The recent arrival of CDK4/6 inhibitor agents, with an approximate doubling of progression-free survival (PFS) associated with their use in hormone receptor-positive, HER2-negative advanced breast cancer (BC), has radically changed the approach to managing this disease. However, resistance to CDK4/6 inhibitors is considered a near-inevitability in most patients. Mechanisms of resistance to these agents are multifactorial, and research in this field is still evolving. Biomarkers with the ability to identify early resistance, or to predict the likelihood of successful treatment using CDK4/6 inhibitors are yet to be identified, and represent an area of unmet clinical need. Here we present selected mechanisms of resistance to CDK4/6 inhibitors, largely focussing on roles of Rb, cyclin E1, and the PIK3CA pathway, with discussion of associated biomarkers which have been investigated and applied in recent pre-clinical and clinical studies. These biological drivers may furthermore influence clinical treatment strategies adopted beyond CDK4/6 resistance.

Palbociclib is a CDK4/6 inhibitor that received FDA approval for treatment of hormone receptor positive (HR+) HER2 negative (HER2neg) advanced breast cancer. To better personalize patients treatment it is critical to identify subgroups that would mostly benefit from it. We hypothesize that complex alterations of the Retinoblastoma (Rb) pathway might be implicated in resistance to CDK4/6 inhibitors and aim to investigate whether signatures of Rb loss-of-function would identify breast cancer cell lines resistant to palbociclib. We established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in breast cancers within The Cancer Genome Atlas. We assessed the RBsig prognostic role in the METABRIC and in a comprehensive breast cancer meta-dataset. Finally, we analyzed whether RBsig would discriminate palbociclib-sensitive and -resistant breast cancer cells in a large RNA sequencing-based dataset. The RBsig was associated with RB1 genetic status in all tumors (p <7e-32) and in luminal or basal subtypes (p < 7e-11 and p < 0.002, respectively). The RBsig was prognostic in the METABRIC dataset (discovery: HR = 1.93 [1.5-2.4] p = 1.4e-08; validation: HR = 2.01 [1.6-2.5] p = 1.3e-09). Untreated and endocrine treated patients with estrogen receptor positive breast cancer expressing high RBsig had significantly worse recurrence free survival compared to those with low RBsig (HR = 2.37 [1.8 − 3.2] p = 1.87e−08 and HR = 2.62 [1.9− 3.5] p = 8.6e−11, respectively). The RBsig was able to identify palbociclib resistant and sensitive breast cancer cells (ROC AUC = 0,7778). Signatures of RB loss might be helpful in personalizing treatment of patients with HR+/HER2neg breast cancer. Further validation in patients receiving palbociclib is warranted.

Randomized clinical trials demonstrated that CDK4/6 inhibitors are highly effective in patients with hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer in combination with endocrine therapy. The use of CDK4/6 inhibitors in clinics is becoming common for patients with HR+/HER2- metastatic breast cancer and will certainly increase in the near future. However, patients might show de novo or acquired resistance to these drugs. Molecular alterations have been suggested as determinants for de novo resistance to CDK4/6 inhibitors, but have never been validated in a clinical setting. In addition, molecular mechanisms of acquired resistance to palbociclib have been analyzed only in preclinical studies. Here we review the current knowledge on the available preclinical data about the mechanisms of de novo and acquired resistance to CDK4/6 inhibitors in breast cancer, and clinical data about potential biomarkers of response.

Purpose: Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC. Experimental Design: Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor–positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the “To Reverse ENDocrine Resistance” (TREnd) trial (n = 46). Results: Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase—correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05). Conclusions: TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.

Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy