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Purpose: In hormone receptor–positive (HR+)/HER2− metastatic breast cancer (MBC), it is imperative to identify patients who respond poorly to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) and to discover therapeutic targets to reverse this resistance. Non-luminal breast cancer subtype and high levels of CCNE1 are candidate biomarkers in this setting, but further validation is needed. Experimental Design: We performed mRNA gene expression profiling and correlation with progression-free survival (PFS) on 455 tumor samples included in the phase III PEARL study, which assigned patients with HR+/HER2− MBC to receive palbociclib+endocrine therapy (ET) versus capecitabine. Estrogen receptor–positive (ER+)/HER2− breast cancer cell lines were used to generate and characterize resistance to palbociclib+ET. Results: Non-luminal subtype was more prevalent in metastatic (14%) than in primary tumor samples (4%). Patients with non-luminal tumors had median PFS of 2.4 months with palbociclib+ET and 9.3 months with capecitabine; HR 4.16, adjusted P value < 0.0001. Tumors with high CCNE1 expression (above median) also had worse median PFS with palbociclib+ET (6.2 months) than with capecitabine (9.3 months); HR 1.55, adjusted P value = 0.0036. In patients refractory to palbociclib+ET (PFS in the lower quartile), we found higher levels of Polo-like kinase 1 (PLK1). In an independent data set (PALOMA3), tumors with high PLK1 show worse median PFS than those with low PLK1 expression under palbociclib+ET treatment. In ER+/HER2− cell line models, we show that PLK1 inhibition reverses resistance to palbociclib+ET. Conclusions: We confirm the association of non-luminal subtype and CCNE1 with resistance to CDK4/6i+ET in HR+ MBC. High levels of PLK1 mRNA identify patients with poor response to palbociclib, suggesting PLK1 could also play a role in the setting of resistance to CDK4/6i.

Descriptive analysis of the clinical characteristics of the two main clusters resulted from the unsupervised hierarchical clustering of fifty-one differentially expressed genes in extreme responders to palbociclib plus ET

List of 51 genes differentially expressed in tumors, grouped by sensitive or refractory to palbociclib plus ET. Controlling the FDR at 5% with the q-value method (SAM)

Purpose:In hormone receptor–positive (HR+)/HER2− metastatic breast cancer (MBC), it is imperative to identify patients who respond poorly to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) and to discover therapeutic targets to reverse this resistance. Non-luminal breast cancer subtype and high levels of CCNE1 are candidate biomarkers in this setting, but further validation is needed.Experimental Design:We performed mRNA gene expression profiling and correlation with progression-free survival (PFS) on 455 tumor samples included in the phase III PEARL study, which assigned patients with HR+/HER2− MBC to receive palbociclib+endocrine therapy (ET) versus capecitabine. Estrogen receptor–positive (ER+)/HER2− breast cancer cell lines were used to generate and characterize resistance to palbociclib+ET.Results:Non-luminal subtype was more prevalent in metastatic (14%) than in primary tumor samples (4%). Patients with non-luminal tumors had median PFS of 2.4 months with palbociclib+ET and 9.3 months with capecitabine; HR 4.16, adjusted P value < 0.0001. Tumors with high CCNE1 expression (above median) also had worse median PFS with palbociclib+ET (6.2 months) than with capecitabine (9.3 months); HR 1.55, adjusted P value = 0.0036. In patients refractory to palbociclib+ET (PFS in the lower quartile), we found higher levels of Polo-like kinase 1 (PLK1). In an independent data set (PALOMA3), tumors with high PLK1 show worse median PFS than those with low PLK1 expression under palbociclib+ET treatment. In ER+/HER2− cell line models, we show that PLK1 inhibition reverses resistance to palbociclib+ET.Conclusions:We confirm the association of non-luminal subtype and CCNE1 with resistance to CDK4/6i+ET in HR+ MBC. High levels of PLK1 mRNA identify patients with poor response to palbociclib, suggesting PLK1 could also play a role in the setting of resistance to CDK4/6i.

Descriptive analysis of extreme responders subpopulation, according to clinical characteristics and tumor subtype

Evaluation of volasertib effects on tumor spheroid growth and on the activation of signal transduction pathways in PalboR and FPR cells

Effects of PLK1 gene modulation on cell sensitivity to palbociclib alone or in combination with fulvestrant in ER+ breast cancer cell lines

PALOMA-3 study as validation dataset

Volasertib overcomes resistance to fulvestrant and palbociclib in PLK1-overexpressing ER+ breast cancer cells

Generation and characterization of ER+ breast cancer cell lines resistant to palbociclib alone or in combination with fulvestrant

Patients’ characteristics and Progression Free Survival comparison. Biomarker Cohort and PEARL Study Population

Progression Free Survival analysis on CCNE1 expression levels under palbociclib plus ET treatment

Progression Free Survival analysis on intrinsic subtypes

Drug combination studies in MCF7, T47D and T47D-FPR cells

CONSORT diagram of the study

Analysis of gene expression associated with treatment benefit

1014 Background: The randomized PEARL trial found no superiority of palbociclib plus endocrine therapy over capecitabine in patients (pts) with metastatic HR-positive, HER2-negative breast cancer resistant to prior aromatase inhibitors (Martin M, Ann Oncol 2020). Gene expression analysis showed high CCNE1 mRNA ( CCNE1) conferring relative resistance to palbociclib in the PALOMA-3 trial (Turner N, JCO 2019), but further validation is needed. Cyclin E1 protein (cyclin E1) expression in this context has not been studied in randomized trials. We explored CCNE1 and cyclin E1 as predictive biomarkers in tumor samples from the PEARL study. Methods: Formalin-fixed paraffin-embeded tumor samples were retrieved from pts enrolled in PEARL cohort 2 (palbociclib (PAL) + fulvestrant (FUL) vs capecitabine (CAPE)). We measured CCNE1 using the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics). Cyclin E1 immunohistochemistry (IHC) staining was performed using specific mouse monoclonal antibody HE12 (Abcam) and scored as percentage of invasive nuclei stained (0-100%). CCNE1 and cyclin E1 correlations were explored using Pearson coefficients. Cox regression models were used for progression free-survival (PFS) analyses using expression levels split by median, to define high ( > median values) vs. low expression. Site of disease and prior chemotherapy were used as confounders in multivariate models. Results: Analyses were conducted in 219 pts (47% receiving PAL + FUL and 53% CAPE) with available tumors, with the analysed patients representative of the overall study. Most samples were from the archival primary (72%), obtained > 5 years before this analysisº (74%). CCNE1 and cyclin E1 were only moderately correlated (r = 0.5). Median CCNE1 was higher in metastatic vs primary (7.37 vs 6.94, p < 0.01), and in luminal B and non-luminal subtypes compared to luminal A (p < 0.001). In patients with high CCNE1 expression, median PFS on CAPE was 10.35 and PAL + FUL was 5.68 (HR = 1.63, 95% CI 1.02-2.59, p = 0.042). In patients with low CCNE1 expression, median PFS on CAPE was 9.43 and PAL + FUL was 8.97 (adjusted HR = 0.93, 95% CI 0.59-1.48, p = 0.762, interaction p = 0.072). Median cyclin E1 protein was higher in luminal B and non-luminal subtypes compared to luminal A (p < 0.01). Cyclin E1 protein expression was not predictive of treatment effect (high cyclin E1 expression CAPE vs PAL + FUL HR = 1.17, low cyclin E1 expression CAPE vs PAL + FUL HR = 1.21, interaction p = 0.977). Conclusions: High tumor CCNE1 mRNA expression identified patients with relative resistance to palbociclib plus fulvestrant, validating prior observations although without statistical significance for interaction. Assessment of Cyclin E1 protein expression did not show predictive value. Investigation treatments to enhance CDK4/6 inhibitor efficacy in tumors with high CCNE1 expression is warranted. Clinical trial information: NCT02028507 .

To examine the nature of the symptom cluster of emotional distress, fatigue, and cognitive difficulties in young and older breast cancer survivors (BCS); To assess the mediating role of subjective stress and coping strategies (emotional control and meaning-focused coping) in the association between age and symptom cluster.Participants were 170 BCS, stages I-III, 1-12 months post-chemotherapy, filled-out the Fatigue, Emotional Control, Meaning-focused Coping, Emotional Distress and the Cognitive Difficulties Questionnaires. Statistical analyses included tests for difference between-groups Pearson correlations and Structural Equation Modeling for the assessment of the study model.Older BCS (aged 60-82) reported lower levels of emotional distress (M = 0.87, SD = 0.87), fatigue (M = 3.85, SD = 2.38), and cognitive difficulties (M = 1.17, SD = 1.07) compared to the younger BCS (aged 24-59) (emotional distress M = 1.17, SD = 0.85, fatigue M = 5.02, SD = 2.32, and cognitive difficulties M = 1.66, SD = 1.23, p .01-,05). The older survivors reported lower levels of subjective stress and used more emotional control strategies compared to the younger BCS. The empirical model had good fit indices (χ2 = 27.60, p = 0.20, χ2/df = 1.26; CFI = 0.98; TLI = 0.98; NFI = 0.95; RMSEA = 0.04 (90% CI = 0.00, 10) and showed that subjective stress, but not coping strategies, mediated the effect of age on symptom cluster severity.Lower levels of subjective stress, but not coping strategies, mediated the association of age with the symptom cluster of emotional distress, fatigue and cognitive difficulties. Further research is needed to explore differences in subjective stress by age.