Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Here, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare subpopulation of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. We find evidence for sub-clonal expression of a PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers.

The androgen receptor (AR) is a ligand-dependent transcription factor, and its effects on breast range from physiological pubertal development and age-related modifications to cancer onset and proliferation. The prevalence of AR in early breast cancer is around 60%, and AR is more frequently expressed in ER-positive than in ER-negative tumors. We offer an overview of AR signaling pathways in different breast cancer subtypes, providing evidence that its oncogenic role is likely to be different in distinct biological and clinical scenarios. In particular, in ER-positive breast cancer, AR signaling often antagonizes the growth stimulatory effect of ER signaling; in triple-negative breast cancer (TNBC), AR seems to drive tumor progression (at least in luminal AR subtype of TNBC with a gene expression profile mimicking luminal subtypes despite being negative to ER and enriched in AR expression); in HER2-positive breast cancer, in the absence of ER expression, AR signaling has a proliferative role. These data represent the rationale for AR-targeting treatment as a potentially new target therapy in breast cancer subset using androgen agonists in some AR-positive/ER-positive tumors, AR antagonists in triple-negative/AR-positive tumors and in combination with anti-HER2 agents or with other signaling pathways inhibitors (including PI3K/MYC/ERK) in HER2-positive/AR-positive tumors. Only the ongoing and future prospective clinical trials will allow us to establish which agents are the best option in every specific condition, keeping in mind that there is evidence of opposite androgens and AR agonist/antagonist drug effects on cell proliferation particularly in AR-positive/ER-positive tumors.

Although gene alterations of the RAS/RAF/MEK/ERK pathway are uncommon in breast cancer, this pathway is frequently activated in breast tumors, implying its role in tumor progression. We describe, after a revision of the literature, the frequency and types of gene alterations affecting this pathway in breast cancer by analyzing some public datasets from cBioPortal. Moreover, we consider their prognostic and predictive impact on treatment response, along with the role of transcriptomic predictors of RAS pathway activation. Our analysis shows that the driver alterations in RAS/RAF/MEK/ERK pathway-related genes are detected in 11% of primary breast cancers. The most frequently mutated genes are NF1 and KRAS, while copy number alterations mainly affect KRAS and BRAF, especially in basal-like tumors. The subgroup of patients carrying these alterations shows a worse prognosis; alterations in NF1 and RAF1 are associated with significantly reduced breast-cancer-specific survival in multivariate analysis. The literature review shows that the pathway is implicated, either by genetic or epigenetic alterations or by signaling network adaptations, in the mechanisms of sensitivity and resistance to a wide range of drugs used in the treatment of breast cancer. A thorough understanding of these alterations is critical for developing combination therapies that can delay or overcome drug resistance.

Deregulation of the cell cycle is a hallmark of cancer, and research on cell cycle control has allowed identification of potential targets for anticancer treatment. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which are involved, with their coregulatory partners cyclin D, in the G1-S transition. Inhibition of this step halts cell cycle progression in cells in which the involved pathway, including the retinoblastoma protein (Rb) and the E2F family of transcription factors, is functioning, although having been deregulated. Among breast cancers, those with functioning cyclin D-CDK4/6-Rb-E2F are mainly hormone-receptor (HR) positive, with some HER2-positive and rare triple-negative cases. Deregulation results from genetic or otherwise occurring hyperactivation of molecules subtending cell cycle progression, or inactivation of cell cycle inhibitors. Based on results of randomized clinical trials, palbociclib was granted accelerated approval by the US Food and Drug Administration (FDA) for use in combination with letrozole as initial endocrine-based therapy for metastatic disease in postmenopausal women with HR-positive, HER2-negative breast cancer, and was approved for use in combination with fulvestrant in women with HR-positive, HER2-negative advanced breast cancer with disease progression following endocrine therapy. This review provides an update of the available knowledge on the cell cycle and its regulation, on the alterations in cyclin D-CDK4/6-Rb-E2F axis in breast cancer and their roles in endocrine resistance, on the preclinical activity of CDK4/6 inhibitors in breast cancer, both as monotherapy and as partners of combinatorial synergic treatments, and on the clinical development of palbociclib in breast cancer.

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients’ surveillance and follow-up.

CDK4/6 inhibitors (CDK4/6i) are recommended in patients with estrogen receptor (ER)-positive, HER2-negative advanced breast cancer (ABC). Up to now, no prognostic biomarkers have been identified in this setting. We retrospectively analyzed the expression of progesterone receptor (PR) and Ki67, assessed by immunohistochemistry, in 71 ABC patients treated with CDK4/6i and analyzed the impact of these markers on progression-free survival (PFS). The majority of patients 63/71 (88.7%) received palbociclib, 4 (5.6%) received ribociclib, and 4 (5.6%) received abemaciclib. A higher median value of Ki67 was observed in cases undergoing second-line treatment (p = 0.047), whereas the luminal B subtype was more prevalent (p = 0.005). In the univariate analysis of the first-line setting, luminal A subtype showed a trend towards a correlation with a longer PFS (p = 0.053). A higher continuous Ki67 value led to a significantly shorter PFS. When the interaction between pathological characteristics and line of treatment was considered, luminal B subtype showed a significantly (p = 0.043) worse outcome (Hazard Ratio (HR) 2.84; 1.03–7.82 95% Confidence Interval (CI)). PFS in patients undergoing endocrine therapy plus CDK4/6i was inversely correlated with Ki67 expression but not with PR, suggesting that tumor proliferation has a greater impact on cell cycle inhibitors combined with endocrine therapy than PR expression.