CDKN1B mutation and copy number variation are associated with tumor aggressiveness in luminal breast cancer.

The CDKN1B gene, encoding for the CDK inhibitor p27kip1 , 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 p27kip1 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 p27kip1 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 p27kip1 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. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

p27kip1 expression and phosphorylation dictate Palbociclib sensitivity in KRAS-mutated colorectal cancer.

In colorectal cancer, mutation of KRAS (RASMUT) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RASMUT colorectal cancer cells, we observed an increased expression of p27kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27kip1 showed variable expression in RASMUT colorectal cancer samples, our study supports the possibility that p27kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.

Plasma-Based Longitudinal Evaluation of ESR1 Epigenetic Status in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer.

BACKGROUND: Endocrine therapy (ET) is the mainstay of treatment for hormone receptor-positive human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer; however, adaptive mechanisms emerge in about 25-30% of cases through alterations in the estrogen receptor ligand-binding domain, with a consequent ligand-independent estrogen receptor activity. Epigenetic-mediated events are less known and potentially involved in alternative mechanisms of resistance. The aim of this study was to test the feasibility of estrogen receptor 1 (ESR1) epigenetic characterization through liquid biopsy and to show its potential longitudinal application for an early ET sensitivity assessment. METHODS: A cohort of 49 women with hormone receptor-positive HER2-negative MBC was prospectively enrolled and characterized through circulating tumor DNA using methylation-specific droplet digital PCR (MS-ddPCR) before treatment start (BL) and after 3 months concomitantly with computed tomography (CT) scan restaging (EV1). ESR1 epigenetic status was defined by assessing the methylation of its main promoters (promA and promB). The most established cell-free tumor DNA (ctDNA) factors associated with ET resistance [ESR1 and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations] were assessed through next-generation sequencing. Associations were tested through Mann-Whitney U test, matched pairs variations through Wilcoxon signed rank test, and survival was analyzed by log-rank test. RESULTS: The ET backbone was mainly based on aromatase inhibitors (AIs) (70.83%) in association with CDK4/6 inhibitors (93.75%). Significantly lower promA levels at baseline were observed in patients with liver metastases (P = 0.0212) and in patients with ESR1 mutations (P = 0.0091). No significant impact on PFS was observed for promA (P = 0.3777) and promB (P = 0.7455) dichotomized at the median while a ≥2-fold increase in promB or in either promA or promB at EV1 resulted in a significantly worse prognosis (respectively P = 0.0189, P = 0.0294). A significant increase at EV1 was observed for promB among patients with PIK3CA mutation (P = 0.0173). A trend was observed for promB in ESR1 wild-type patients and for promA in the ESR1 mutant subgroup. CONCLUSION: The study proofed the concept of an epigenetic characterization strategy based on ctDNA and is capable of being integrated in the current clinical workflow to give useful insights on treatment sensitivity.