin, Martina Cusan, Monica Schiappacassi, Lorenzo Gerratana, Sara D'Andrea, Francesca Citron, Filippo Vit, Lorena Musco, Maria Chiara Mattevi, Giorgia Mungo, Milena S Nicoloso, Maura Sonego, Samuele Massarut, Roberto Sorio, Luigi Barzan, Giovanni Franchin, Giorgio Giorda, Emilio Lucia, Sandro Sulfaro, Vittorio Giacomarra, Jerry Polesel, Federica Toffolutti, Vincenzo Canzonieri, Fabio Puglisi, Valter Gattei, Andrea Vecchione, Barbara Belletti, Gustavo Baldassarre